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https://openalex.org/W3194629514	https://www.spractice.ru/jour/article/download/222/172	Russian	null	THERAPEUTIC TACTICS FOR PEPTIC STRICTURES OF THE ESOPHAGUS. LITERATURE REVIEW	Hirurgičeskaâ praktika	2,021	cc-by	7,195	THERAPEUTIC TACTICS FOR PEPTIC STRICTURES OF THE ESOPHAGUS. LITERATURE REVIEW GANKOV V.A., ANDREASYAN A.R., MASLIKOVA S.A., BAGDASARYAN G.I. Federal State Budgetary Educational Institution of Higher Education "Altai State Medical University" of the Ministry of Health of the Russian Federation, Lenin Avenue, 40, 656038, Altai Krai, Barnaul, Russian Federation. Federal State Budgetary Educational Institution of Higher Education "Altai State Medical University" of the Ministry of Health of the Russian Federation, Lenin Avenue, 40, 656038, Altai Krai, Barnaul, Russian Federation. ЛЕЧЕБНАЯ ТАКТИКА ПРИ ПЕПТИЧЕСКИХ СТРИКТУРАХ ПИЩЕВОДА. ОБЗОР ЛИТЕРАТУРЫ ГАНКОВ В.А., АНДРЕАСЯН А.Р., МАСЛИКОВА С.А., БАГДАСАРЯН Г.И., НДРЕАСЯН А.Р., МАСЛИКОВА С.А., БАГДАСАРЯН Г.И., ШЕСТАКОВ Д.Ю Федеральное государственное бюджетное образовательное учреждение высшего образования «Алтайский государствен ный медицинский университет» Министерства здравоохранения Российской Федерации, проспект Ленина, д. 40, 656038, Алтайский край, г. Барнаул, Российская Федерация. Keywords: peptic stricture, bougienage, antireflux surgery, review. Реферат: ф р Работа основана на анализе литературных данных, посвященных проблеме выбора лечения пептических стриктур пищевода. Главная цель данного обзора – выявить тактику лечения больных со стенозирующим рефлюкс-эзофагитом. ота основана на анализе литературных данных, посвященных проблеме выбора лечения пептических стри ного обзора – выявить тактику лечения больных со стенозирующим рефлюкс-эзофагитом. Исследователи указывают, что основными причинами возникновения ГЭРБ является уменьшение давления в нижнем пищеводном сфинктере, действие повреждающих свойств рефлюктанта. Несвоевременное лечение ГЭРБ может привести к таким осложнениям как пептическая стрик тура, пищевод Баррета. Появлению стриктуры ГЭРБ чаще всего способствуют: постоянная изжога после бужирования, эрозии нижней трети пищевода, укорочение пищевода II степени, а также неадекватная антисекреторная терапия. у р р р р Приведены различные методы лечения на всех этапах появления пептической стриктуры, в зависимости от степени дисфагии и протяжен ности стриктуры, применение адекватных схем консервативной терапии ИПП, бужирования, а также описание различных методик антиреф люксных операций. Эндоскопическая дилатация является первым вариантом лечения всех симптоматических доброкачественных стриктур пищевода. Существуют такие методы лечения доброкачественных рефрактерных стриктур пищевода как эндоскопическая дилатация с внутрипросветной инъекцией стероидов, эндоскопическая послеоперационная терапия или стриктуропластика, стентирование пищевода, самобужирование, а также хи рургическое вмешательство – атирефлюксная операция, эзофагэктомия с замещение пищевода желудком или толстой кишкой [1]. Эндоскопическая дилатация является первым вариантом лечения всех симптоматических доброкачественных стриктур пищевода. Существуют такие методы лечения доброкачественных рефрактерных стриктур пищевода как эндоскопическая дилатация с внутрипросветной инъекцией стероидов, эндоскопическая послеоперационная терапия или стриктуропластика, стентирование пищевода, самобужирование, а также хи рургическое вмешательство – атирефлюксная операция, эзофагэктомия с замещение пищевода желудком или толстой кишкой [1]. Основной целью в лечении пептических стриктур пищевода по мнению большинства авторов является устранение прогрессирования ГЭРБ, проведение бужирования или баллонной дилатации, стентирование, выбор оптимальной антирефлюксной операции. Лечебная тактика при пептических стриктурах должна минимизировать риск повторной стриктуры пищевода. рур р ф р ф у Основной целью в лечении пептических стриктур пищевода по мнению большинства авторов является устранение прогрессирования ГЭРБ, проведение бужирования или баллонной дилатации, стентирование, выбор оптимальной антирефлюксной операции. Лечебная тактика при пептических стриктурах должна минимизировать риск повторной стриктуры пищевода. Хирургическая практика Хирургическая практика №2 (44), 2021 Основная часть На протяжении многих лет терапевты, гастроэнтеро логи и хирурги пытаются предупредить прогрессирова ние гастроэзофагеальной рефлюксной болезни (ГЭРБ), которая приводит к различным осложнениям. Одним из грозных осложнений ГЭРБ является пептическая стрик тура пищевода, которая возникает вследствие воздей ствия повреждающих факторов содержимого желудка на слизистую оболочку, мышечный слой пищевода и не редко воспалительный процесс переходит на параэзофа геальную клетчатку [5, 10, 11, 12]. Частота встречаемости данной патологии по данным разных авторов составляет от 7 до 13%. [10, 11, 13]. Согласно клиническим рекомендациям больные с рефрактерным течением заболеваниям и при ослож нении ГЭРБ (стриктуры, пищевод Баррета) рекоменду ется проведение обследования: внутрипищеводной рН-метрии, манометрии пищеводно-желудочного пере хода, рентгеноскопии пищевода и желудка, УЗИ органов брюшной полости. При осложнениях ГЭРБ применяются курсы внутри венных форм ингибиторов протонной помпы. Для лече ния пептических стриктур необходимо бужирование пищевода, баллонная дилатация, инъекция стероидов в месте стриктуры во время дилатации, аргоно-плазмен ная коагуляция стриктуры, стентирование и оператив ное лечение [4]. Многие авторы отмечают появление пептических стриктур вследствие неадекватной консер вативной терапии. Рецидив ГЭРБ возникает у 80% боль ных, которые получают поддерживающую консер- вативную терапию [5]. Зарубежные коллеги отмечают положительные результаты после самобужирования па циента [6, 7]. Д.И. Василевский и его коллеги изучили распростра ненность эрозивного эзофагита и пептических стриктур пищевода у жителей Ленинградской области и получили результаты, что пик заболеваемости возникает от 61 года до 75 лет и отметили, что пептические стриктуры встре чаются у 0,2% россиян. При этом данное осложнение возникает у 3,7% больных, страдающих эрозивным эзо фагитом степени C и D согласно Лос-Анджелесской клас сификации [14]. Пептическая стриктура сочетается у некоторых паци ентов с эрозивно-язвенным эзофагитом на фоне ГЭРБ, что более утяжеляет течение заболевания [15]. Чаще всего эрозивные формы ГЭРБ встречаются у больных, принимающих нестероидные противовоспалительные препараты и аспирин [16]. Протяженность пептической стриктуры, а также глубина воспалительных изменений зависит от определенных факторов: состав рН желудка, частота гастроэзофагеальных рефлюксов, дуоденогаст ральных рефлюксов, защитная реакция слизистой обо лочки на повреждающие факторы [17, 18, 19]. Как европейские, так и британские руководства реко мендуют использовать стентирование в лечении рефрак терных стриктур после неудачных попыток бужирова- ния и баллонной дилатации. Стент должен быть удален в течение 3х месяцев, чтобы избежать гипертрофии тка ней [4]. Антирефрюксная хирургия направлена на восстанов ление нормальной функции нижнего пищеводного сфинктера, эффективность хирургического лечения ГЭРБ была подтверждена результатами многих исследо ваний. Чаще всего проводится эзофагофундопликация по Ниссен, но данная операция не является оптималь ной, т.к. возникает большое число осложнений [3, 8]. По данным Королева М.П. Хирургическая практика Хирургическая практика №2 (44), 2021 Введение ционной манжеты в средостение (до 30%), а также воз никновение параэзофагеальной грыжи (до 6%) [9]. Пептическая стриктура пищевода как осложнение ГЭРБ – рубцовое сужение пищевода вследствие рефлюкс- эзофагита. По классификации Годжелло Э.А. и Галлингера Ю.И. стеноз пищевода делится в зависимости от диаметра пищевода на участке сужения: I степень – 9-11 мм; II сте пень – 6-8 мм; III степень – 3-5 мм; IV степень – 1-2 мм или просвет пищевода полностью облитерирован [1,2]. В настоящее время является достаточно актуальной тема выбора антирефлюксных операций, так как под ходы бывают разные, перед операцией в большинстве случаев проводится бужирование или баллонная диля тация, некоторые авторы придерживаются методики бу жирования во время операции эзофагофундопликации перед формированием фундопликационной манжеты, с целью уменьшения риска возникновения рецидива пеп тической стриктуры в послеоперационном периоде. По данным института Gallop (США) от 4 до 20% боль ных страдает стриктурой пищевода [3]. ГЭРБ чаще всего возникает у лиц старше 50 лет, курильщиков, людей с ожирением. Распространенность ГЭРБ в России состав ляет до 24%. [4]. Abstract: The work is based on the analysis of literature data devoted to the choice of treatment for peptic esophageal stricture dentify treatment tactics for patients with stenosing reflux esophagitis. Researchers point out that the main causes of GERD are a decrease in pressure in the lower esophageal sphincter, the action of the damaging properties of the refluctant. Untimely treatment of GERD can lead to complications such as peptic stricture, Barrett's esophagus. The appearance of GERD stricture is most often promoted by: persistent heartburn after bougienage, erosion of the lower third of the esophagus, shortening of the II degree esophagus, and inadequate antisecretory therapy. q y py Various methods of treatment at all stages of the appearance of peptic stricture are presented, depending on the degree of dysphagia and the length of the stricture, the use of adequate conservative therapy regimens for PPIs, bougienage, as well as a description of various methods of antireflux operations. Endoscopic dilation is the first treatment option for all symptomatic benign esophageal strictures. There are treatments for benign refractory esophageal strictures such as endoscopic dilatation with intraluminal steroid injection, endoscopic postoperative therapy or stricturoplasty, esophageal stenting, self-bougienage, as well as surgery - antireflux surgery, esophagectomy with replacement of the esophagus by the stomach or colon [1]. Various methods of treatment at all stages of the appearance of peptic stricture are presented, depending on the degree of dysphagia and the length of the stricture, the use of adequate conservative therapy regimens for PPIs, bougienage, as well as a description of various methods of antireflux operations. Endoscopic dilation is the first treatment option for all symptomatic benign esophageal strictures. There are treatments for benign refractory esophageal strictures such as endoscopic dilatation with intraluminal steroid injection endoscopic postoperative therapy or stricturoplasty esophageal stenting The main goal in the treatment of peptic esophageal strictures, according to most authors, is to eliminate the progression of GERD, conduct bougienage or balloon dilatation, and select the optimal antireflux surgery. Treatment for peptic strictures should minimize the risk of re-stricture of the esophagus. Keywords: peptic stricture, bougienage, antireflux surgery, review. 14 Хирургическая практика Хирургическая практика №2 (44), 2021 желудка, длительное назоинтестинальное зондирование верхних отделов желудочно-кишечного тракта, заболе вания панкреатобилиарной зоны. клинических и функциональных признаков гастроэзо фагеального рефлюкса необходимо выполнение анти рефлюксных операций. Этот подход позволяет избежать очень травматичной операции - резекции пищевода [12]. клинических и функциональных признаков гастроэзо фагеального рефлюкса необходимо выполнение анти рефлюксных операций. Этот подход позволяет избежать очень травматичной операции - резекции пищевода [12]. Особенность этого заболевания кроется в частом ре цидивировании стриктуры после лечения. Аллахвердян А.С. и его соавторы [13] определили факторы, влияющие на частоту возникновения ранних рестенозов: молодой возраст, регулярная изжога после бужирования, наличие дуодено-гастрального рефлюкса, наличие эрозий в пи щеводе, протяженность стриктуры более 3 см, первона чальный диаметр стриктуры менее 5 мм, дисфагия более 6 месяцев, укорочение пищевода II степени, грыжа пи щеводного отверстия диафрагмы, неадекватная антисек реторная терапия. Ряд авторов другими причинами рецидива пептических стриктур у части больных назы вают предшествующие операции по поводу язвенной бо лезни желудка и ахалазии кардии. Коллеги из военно-медицинской академии имени С.М. Кирова (Санкт-Петербург) рекомендуют проводить бужирование или баллонную дилатацию при лечении нижних пищеводных стриктур, а также отмечают, что пептические стриктуры пищевода являются показанием к местной эзофагопластике [27]. Крупное рандомизированное исследование клиниче ское исследование из Великобритании показало значи тельное уменьшение рефлюксов у пациентов, пере- несших лапароскопическую фундопликацию Ниссена, чем у пациентов, получавших поддерживающую терапию ИПП [28, 29]. Согласно клиническим рекомендациям Мексикан ской ассоциации гастроэнтерологов первым этапом лечения пептических стриктур пищевода является бужи рование и баллонная дилатация. Все пациенты с пепти ческими стриктурами получают консервативную тера- пию ИПП, а при рефрактерных стриктурах пищевода проводят стентирование [30]. Пептическая стриктура как осложнение ГЭРБ является показанием к антиреф люксной операциям [31]. По мнению многих коллег применение ингибиторов протонной помпы (ИПП) при лечении ГЭРБ в последние годы снизило распространенность и заболеваемость пеп тической стриктурой, но это также может свидетель ствовать о большом распространении антирефлюксных операций [20]. По данным клинических исследований ИПП уменьшают симптомы эзофагита и снижают коли чество процедур бужирования [21]. Большинство авто ров считает, что антирефлюксные вмешательства явля- ются необходимыми в лечении пациентов с пептической стриктурой [22, 23, 24, 25]. Выполнение фундопликации уменьшает вероятность развития ранних рестенозов в 3 раза [13]. В последнее время имеет актуальность такой метод лечения, как инъекция стероидов в месте стриктуры во время дилатации обеспечивает снижение отложения коллагена и фиброза, связанных с хроническим воспале нием и, следовательно, снижает вероятность рецидива стриктуры [4]. Основная часть [18] и его соавторов основ ными причинами появления пептических стриктур яв ляются: грыжа пищеводного отверстия диафрагмы, пищевод Барретта, язвенный стеноз выходного отдела Наиболее частыми осложнениями лапароскопиче ской фундопликации является миграция фундоплика 15 Хирургическая практика Хирургическая практика №2 (44), 2021 №2 (44), 2021 никновения). У больных с рефрактерной пептической стриктуры проводят внутрипросветную стероидную те рапию, которая включает инъекцию 0,5 мл триамцино лона во все четыре квадранта стриктуры. А также используют внутрипросветную стероидную терапию в сочетании с дилатацией при рефрактерных стриктурах, если ранее проводилась консервативная терапия ИПП. При лечении большинства рефрактерных стриктур про водят стентирование от 4 до 8 недель, не рекомендуют оставлять стенты более 3х месяцев, так как имеется риск врастания стента. Частота рецидивов рефрактерных стриктур после стентирования составляет 69%, осо бенно у пациентов с протяженными стриктурами (более 7 см) [34]. Согласно клиническим рекомендациям Российской гастроэнтерологической ассоциации по диагностике и лечению гастро-эзофагеальной рефлюксной болезни [37] антирефлюксное хирургическое лечение считают пока занным при осложненном течении заболевания (повтор ные кровотечения, пептические стриктуры пищевода, развитие пищевода Баррета с дисплазией эпителия вы сокой степени, доказанной двумя морфологами, частые аспирационные пневмонии). В некоторых случаях если пациенту по тем или иным объективным либо субъ ективным причинам не может быть проведена консерва тивная терапия ГЭРБ, следует рассмотреть вопрос о хирургическом лечении и при неосложненном течении. Хирургическое лечение может быть более эффективным у тех пациентов с ГЭРБ, у которых имеются типичные проявления заболевания и лечение которых с помощью ИПП тоже эффективно [38, 39]. При неэффективности ИПП, а также при наличии внепищеводных проявлений хирургическое лечение будет менее эффективным. Petra G. A. van Boeckel и его соавторы предложили определенный алгоритм лечения пациентов с доброкаче ственной дисфагией. Дилатация остается первым выбо ром как наиболее инвазивный подход с низкой частотой осложнений. Следующий шаг в алгоритме должен об суждаться с пациентом: самобужирование, дилатация стероидами, послеоперационная терапия или стентиро вание. Конечным этапом в лечении рефрактерных доб рокачественных стриктур пищевода является хирур- гическое вмешательство, которое не гарантирует появле ния рестеноза в послеоперационном периоде [6]. Свои данные приводят Морошек А.А. [10], Королева М.П. [18], Оглоблина А.П. [40] и другие исследователи [13, 22], которые в предоперационном и послеопера ционном периоде у больных пептической стриктурой пищевода проводили бужирование и баллонную дилата цию, помимо этого больные получали медикаментозную терапию антацидными препаратами, блокаторами инги биторов протонной помпы и прокинетиками. Несмотря на это у части больных возник рецидив стриктуры пи щевода. Значительному числу больных (83%) были про ведены антирефлюксные операции в виде лапаро- скопической фундопликации по Ниссену-Розетти или Ниссену. Рецидив возник у 16% больных, при этом ста тистически значимой связи между рецидивом пептиче ской стриктуры и рецидивом ГЭР также отмечено не было. Хирургическая практика Исследования, проведенные в Нидерлан дах в 2013 году, не показало большого влияния инъекции стероидов в область стриктуры во время дилатации на интервал без дисфагии и не сократило время для повтор ной дилатации [32]. Zhang Y.W. и его коллеги имеют не большой опыт наблюдения с пептическими стриктурами пищевода и отметили значительное снижение количе ства последующих дилатаций после инъекций триамци нолона [33]. Результаты этих двух исследований сущест- венно различаются между собой, но в целом эти иссле дования поддерживают инъекцию стероидов (в область стриктуры во время дилатации) в качестве терапевтиче ского метода при резистентных стриктурах. Мелкуманян Р.А. проанализировал опыт наблюдения и лечения 32 пациентов с пептической стриктурой пи щевода. Пациентам проводилось бужирование, у 75% больных была ГПОД, 25% из них выполнена фундопли кация по Ниссену. У данных пациентов в дальнейшем не наблюдалось пептической стриктуры. Автор по резуль татам своей работы определил наиболее приемлемый способ лечения пептической стриктуры, который имеет такие этапы лечения, как бужирование пептической стриктуры по струне-проводнику, далее пациенты об учались самобужированию в домашних условиях по 3 раза в день, получали консервативную терапию, а за ключительный этап лечения – это хирургическая коррек ция ГЭРБ (сочетание пластики диафрагмы и фундо- пликации по Ниссену) [26]. Согласно клиническим стандартам Британского об щества гастроэнтерологов (BSG) в лечении пептических стриктур пищевода проводится либо бужирование, либо баллонная дилатация в зависимости от характера стриктуры (протяженности, локализации, причины воз По данным российских и зарубежных авторов мето дом выбора лечения пептических стриктур пищевода яв ляется бужирование пищевода. Однако при сохранении 16 Хирургическая практика плексному лечебному алгоритму применяли методы лечения – дилатация пищевода в виде бужирования под эндоскопическим контролем в до- и послеоперационном периоде при пептической стриктуре. В раннем послеопе рационном периоде у 37% пациентов отмечалось разви тие дисфагии и gas-bloat-синдрома, которое носило транзиторный характер [41, 42]. Шарапов Т.Л. и соавт. внедрили в практику в отделении хирургии пищевода РКОД трансиллюминационный фототензометрический зонд, применение которого способствовало профилак тике интраоперационных осложнений [43]. Королев М.П. [18] имеет опыт наблюдений 55 боль ных с пептическим стенозирующим эзофагитом. Опера тивное лечение провели 12 больным. Были выполнены крурорафия с эзофагофундопликацией и проксимальной селективной ваготомией, резекция желудка по Бильрот- II в модификации Гофмейстера- Финстерера с крурора фией, резекция нижней трети пищевода и прокси- мального отдела желудка с формированием инвагина ционного анастомоза по Цоцаниди, резекция желудка по Бильрот – II в модификации Гофмейстера-Финстерера. При этом рецидив стриктуры обнаружен у большинства больных (73%). Причиной рецидива являлся преимуще ственно гиперацидный тип желудочной секреции и отказ больных от постоянного приема медикаментозных пре паратов. Котив Б.Н. и соавторы [17] в 16 наблюдениях выпол няли парциальную фундопликацию на 240© по Toupet, и нашли определенные преимущества данной методики: повышение внутрижелудочного давления вызывает уве личение компрессионного эффекта манжеты, что пре пятствует гастро-эзофагеальным рефлюксам. Коллеги из московского областного научно-исследо вательского клинического института им. М.Ф. Влади мирского имеют опыт лечения 204 больных с пепти- ческими стриктурами пищевода, которые включали ди латацию (бужирование) в комбинации с консервативной терапией, антирефлюксную операцию, восстановление пассажа из желудка, резекцию пищевода. Показаниями к резекции пищевода явились невозможность бужиро вания стриктуры, частые рецидивы стриктуры после бу жирования, подозрение на рак. Резекция пищевода была выполнена 11% больных. После бужирования пептиче ских стриктур пищевода были выполнены антирефлюкс ные операции 39% больных. Показаниями к ним послу- жили некупируемый медикаментозно патологический рефлюкс, частые рестенозы стриктуры на фоне лечения ИПП, а также молодой возраст больных. (Pascalescu C., Bulat С.) Некоторые авторы считают абсолютными про тивопоказаниями к фундопликации – недилатируемая стриктура, предшествующая резекция желудка, значи тельное укорочение пищевода [44]. Аллахвердян А.С. [13] и соавторы описали собствен ную оригинальную методику циркулярной фундоплика ции (68% наблюдений). Оригинальная методика вклю- чала в себя перемещение задней стенки желудка позади пищевода, формирование фундопликационной манжеты (360©) вне ветвей вагуса, по медиальной стенке пище вода на протяжении 5-6 см. Рестенозы были у 9% - после частичной фундопликации. Но при циркулярной протя женной фундопликации по собственной методике ресте нозов в сроки более 12 месяцев не было выявлено [13]. Хирургическая практика Значительная часть ранних послеоперационных осложнений относилась к синдрому гиперфункции фун допликационной манжеты, который проходил самостоя тельно в течение 1-2 месяцев [10]. В период 2012 по 2016 год в Академическом медицин ском центре Амстердама Emo E. van Halsema и его кол леги провели ретроспективный анализ наблюдений пациентов с резистентными доброкачественными стрик турами пищевода, у которых проводилось самобужиро вание пищевода, которое оказалось успешным у 94% пациентов и происходило под строгим руководством. Все пациенты, выполнявшие самостоятельное бужиро вание, достигли стабильного результата, смогли есть твердую пищу без необходимости эндоскопической ди латации. Кроме бужирования и баллонной дилатации 47% пациентов получили эндоскопическое лечение эн доскопическое лечение в виде внутрипросветной инъ екции стероидов, инцизионной терапии или стенти- рования [35]. Хирурги из Национального медицинского исследова тельского центра онкологии им. Н.Н. Блохина (Морошек А.А.) и Республиканской клинической больницы, г.Ка зань (Бурмистров М.В.) разработали комплексный ле чебный алгоритм, который включал консервативную антирефлюксную терапию до 4-8 недель, антирефлюкс ную хирургическую операцию, а также диспансерное на блюдение, включающее ФЭГДС с двойной хромоскопией и биопсией, рентгеноскопии пищевода и желудка 2 раза в год, с частотой 1 раз в 6 месяцев. Дополнительно к ком Самобужирование пищевода также положительно влияет на показатели качества жизни пациентов. Иссле дования клиники Mayo в Аризоне показало, что при рет роспективном анализе с помощью самостоятельно разработанного опросника показатели дисфагии и об щего качества жизни значительно улучшились при само бужировании по сравнению с периодом эндоскопически выполненных дилатаций [36]. 17 Хирургическая практика №2 (44), 2021 №2 (44), 2021 эндоскопический метод бужирования с атравматиче ским кончиком полыми бужами типа Savary-Gilliard, при этом у некоторых больных проводилось эндоскопиче ское и рентгенологическое исследование, что являлось методом двойного контроля положения металлического проводника и бужа. Метод «блокирования» произво дился курсами (5-6 сеансов за 1 курс). Поддерживающие бужирования проводились на протяжении 6 месяцев с интервалами в 2-4 недели. При пептических стриктурах были произведены стандартные методики фундоплика ции по Ниссену, Тоупе, Белси и по Дору. С целью пред упреждения послеоперационной дисфагии больными с нарушенной перистальтикой выполнялась частичная фундопликация. плексному лечебному алгоритму применяли методы лечения – дилатация пищевода в виде бужирования под эндоскопическим контролем в до- и послеоперационном периоде при пептической стриктуре. В раннем послеопе рационном периоде у 37% пациентов отмечалось разви тие дисфагии и gas-bloat-синдрома, которое носило транзиторный характер [41, 42]. Шарапов Т.Л. и соавт. внедрили в практику в отделении хирургии пищевода РКОД трансиллюминационный фототензометрический зонд, применение которого способствовало профилак тике интраоперационных осложнений [43]. Хирургическая практика Авторы считают оптимальными сроками выполнения данного оперативного вмешательства после завершения курса бужирования спустя 3-6 месяцев (97% наблюде ний). Невозможность бужирования, частые рецидивы стриктуры или подозрение на малигнизацию на фоне длительно существующей пептической стриктуры явля лись показанием к резекции с внутриплевральной пла стикой желудком (11%). В отделении эндоскопии Российского научного центра хирургии им. акад. Б.В. Петровского РАМН имеют опыт наблюдения и лечения 526 больных с рубцовыми суже ниями пищевода, используя эндоскопические вмеша тельства, из них 31% больных с пептическими стрик- турами пищевода. Годжелло Э.А. и Ю.И. Галлингер счи тают, что абсолютными показаниями к эндоскопическим методам лечения рубцовых стриктур пищевода яв Аллахвердян А.С. [22] проанализировал результаты лечения 310 больных с рубцовыми стриктурами пище вода, среди них 151 больной с пептической стриктурой пищевода. Автор описывает метод «блокирования» – это 18 Хирургическая практика №2 (44), 2021 применяется в Краевой клинической больнице г. Читы. После использования данного метода у больных с пепти ческими стриктурами были отмечены хорошие в 58% случаев и удовлетворительные результаты – 11,8%, по сравнению с больными, которые выполняли бужирова ние сужения под рентген-контролем. Авторы отмечают, что внутрипросветное эндоскопическое лечение с при менением аргоновой плазмы сопровождается купирова нием локального воспаления наряду со снижением уровня противовоспалительных цитокинов, обладаю щих хемоаттрактантным действием, и структурно-функ циональным редепонированием. Проводили от 2 до 5 сеансов лечения, в среднем 2,8±0,5 [46, 47, 48, 49]. ляются: стенозы пищевода III-IV степени, высокая лока лизация верхнего края сужения, большая протяжен ность и извитой ход стриктуры, эксцентричное распо- ложение входа в стриктуру, деформация супрастеноти ческого расширения пищевода, двойные и множествен ные сужения, стриктуры пищеводных анастомозов любой локализации и любой степени выраженности. Применяли различные эндоскопические методы: бужи рование по струне-направителю, баллонная гидродила тация, рассечение рубца током высокой частоты, инъекции лидазы и кортикостероидов в зону рубца (в комбинации с дилатационными методиками), эндопро тезирование пластиковыми трубчатыми стентами и в одном наблюдении стентирование саморасправляю щимся биодеградирующим стентом. Осложнения воз никли у 5 (1%) из 514 больных: перфорация – у 4 (0,8%), кровотечение – у 1 (0,2%). Ближайшие результаты были отличными (14 мм и более) у 39,5%, хорошие (10-13 мм) – у 21%. После окончания основного курса эндоскопиче ского лечения были оперированы у 21% (из 514). Паци ентам с пептическими стриктурами не выполнялось стентирование. Многолетний опыт работы Э.А. Годжи елло и Галлингера О.И. говорит о том, что при рубцовых стриктурах пищевода эндоскопическое лечение будет бесперспективным при невозможности проведения через сужение бужа диаметром 9 мм [2]. Дробязгин Е.А. и Чикинев Ю.В. Хирургическая практика [50] описывали на блюдения и лечение пациентов после эзофагогастропла стики у больных с пептической стриктурой и пост- ожоговыми стриктурами пищевода, с рубцовым суже нием пищевода после ушивания дефекта стенки, а также больных с ахалазией пищевода IV стадии. Показанием к оперативному лечению у данных пациентов была невоз можность эндоскопического лечения или отсутствие эф фекта от него в течение 4-6 месяцев. В послеопера- ционном периоде у некоторых пациентов развился сте ноз пищеводного анастомоза, критическими сроками были первые 3 месяца и 1 год после вмешательства. Чаще всего развивался стеноз пищеводного анастомоза у па циентов, оперированных по поводу рубцового после ожогового сужения пищевода. Коллеги из Российского научного центра хирургии имени академика Б.В. Петровского (РНЦХ) имеют опыт на блюдения и лечения 209 больнфых с рефлюкс-эзофагитом, которым была выполнена фундопликациия по методитке РНЦХ из лапаротомного и лапароскопического доступов. Среди них 47 пациентов с пептитческими стриктурами пи щевода. Всем пациентам с выявленными пептическими стриктурами пищевода в предоперационном периоде про водились сеансы бужирования стриктуры. При анализе ближайших результатов обычного и модифицированного лапароскопических доступов и способов формирования фундопликационной манжеты по методике РНЦХ показан положительный результат у 95% больных. При анализе бли жайших результатов оперативного лечения дисфагия на блюдалась у 3,2% больных, при этом у 1% в предопе- рационном периоде проводили бужирование по поводу пептической стриктуры нижней трети пищевода. При эн доскопическом исследовании отдаленных результатов лече ния не было выявлено пептических стриктур пищевода [45]. По данным Орынбасар Н.Т. пациентам, у которых имелся стенозирующий эзофагит в предоперационном периоде выполнялось эндоскопическое бужирование пи щевода. Во время выполнения лапароскопической фун допликации формировали фундопликационную манже- ту с использованием калибровочного зонда диаметром 12,0 мм. Бужирование пищевода было выполнено 9% па циентов контрольной группы с пептическим стенози рующим эзофагитом, и было выполнено под эндоско- пическим контролем по струне-проводнику. Показанием были стриктуры протяженностью более 3 мм [9]. Таким образом, большинство авторов считают, что лечение больных с пептической стриктурой пищевода должно быть разнонаправленным. Выводы Хирургическая практика №2 (44), 2021 Gastrointestinal Endoscopy. 2019;12:1-12. https://doi.org/ 10/1177/2631774519862134. Gastrointestinal Endoscopy. 2019;12:1-12. https://doi.org/ 10/1177/2631774519862134. 2. Европейские и британские руководства рекомен дуют использовать стентирование в лечении рефрактер ных стриктур, при этом стент удаляют в течение 3х месяцев, чтобы не возникло гипертрофии тканей. 5. Дурлештер В.М., Сиюхов Р.Ш., Щупляк С.В. Диаг ностика и лечение доброкачественных стриктур пище вода и желудка. Вестник хирургической гастроэнтеро- логии. 2007;3:114. [Durleshter V.M., Sijuhov R.Sh., Shhupljak S.V. Diagnostika i lechenie dobrokachestvennyh striktur pishhevoda i zheludka. Vestnik hirurgicheskoj gastrojenterol ogii. 2007;3:114. (In Russian)] 3. Российские и зарубежные коллеги довольно эффек тивно используют у больных с рефрактерной пептиче ской стриктурой пищевода внутрипросветную сте- роидную терапию, которая включает инъекцию 0,5 мл триамцинолона во все четыре квадранта стриктуры. 4. Зарубежные коллеги описывают положительный опыт наблюдения и лечения пациентов с пептическими стриктурами пищевода с выполнением пациентом само буживания в домашних условиях. 6. Petra G. A.an Boeckel, Siersema P.D. Refractory Esoph ageal Strictures: What To Do When Dilation Fails. Siersema. Current Treatment Options in Gastroenterology. 2015;13:47–58. https://doi.org/10.1007/s11938-014-0043-6 7. Van Halsema Emo E., Chantal A. et Hoen, Patricia S. de Koning, Wilda D. Rosmolen, Jeanin E. van Hooft, Jacques J. Bergman Self-dilation for therapy-resistant benign esoph ageal strictures: towards a systematic approach., 4 July 2017 / Accepted: 3 January 2018. Surgical Endoscopy https://doi.org/10.1007/s00464-018-6037-z 7. Van Halsema Emo E., Chantal A. et Hoen, Patricia S. de Koning, Wilda D. Rosmolen, Jeanin E. van Hooft, Jacques J. Bergman Self-dilation for therapy-resistant benign esoph ageal strictures: towards a systematic approach., 4 July 2017 / Accepted: 3 January 2018. Surgical Endoscopy https://doi.org/10.1007/s00464-018-6037-z 5. При неэффективности эндоскопического лечения пептических стриктур пищевода выполняют антиреф люксные операции в различных модификациях (по Нис сену, Дору, Тупе, собственные оригинальные методики различных авторов), резекцию пищевода. 6. Лечебная тактика при пептических стриктурах должна быть определена в зависимости от протяженно сти стриктуры пищевода. 8. Буслаев О.А., Куликов Л.К., Привалов Ю.А., Михайлов А.Л., Егоров И.А., Соботович В.Ф. Осложнения и результаты хирургического лечения гастроэзофагеальной рефлюксной болезни. Acta Biomedica Scientifica. 2017; 2(6): 100-103. https://doi.org/10.12737/article_5a0a898361d4a4.71143035 [Buslaev O. A., Kulikov L. K., Privalov Yu. A., Mikhailov A. L., Egorov I. A., Sobotovich V. F. Complications and results of surgical treatment of gastroesophageal reflux disease. Acta Biomedica Scientifica. 2017; 2(6): 100-103. (In Russian)] https://doi.org/10.12737/article_5a0a898361d4a4.71143035 Выводы 1. Основными методами лечения пептических стрик тур пищевода являются бужирование, баллонная дила тация, внутрипросветная инъекция стероидов в месте дилатации, стентирование, а также выбор оптимальной антирефлюксной операции. В Читинской медицинской академии в 2012 году раз работали метод эндоскопической аргоноплазменной ре канализации рубцовых стриктур пищевода, который 19 С п ис ок л и т е р ат у ры 1. Boregowdaa U., Goyalb H., Mannc R., Gajendrand M., Patele S., Echavarriae J., Sayanae H., Saligrame S. Endoscopic management of benign recalcitrant esophageal strictures. An nals of Gastroenterology 2021;34:1-13. https://doi.org/10.20524/ aog.2021.0585. 1. Boregowdaa U., Goyalb H., Mannc R., Gajendrand M., Patele S., Echavarriae J., Sayanae H., Saligrame S. Endoscopic management of benign recalcitrant esophageal strictures. An nals of Gastroenterology 2021;34:1-13. https://doi.org/10.20524/ aog.2021.0585. [Buslaev O. A., Kulikov L. K., Privalov Yu. A., Mikhailov A. L., Egorov I. A., Sobotovich V. F. Complications and results of surgical treatment of gastroesophageal reflux disease. Acta Biomedica Scientifica. 2017; 2(6): 100-103. (In Russian)] https://doi.org/10.12737/article_5a0a898361d4a4.71143035 2. 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Fundoplikacija v modifika cii Rossijskogo nauchnogo centra hirurgii v lechenii pacien tov s gastrojezofageal'noj bolezn'ju. Moskovskij hirurgicheskij zhurnal. 2014;5(39):24-28 (In Russian)] 50. Е.А. Дробязгин, Ю.В. Чикинев. Эзофагогастропла стика при доброкачественных стенозирующих забо- леваниях пищевода: оценка результатов. Доказа- тельная гастроэнтерология. 2020;9(2):10-19. https://doi.org/ 10.35177/1994-5191-2020-2-24-27. [E.A. Drobjazgin, Ju.V. Chi kinev. Jezofagogastroplastika pri dobrokachestvennyh stenozi rujushhih zabolevanijah pishhevoda: ocenka rezul'tatov. Dokazatel'naja gastrojenterologija. 2020;9(2):10-19. (In Russian)] https://doi.org/10.35177/1994-5191-2020-2-24-27 46. Скажутина Т.В., Цепелев В.Л., Степанов А.В. Внут ирипросветные дилатирующие методы лечения доброкаче ственных стриктур пищевода. Журнал Современные проблемы науки и образования. 2015;5. https://doi.org/10.4213/ book1602. [Skazhutina T.V., Cepelev V.L., Stepanov A.V. 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Laparos copic antireflux surgery vs esomeprazole treatment for chronic GERD: The LOTUS randomized clinical trial. JAMA. 2011;305(19):1969-1977. https://doi.org/10.1001/jama.2011.626 39. Wileman S.M., McCann S., Grant A.M. Medical versus surgical management for GERD in adults. Cochrane Database Syst Rev. 2010; pp CD003243. https://doi.org/10.1002/14651858.cd003243.pub2 32. Hirdes M.M.C., Van Hooft J.E., Koornstra J.J. Endo scopic corticosteroid injections do not reduce dysphagia after endoscopic dilation therapy in patients with benign esopha gogastric anastomotic strictures. Clin Gast Hep. 2013;11:795– 801. https://doi.org/10.1016/j.cgh.2013.01.016 40. Оглоблин А.Л., Федотов Л.Е., Федотов Б.Л., Па шков Д.В., Конанов Е.В. Пептическая стриктура пище вода, диагностика и лечение. Эндоскопия желудка. 2014. [Ogloblin A.L., Fedotov E.L., Fedotov B.L., Pashkov D.V., Ko nanov E.V. Pepticheskaja striktura pishhevoda, diagnostika i lechenie. Jendoskopija zheludka. 2014. (In Russian)] 33. Zhang Y.W., Wei F.X., Qi X.P. Efficacy and safety of endoscopic intralesional triamcinolone injection for benign esophageal strictures. Gastroenterol Res Pract. 2018;761:1-7. https://doi.org/10.1155/2018/7619298 34. Sami S.S., Haboubi H.N., Ang Y., Ph. Boger. UK guidelions on oesophageal dilatation in clinical practice. Additional material is published online only. To view please visit the journal online. Gut 2018;67:1000–1023. https://doi.org/10.1136/gutjnl-2017-315414 34. Sami S.S., Haboubi H.N., Ang Y., Ph. Boger. UK guidelions on oesophageal dilatation in clinical practice. Additional material is published online only. To view please visit the journal online. Gut 2018;67:1000–1023. https://doi.org/10.1136/gutjnl-2017-315414 41. Морошек А.А., Бурмистров М.В. Комплексный подход в лечении пациентов с осложненными формами гастроэзофагеальной рефлюксной болезни. Казанский медицинский журнал. 2020;101(4):595-602. https://doi.org/ 22 Хирургическая практика №2 (44), 2021 10.37882/2223-2966.2020.04.29. [Moroshek A.A., Burmistrov M.V. Kompleksnyj podhod v lechenii pacientov s oslozhnen nymi formami gastrojezofageal'noj refljuksnoj bolezni. Ka zanskij medicinskij zhurnal. 2020;101(4):595-602. (In Russian)] https://doi.org/10.37882/2223-2966.2020.04.29 10.37882/2223-2966.2020.04.29. [Moroshek A.A., Burmistrov M.V. Kompleksnyj podhod v lechenii pacientov s oslozhnen nymi formami gastrojezofageal'noj refljuksnoj bolezni. Ka zanskij medicinskij zhurnal. 2020;101(4):595-602. (In Russian)] https://doi.org/10.37882/2223-2966.2020.04.29 пищевода методом бужирования. Современные проблемы науки и образования. 2016;2. https://doi.org/10.17116/hi rurgia20161016-20. [Skazhutina T.V., Cepelev V.L. Rezul'taty lechenija pacientov s dobrokachestvennymi rubcovymi stenozami pishhevoda metodom buzhirovanija. Sovremennye problemy nauki i obrazovanija. 2016;2. (In Russian)] https://doi.org/10.17116/hirurgia20161016-20 пищевода методом бужирования. Современные проблемы науки и образования. 2016;2. https://doi.org/10.17116/hi rurgia20161016-20. [Skazhutina T.V., Cepelev V.L. Rezul'taty lechenija pacientov s dobrokachestvennymi rubcovymi stenozami pishhevoda metodom buzhirovanija. Sovremennye problemy nauki i obrazovanija. 2016;2. (In Russian)] https://doi.org/10.17116/hirurgia20161016-20 42. Морошек А.А., Бурмистров М.В., Иванов А.И., Сигал Е.И. Диагностические особенности и комплексное патогенетическое лечение пептических стриктур пищевода. Вестник хирургической гастроэнтерологии. 2014;3-4:74-83. [Moroshek A.A., Burmistrov M.V., Ivanov A.I., Sigal E.I. Diagnosticheskie osobennosti i kompleksnoe patogenetiches koe lechenie pepticheskih striktur pishhevoda. Vestnik hirur gicheskoj gastrojenterologii. 2014;3-4:74-83 (In Russian)] 48. Скажутина Т.В., Цепелев В.Л. Диагностическая и лечебная эндоскопия при гастроэзофагеальной рефлюкс ной болезни и ее осложнениях. ЭНИ Забайкальский меди цинский вестник. 2020;2:126-139. [Skazhutina T.V., Cepelev V.L. Diagnosticheskaja i lechebnaja jendoskopija pri gastroje zofageal'noj refljuksnoj bolezni i ee oslozhnenijah. JeNI Za bajkal'skij medicinskij vestnik. 2020;2:126-139 (In Russian)] 43. Шарапов Т.Л., Бурмистров М.В., Сигал Е.И. Про филактика осложнений при лапароскопических анти рефлюксных операциях. Эндоскопия хирургия. 2012;3:9-14. [Sharapov T.L., Burmistrov M.V., Sigal E.I. Pro filaktika oslozhnenij pri laparoskopicheskih antirefljuksnyh operacijah. Jendoskopija hirurgija. 2012;3:9-14. (In Russian)] 49. Пензина О.Ж., Потхоева С.С., Суворова В.В. Це пелев В.Л., Крюкова В.В. Распространенность эрозивного эзофагита и пептических стриктур пищевода по данным эндоскопических исследований. Материалы XVII меж региональной научно-практической конференции сту дентов и молодых ученых, посвященной 65-летию Читинской государственной медицинской академии. Ме дицина завтрашнего дня. 2018:93-94. [Penzina O.Zh., Pot hoeva S.S., Suvorova V.V. Cepelev V.L., Krjukova V.V. Rasprostranennost' jerozivnogo jezofagita i pepticheskih striktur pishhevoda po dannym jendoskopicheskih issledo vanij. Materialy XVII mezhregional'noj nauchno-praktiches koj konferencii studentov i molodyh uchenyh, posvjashhennoj 65-letiju Chitinskoj gosudarstvennoj medi cinskoj akademii. Medicina zavtrashnego dnja. 2018:93-94 (In Russian)] 44. Аллахвердян А.С., Мазурин В.С., Шестаковская Е.Е., Титов А.Г. Лапароскопическая фундопликация в профилактике рестенозов пептических стриктур пище вода. Вестник Санкт-Петербурского университета. 2012;11(4):104-113. [Allahverdjan A.S., Mazurin V.S., Shes takovskaja E.E., Titov A.G. Laparoskopicheskaja fundoplika cija v profilaktike restenozov pepticheskih striktur pishhevoda. Vestnik Sankt-Peterburskogo universiteta. 2012;11(4):104-113 (In Russian)] 45. Юрасов А.В., Шестаков А.Л., Битаров Т.Т., Сели ванова И.М. Д л я ко р р е с пон де н ц и и Масликова Светлана Анатольевна – кафедра общей хирургии, оперативной хирургии и топографической анатомии АГМУ, проспект Ленина, д. 40, 656038, Алтайский край, г. Барнаул, Российская Федерация; maslikova18@mail.ru The authors declare no conflicts of interest. Хирургическая практика №2 (44), 2021 Operative Surgery and Topographic Anatomy of the Altai State Medical University, 40 Lenin Avenue, 656038, Altai Krai, Barnaul, Russian Federation; maslikova18@mail.ru; http://orcid.org/0000-0002-5542-9707 Operative Surgery and Topographic Anatomy of the Altai State Medical University, 40 Lenin Avenue, 656038, Altai Krai, Barnaul, Russian Federation; maslikova18@mail.ru; http://orcid.org/0000-0002-5542-9707 Андреасян Армен Ромикович – к.м.н., доцент кафедры общей хирургии, оперативной хирургии и топографической анатомии Алтайского государственного медицинского уни верситета, проспект Ленина, д. 40, 656038, Алтайский край, г. Барнаул, Российская Федерация; andreasyanarm@rambler.ru; http://orcid.org/0000-0003-3113-8734 Gayane I. Bagdasaryan – Assistant of the Department of General Surgery, Operative Surgery and Topographic Anat omy of the Altai State Medical University, Lenin Avenue, 40, 656038, Altai Krai, Barnaul, Russian Federation; ms.bag1973@mail.ru; http://orcid.org/0000-0001-8599-825X Масликова Светлана Анатольевна – к.м.н., доцент кафедры общей хирургии, оперативной хирургии и то пографической анатомии Алтайского государственного медицинского университета, проспект Ленина, д. 40, 656038, Алтайский край, г. Барнаул, Российская Федерация; maslikova18@mail.ru; http://orcid.org/0000-0002-5542-9707 Dmitry Yu. Shestakov – Assistant of the Department of General Surgery, Operative Surgery and Topographic Anat omy of the Altai State Medical University, 40 Lenin Avenue, 656038, Altai Krai, Barnaul, Russian Federation; shestago@gmail.com; http://orcid.org/0000-0001-9861-3697 Багдасарян Гаяне Игитовна – ассистент кафедры общей хирургии, оперативной хирургии и топографиче ской анатомии Алтайского государственного медицин ского университета, проспект Ленина, д. 40, 656038, Алтайский край, г. Барнаул, Российская Федерация; ms.bag1973@mail.ru; http://orcid.org/0000-0001-8599-825X Багдасарян Гаяне Игитовна – ассистент кафедры общей хирургии, оперативной хирургии и топографиче ской анатомии Алтайского государственного медицин ского университета, проспект Ленина, д. 40, 656038, Алтайский край, г. Барнаул, Российская Федерация; ms.bag1973@mail.ru; http://orcid.org/0000-0001-8599-825X Св едения о б а втор а х Ганков Виктор Анатольевич – д.м.н., профессор ка федры общей хирургии, оперативной хирургии и топо графической анатомии Алтайского государственного медицинского университета, проспект Ленина, д. 40, 656038, Алтайский край, г. Барнаул, Российская Федерация; viktorgankov@yandex.ru; http://orcid.org/0000-0001-9314-7180 47. Скажутина Т.В., Цепелев В.Л. Результаты лечения пациентов с доброкачественными рубцовыми стенозами 23 For cor resp ondence Svetlana A. Maslikova – Department of General Surgery, Operative Surgery and Topographic Anatomy, ASMU, 40 Lenin Avenue, 656038, Altai Krai, Barnaul, Russian Feder ation; maslikova18@mail.ru Шестаков Дмитрий Юрьевич - ассистент кафедры общей хирургии, оперативной хирургии и топографиче ской анатомии Алтайского государственного медицин ского университета, проспект Ленина, д. 40, 656038, Алтайский край, г. Барнаул, Российская Федерация; shestago@gmail.com; http://orcid.org/0000-0001-9861-3697 In for mat i on ab out aut hors Viktor A. Gankov – MD, Professor of the Department of General Surgery, Operative Surgery and Topographic Anatomy of the Altai State Medical University, 40 Lenin Avenue, 656038, Altai Krai, Barnaul, Russian Federation; viktorgankov@yandex.ru; http://orcid.org/0000-0001-9314-7180. Armen R. Andreasyan – PhD, Associate Professor of the Department of General Surgery, Operative Surgery and Topographic Anatomy of the Altai State Medical University, 40 Lenin Avenue, 656038, Altai Krai, Barnaul, Russian Federation; andreasyanarm@rambler.ru; http://orcid.org/0000-0003-3113-8734 Svetlana A. Maslikova – Candidate of Medical Sciences, Associate Professor of the Department of General Surgery, Svetlana A. Maslikova – Candidate of Medical Sciences, Associate Professor of the Department of General Surgery, 24
https://openalex.org/W4391176631	https://www.degruyter.com/document/doi/10.1515/dema-2023-0125/pdf	English	null	Solutions of a coupled system of hybrid boundary value problems with Riesz-Caputo derivative	Demonstratio Mathematica	2,024	cc-by	20,017	Dehong Ji, Shiqiu Fu, and Yitao Yang* Solutions of a coupled system of hybrid boundary value problems with Riesz-Caputo derivative https://doi.org/10.1515/dema-2023-0125 received March 6, 2023; accepted October 6, 2023 Abstract: Riesz-Caputo fractional derivative refers to a fractional derivative that reﬂects both the past and the future memory eﬀects. This study gives suﬃcient conditions for the existence of solutions for a coupled system of fractional order hybrid diﬀerential equations involving the Riesz-Caputo fractional derivative. For this motive, the results are obtained via classical results due to Dhage. Keywords: ﬁxed point theorem, coupled system of hybrid boundary value problems, existence of solutions, Riesz-Caputo fractional derivative MSC 2020: 34A08, 34A12 MSC 2020: 34A08, 34A12 Dehong Ji: School of Science, Tianjin University of Technology, Tianjin 300384, China Shiqiu Fu: School of Mathematical Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013, China  * Corresponding author: Yitao Yang, School of Science, Tianjin University of Technology, Tianjin 300384, China, e-mail: yitaoyangqf@163.com Dehong Ji: School of Science, Tianjin University of Technology, Tianjin 300384, China Shiqiu Fu: School of Mathematical Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013, China  * Corresponding author: Yitao Yang, School of Science, Tianjin University of Technology, Tianjin 300384, China, e-mail: yitaoyangqf@163.com Open Access. © 2024 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. Open Access. © 2024 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. Demonstratio Mathematica 2024; 57: 20230125 Houas [21] studied the results for the coupled hybrid system that contains integral boundary conditions as follows: ( ) ( ( ) ( )) ( ( ) ( )) [ ] ( ) ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ = ∈ ∈ D u t g t u t u t f t u t u t t α , , , , , 0, 1 , 0, 1 , α 1 1 1 2 1 1 2 1 1 ( ) ( ( ) ( )) ( ( ) ( )) [ ] ( ) ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ = ∈ ∈ D u t g t u t u t f t u t u t t α , , , , , 0, 1 , 0, 1 , α 2 2 1 2 2 1 2 1 2 ( ) ( ) ( ) ( ) ∫ = ∈ u A s u s s θ 0 d , 0, 1 , θ 1 0 1 1 1 1 ( ) ( ) ( ) ( ) ∫ = ∈ u A s u s s θ 0 d , 0, 1 , θ 2 0 2 2 2 2 ( ) ( ) ( ) ( ) ∫ = ∈ u A s u s s θ 0 d , 0, 1 , θ 2 0 2 2 2 2 where ( ) = D i 1, 2 αi stands for the Caputo’s derivative. where ( ) = D i 1, 2 αi stands for the Caputo’s derivative. Derbazi et al. [22] considered the solutions for the fractional boundary value problem as follows: where ( ) = D i 1, 2 αi stands for the Caputo’s derivative. where ( ) = D i 1, 2 αi stands for the Caputo’s derivative. Derbazi et al. [22] considered the solutions for the fractional boundary value problem as follows: where ( ) = D i 1, 2 αi stands for the Caputo’s derivative. Derbazi et al. [22] considered the solutions for the bazi et al. 1 Introduction The fractional diﬀerential equations have become a signiﬁcant ﬁeld of investigation due to their frequent use in biophysics, economics, chemistry, mechanics, control theory, image processing and signal, etc. [1–3]. Recently, existence theory of solutions to initial and boundary value problems for fractional system has received considerable attention from many researchers [4–11]. As is known to all, Riesz-Caputo derivative refers to a fractional derivative that reﬂects both the future and the past memory eﬀects; consequently, the Riesz fractional operator plays an important role in characterizing anomalous diﬀusion, owing to successful applications to subdiﬀusive, superdiﬀusive, and evolution problems. Space fractional quantum mechanics is a natural generalization of standard quantum mechanics, which arises when the Brownian trajectories in Feynman path integrals are replaced by Levy ﬂights. The classical Levy ﬂight is a stochastic processes, which in one dimension, is described by a jump length probability density function. The position space representation of the αth power of the momentum operator is given by: ⟨∣ ∣⟩ ( ) = − ∣∣ x P ψ h D ψ x ˆ , α α x α where ∣∣ D x α is the Riesz fractional derivative operator of order α. The Riesz fractional derivative is regarded as an eﬀective tool for studying nonlocal and memory eﬀects in physics, engineering, and applied sciences. Therefore, many scholars are engaged in the study of the solutions of diﬀerential equations with the Riesz fractional derivative. Many works carried out so far discuss the numerical solutions of diﬀusion equations, which contain the Riesz derivative, and fractional variational problems, which contain the Riesz-Caputo derivative, and only a few works report on the existence results of fractional boundary value problems, which contain the Riesz-Caputo derivative. [22] considered the solutions for the fractional boundary value problem as follows: ( ) ( ( )) ( ( )) ( ( )) [ ] ⎡ ⎣⎢ − ⎤ ⎦⎥= < ≤ ∈ = + D x t f t x t g t x t h t x t α t J T , , , , 1 2, 0, , α C 0 ( ) ( ( )) ( ( )) ( ) ( ( )) ( ( )) ⎡ ⎣⎢ − ⎤ ⎦⎥ + ⎡ ⎣⎢ − ⎤ ⎦⎥ = = = a x t f t x t g t x t b x t f t x t g t x t λ , , , , , t t T 1 0 1 1 ( ) ( ( )) ( ( )) ( ) ( ( )) ( ( )) ⎡ ⎣⎢ − ⎤ ⎦⎥ + ⎡ ⎣⎢ − ⎤ ⎦⎥ = < < = = + + a D x t f t x t g t x t b D x t f t x t g t x t λ η T , , , , , 0 , β C t η β C t T 2 0 2 0 2 where + D C α 0 and + D β C 0 stand for the Caputo fractional derivative. Recently, Baleanu et al. [23] discussed the hybrid fractional where + D C α 0 and + D β C 0 stand for the Caputo fractional derivative. Recently, Baleanu et al. [23] discussed the hybrid fractional coupled system as follows: where + D C α 0 and + D β C 0 stand for the Caputo fractional derivative. Recently, Baleanu et al. 2  Dehong Ji et al. 2  Dehong Ji et al. [23] discussed the hybrid fractional coupled system as follows: ( ) ( ( ) ( )) ( ( ) ( )) ( ] ⎜ ⎟ ⎛ ⎝ ⎞ ⎠= − ∈ D x t H t x t z t K t x t z t ω , , , , , 2, 3 , ω 1 ( ) ( ( ) ( )) ( ( ) ( )) ( ] ⎜ ⎟ ⎛ ⎝ ⎞ ⎠= − ∈ D z t g t x t z t K t x t z t ε , , , , , 2, 3 , ε 2 ( ) ( ( ) ( )) ( ) ( ( ) ( )) ( ) ( ) ⎜ ⎟ = ⎛ ⎝ ⎞ ⎠ = = = = x t H t x t z t D x t H t x t z t x , , 0, , , 0, 0 0, t μ t δ 1 2 1 ( ) ( ( ) ( )) ( ) ( ( ) ( )) ( ) ( ) ⎜ ⎟ = ⎛ ⎝ ⎞ ⎠ = = = = z t g t x t z t D z t g t x t z t x , , 0, , , 0, 0 0, t ν t δ 1 2 2 ( ) ( ( ) ( )) ( ) ( ( ) ( )) ( ) ( ) ⎜ ⎟ = ⎛ ⎝ ⎞ ⎠ = = = = x t H t x t z t D x t H t x t z t x , , 0, , , 0, 0 0, t μ t δ 1 2 1 ( ) ( ( ) ( )) ( ) ( ( ) ( )) ( ) ( ) ⎜ ⎟ = ⎛ ⎝ ⎞ ⎠ = = = = z t g t x t z t D z t g t x t z t x , , 0, , , 0, 0 0, t ν t δ 1 2 2 where D D D , , ω ε μ, and Dν stand for the Caputo’s fractional derivative. where D D D , , ω ε μ, and Dν stand for the Caputo’s fractional derivative. Gu et al. [12] discussed a new class of diﬀerential equations that contains the Riesz-Caputo fractional derivative: Gu et al. [12] discussed a new class of diﬀerential equations that contains the Riesz-Caputo fractional derivative: ( ) ( ( )) [ ] = ∈ < ≤ D x ξ h ξ x ξ ξ α , , 0, 1 , 0 1, α RC 1 0 ( ) ( ) = = x x x x 0 , 1 . 0 1 ( ) ( ) = = x x x x 0 , 1 . 0 1 On the other hand, hybrid diﬀerential equation is a class of dynamical systems with quadratic perturba- tion. Nowadays, due to the wide range of application of hybrid diﬀerential equations in several areas of real- life problems, more researchers began to study the existence of solutions for hybrid diﬀerential equations with diﬀerent perturbations (readers can refer to [13–20]). where < ≤ < ≤ ≤ ≤ γ ν t D 0 1, 0 1, 0 1, γ RC 1 0 , and Dν RC 1 0 are Riesz-Caputo’s fractional derivative of orders ([ ] ) ([ ] { }) ∈ × ∈ × γ ν f f h h C R R g g C R R , , , , , 0, 1 , , , 0, 1 , \ 0 1 2 1 2 2 1 2 2 and a a b , , 1 2 1, and b2 are the real constants. We will use “Dhage’s ﬁxed point theorem” to ﬁnd the suﬃcient conditions for the existence of solutions. This is the ﬁrst time for us to present the existence of solutions of Systems (1)–(4). Solutions of a system of hybrid fractional boundary value problems  3 3 By virtue of the aforementioned documents, in this work, the authors present the solutions for the hybrid fractional coupled system that contains the Riesz-Caputo derivative as follows: By virtue of the aforementioned documents, in this work, the authors present the solutions for the hybrid fractional coupled system that contains the Riesz-Caputo derivative as follows: ( ) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) ⎜ ⎟ ⎛ ⎝ − ⎞ ⎠ = < ≤ D x t f t x t y t g t x t y t h t x t y t γ , , , , , , , 0 1, γ RC 1 0 1 1 1 (1) ( ) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) ⎜ ⎟ ⎛ ⎝ − ⎞ ⎠ = < ≤ D y t f t x t y t g t x t y t h t x t y t ν , , , , , , , 0 1, ν RC 1 0 2 2 2 (2) (1) (2) ( ) ( ) = = x a x b 0 , 1 , 1 1 (3) ( ) ( ) = = y a y b 0 , 1 , 2 2 (4) (3) (3) (4) (4) where < ≤ < ≤ ≤ ≤ γ ν t D 0 1, 0 1, 0 1, γ RC 1 0 , and Dν RC 1 0 are Riesz-Caputo’s fractional derivative of orders ([ ] ) ([ ] { }) ∈ × ∈ × γ ν f f h h C R R g g C R R , , , , , 0, 1 , , , 0, 1 , \ 0 1 2 1 2 2 1 2 2 and a a b , , 1 2 1, and b2 are the real constants. We will use “Dhage’s ﬁxed point theorem” to ﬁnd the suﬃcient conditions for the existence of solutions. This is the ﬁrst time for us to present the existence of solutions of Systems (1)–(4). 2 Some lemmas Suppose − < ≤ ∈ n α n n N 1 , for > α 0, and [ ] = n ω and [ ]⋅stands for the ceiling of a number. Deﬁnition 2.1. [24] The Riesz-Caputo fractional derivative for a function ( ) ≤ ≤ x t t T , 0 , can be written as follows: ( ) ( ) ( ) ∣ ∣ ( ) ( ) ( ) ∫ ( ) = − − = + − + − D x t n α x u t u u D t D x t 1 Γ d 1 2 1 , T α RC T n α n t α C n C T α 0 0 1 0 ( ) ( ) ( ) ∣ ∣ ( ) ( ) ( ) ∫ ( ) = − − = + − + − D x t n α x u t u u D t D x t 1 Γ d 1 2 1 , T α RC T n α n t α C n C T α 0 0 1 0 where Dt α C 0 is the left Caputo derivative and DT α C t is the right Caputo derivatives ( ) ( ) ( ) ( ) ( ) ∫ = − − + − D x t n α x u t u u 1 Γ d t α C t n α n 0 0 1 ( ) ( ) ( ) ( ) ( ) ∫ = − − + − D x t n α x u t u u 1 Γ d t α C t n α n 0 0 1 and and and ( ) ( ) ( ) ( ) ( ) ( ) ∫ = − − − + − t D x t n α x u u t u 1 Γ d . C T α n t T n α n 1 ( ) ( ) ( ) ( ) ( ) ( ) ∫ = − − − + − t D x t n α x u u t u 1 Γ d . where D D D , , ω ε μ, and Dν stand for the Caputo’s fractional derivative. From the aforementioned equations, thus we have [25] Let S be a closed convex, bounded, and nonempty subset of a Banach algebra E, and let → A C E E , : and → B S E : be three operators such that (a) A and C are Lipschitzian with Lipschitz constants δ and ρ, respectively, (b) B is compact and continuous, (c) = + ⇒ ∈ x AxBy Cx x S for all ∈ y S, (d) + < δM ρ 1, where ‖ ( )‖ = M B S . Lemma 2.2. [25] Let S be a closed convex, bounded, and nonempty subset of a Banach algebra E, and let → A C E E , : and → B S E : be three operators such that p (a) A and C are Lipschitzian with Lipschitz constants δ and ρ, respectively, Then, there is a solution for the operator equation + = AxBx Cx x in S. Then, there is a solution for the operator equation + = AxBx Cx x in S. 2 Some lemmas C T α n t T n α n 1 Particularly, for ( ) ( ) ∈ x t C T 0, , then Particularly, for ( ) ( ) ∈ x t C T 0, , then ( ) ( ) ( ) = − D x t D D x t 1 2 , T α RC t α C t C T α 0 0 ( ) ( ) ( ) = − D x t D D x t 1 2 , T α RC t α C t C T α 0 0 if < ≤ α 0 1. if < ≤ α 0 1. Deﬁnition 2.2. [24] The right, left, and fractional Riemann-Liouville integrals of order α can be written as follows: ( ) ( ) ( ) ( ) ∫ = − − I x t α u t x u u 1 Γ d , T α t t T α 1 4  Dehong Ji et al. 4  Dehong Ji et al. ( ) ( ) ( ) ( ) ∫ = − − I x t α t u x u u 1 Γ d , t α t α 0 0 1 ( ) ( ) ∣ ∣ ( ) ∫ = − − I x t α u t x u u 1 Γ d . T α T α 0 0 1 ( ) ( ) ( ) ( ) ∫ = − − I x t α t u x u u 1 Γ d , t α t α 0 0 1 ( ) ( ) ∣ ∣ ( ) ∫ = − − I x t α u t x u u 1 Γ d . T α T α 0 0 1 Lemma 2.1. [24] Let ( ) [ ] ∈ x t C T 0, , n we have Lemma 2.1. [24] Let ( ) [ ] ∈ x t C T 0, , n we have ( ) ( ) ( )( ) ( ) ∑ = − − = − I D x t x t x l t 0 ! 2 Some lemmas 0 t α C t α l n l l 0 0 0 1 ( ) ( ) ( )( ) ( ) ∑ = − − = − I D x t x t x l t 0 ! 0 t α C t α l n l l 0 0 0 1 and ( ) ( ) ( ) ( ) ( ) ( ) ( ) ∑ = − ⎡ ⎣⎢ − − − ⎤ ⎦⎥ = − I D x t x t x T l T t 1 1 ! . T t α C t T α n l n l l l 0 1 and ( ) ( ) ( ) ( ) ( ) ( ) ( ) ∑ = − ⎡ ⎣⎢ − − − ⎤ ⎦⎥ = − I D x t x t x T l T t 1 1 ! . T t α C t T α n l n l l l 0 1 From the aforementioned equations, thus we have From the aforementioned equations, thus we have From the aforementioned equations, thus we have From the aforementioned equations, thus we have ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) = + + − + = + − I D x t I D I D x t I D I D x t I D I D x t 1 2 1 1 2 1 2 1 . T α RC T α t α C t α t T αC t α n t t α C T α T t α C t T α t α C t α n T t α C t T α 0 0 0 0 0 0 0 0 Particularly, for ( ) ( ) ∈ x t C T 0, , then Particularly, for ( ) ( ) ∈ x t C T 0, , then Particularly, for ( ) ( ) ∈ x t C T 0, , then ( ) ( ) ( ( ) ( )) = − + I D x t x t x x T 1 2 0 , T αRC T α 0 0 (5) ( ) ( ) ( ( ) ( )) = − + I D x t x t x x T 1 2 0 , T αRC T α 0 0 (5) ( ) ( ) ( ( ) ( )) = − + I D x t x t x x T 1 2 0 , T αRC T α 0 0 (5) if < ≤ α 0 1. if < ≤ α 0 1. if < ≤ α 0 1. Lemma 2.2. [25] Let S be a closed convex, bounded, and nonempty subset of a Banach algebra E, and let → A C E E , : and → B S E : be three operators such that (a) A and C are Lipschitzian with Lipschitz constants δ and ρ, respectively, (b) B is compact and continuous, (c) = + ⇒ ∈ x AxBy Cx x S for all ∈ y S Lemma 2.2. Lemma 3.1. Let [ ] ∈ h C 0, 1 . Then, the equation ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ∫ = ⎡ ⎣ ⎢ − + − + − − x t g t x t y t a f a a g a a b f b b g b b γ t s h s s , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ d t γ 1 1 2 1 2 1 1 2 1 2 0 1 ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ∫ ∫ = ⎡ ⎣ ⎢ − + − + − + − ⎤ ⎥+ − − x t g t x t y t a f a a g a a b f b b g b b γ t s h s s s t h s s f t x t y t , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ d 1 d t γ γ 1 1 2 1 2 1 1 2 1 2 0 1 1 1 ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ∫ ∫ ⎣ ⎢ + − ⎤ ⎦ ⎥+ − g a a g b b γ γ s t h s s f t x t y t 2 0, , 2 1, , Γ 1 Γ d , , t γ 1 2 1 2 0 1 1 (6) (6) ( ) ( ) ( ) ∫ ⎣ ⎤ g g γ 1 0 1 ( ( ) ( ) ( ) ( ( ) ( )) ∫ + − ⎤ ⎦ ⎥+ − γ s t h s s f t x t y t 1 Γ d , , t γ 1 1 is the solution of the boundary value problem: ( ) ( ( ) ( )) ( ( ) ( )) ( ) ⎜ ⎟ ⎛ ⎝ − ⎞ ⎠= < ≤ D x t f t x t y t g t x t y t h t γ , , , , , 0 1, γ RC 1 0 (7) ( ) ( ) = = x a x b 0 , 1 , 1 1 (8) ( ) ( ) = = y a y b 0 , 1 . γ 0 1 1 1 2 1 2 1 1 2 1 2 Consequently, we ﬁnd that Consequently, we ﬁnd that Consequently, we ﬁnd that ( ) ( ( ) ( )) ( ( ) ( )) ( ) ( ) ( ) ( ) − = − + − x t f t x t y t g t t t a f a a g b f b b g b b , , 1 2 0, , 0 1 2 1, , 1 1 1 2 1 1 2 ( ) ( ( ) ( )) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ∫ ∫ − = − + − + − + − − − x t f t x t y t g t x t y t a f a a g a a b f b b g b b γ t s h s s γ s t h s s , , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ d 1 Γ d . t γ t γ 1 1 2 1 2 1 1 2 1 2 0 1 1 1 lds. □ Therefore, (6) holds. □ Therefore, (6) holds. Deﬁne the operator T mapping E to E Deﬁne the operator T mapping E to E Deﬁne the operator T mapping E to E Deﬁne the operator T mapping E to E Deﬁne the operator T mapping E to E g ( )( ) ( ( )( ) ( )( )) = T x y t T x y t T x y t , , , , , 1 2 (10) ( )( ) ( ( )( ) ( )( )) = T x y t T x y t T x y t , , , , , 1 2 (10) ( )( ) ( ( )( ) ( )( )) = T x y t T x y t T x y t , , , , , 1 2 (10) (10) where ( )( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ( ) ( )) ∫ ∫ = ⎡ ⎣ ⎢ − + − + − + − ⎤ ⎦ ⎥+ − − T x y t g t x t y t a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s f t x t y t , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , , , t γ t γ 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 (11) ( )( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ( ) ( )) ∫ ∫ = ⎡ ⎣ ⎢ − + − + − + − ⎤ ⎦ ⎥+ − − T x y t g t x t y t a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s f t x t y t , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , , . t ν t ν 2 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 (12) )( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ∫ = ⎡ ⎣ ⎢ − + − + − − y t g t x t y t a f a a g a a b f b b g b b γ t s h s x s y s s , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d t γ 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 ( )( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ∫ = ⎡ ⎣ ⎢ − + − + − ⎤ − T x y t g t x t y t a f a a g a a b f b b g b b γ t s h s x s y s s , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d t γ 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 (11) ( )( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ( ) ( )) ∫ ∫ = ⎡ ⎣ ⎢ − + − + − + − ⎤ ⎦ ⎥+ − − T x y t g t x t y t a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s f t x t y t , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , , , t γ t γ 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 (11) (11) (11) ( )( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ( ) ( )) ∫ ∫ = ⎡ ⎣ ⎢ − + − + − + − ⎤ ⎦ ⎥+ − − T x y t g t x t y t a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s f t x t y t , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , , . 2 2 (9) ( ) ( ( ) ( )) ( ( ) ( )) ( ) ⎜ ⎟ ⎛ ⎝ − ⎞ ⎠= < ≤ D x t f t x t y t g t x t y t h t γ , , , , , 0 1, γ RC 1 0 (7) (9) Proof. Integrating on both sides of equation (7) and considering (5), (8), and (9), we obtain Proof. Integrating on both sides of equation (7) and considering (5), (8), and (9), we obtain ( ) ( ( ) ( )) ( ) ( ) ( ) − = + − + − x t f t x t y t I h t a f a a b f b b , , 1 0, , 1 1, , . γ 0 1 1 1 2 1 1 2 Proof. Integrating on both sides of equation (7) and considering (5), (8), and (9), we obtain ( ) ( ( ) ( )) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) − = + − + − x t f t x t y t g t x t y t I h t a f a a g a a b f b b g b b , , , , 1 2 0, , 0, , 1 2 1, , 1, , . g yp y ( ) H1 [ ] × → f f h h R R , , , : 0, 1 1 2 1 2 2 and [ ] { } × → g g R R , : 0, 1 \ 0 1 2 2 are continuous functions; 3 Main results The Banach space [ ] C 0, 1 is recorded as X. The norm is deﬁned as follows: ‖ ‖ ∣( )∣ [ ] = ∈ x x t max . t 0,1 Under the following norm deﬁnition ‖( )‖ ‖ ‖ ‖ ‖ = + x y x y , , we have = × E X X, which is a Banach space. Deﬁnition 3.1. [26] If there is a constant > μ 0 that satisﬁes ‖ ( ) ( )‖ (‖ ‖ ‖ ‖) − ≤ − + − F w z F w z μ w w z z , ¯, ¯ ¯ ¯ for all ( ) ( ) ∈ w z w z E , , ¯, ¯ , we call → F E E : as μ-Lipschitz. Moreover, if < μ 1, we call F as a strict contraction. Solutions of a system of hybrid fractional boundary value problems  5 Solutions of a system of hybrid fractional boundary value problems  5 Solutions of a system of hybrid fractional boundary value problems  5 Solutions of a system of hybrid fractional boundary value problems  5 t 2 0,1 2 and ‖ ‖ ‖ ‖ + < ϕ ϕ Λ 1 0 1 2, ‖ ‖ ‖ ‖ + < ϕ ϕ Λ 1 0 1 2, ∣( )∣ [ ] = ∈ f f t sup , 0, 0 t 1 0,1 1 , ∣( )∣ [ ] = ∈ f f t sup , 0, 0 t 2 0,1 2 , ∣( )∣ [ ] = ∈ g g t sup , 0, 0 t 1 0,1 1 , ∣( )∣ [ ] = ∈ g g t sup , 0, 0 . t 2 0,1 2 Theorem 3.2. Problems (1)–(4) have a solution, if Conditions (H1)–(H4) are true. Proof. Let ∣( ) ( )∣ ( )[∣ ∣ ∣ ∣] − ≤ − + − f t x y f t x y ϕ t x x y y , , , ¯, ¯ ¯ ¯ , 2 2 0 (14) ∣( ) ( )∣ ( )[∣ ∣ ∣ ∣] − ≤ − + − g t x y g t x y ϕ t x x y y , , , ¯, ¯ ¯ ¯ , 1 1 1 (15) ∣( ) ( )∣ ( )[∣ ∣ ∣ ∣] − ≤ − + − g t x y g t x y ϕ t x x y y , , , ¯, ¯ ¯ ¯ , 2 2 1 (16) (16) for all [ ] ∈ t x x y 0, 1 , , ¯, , and y¯ are the elements in R; for all [ ] ∈ t x x y 0, 1 , , ¯, , and y¯ are the elements in R; for all [ ] ∈ t x x y 0, 1 , , , , and y are the elements in R; ( ) H3 Functions ( ) ∈ ∞ + p p L J R , , 1 2 and four continuous nondecreasing functions [ ) ( ) ∞→ ∞ ψ ψ ξ ξ , , , : 0, 0, 1 2 1 2 i f [ ] y y ( ) H3 Functions ( ) ∈ ∞ + p p L J R , , 1 2 and four continuous nondecreasing functions [ ) ( ) ∞→ ∞ ψ ψ ξ ξ , , , : 0, 0, 1 2 1 2 satisfy unctions ( ) ∈ ∞ + p p L J R , , 1 2 and four continuous nondecreasing functions [ ) ( ) ∞→ ∞ ψ ψ ξ ξ , , , : 0, 0, 1 2 1 2 ( ) H3 Functions ( ) ∈ ∞ + p p L J R , , 1 2 and four continuous nondecreasing functions [ ) ( ) ∞→ ∞ ψ ψ ξ ξ , , , : 0, 0, 1 2 1 2 satisfy ∣ ( )∣ ( ) (∣∣) (∣∣) ≤ h t x y p t ψ x ξ y , , , 1 1 1 1 (17) ∣ ( )∣ ( ) (∣∣) (∣∣) ≤ h t x y p t ψ x ξ y , , , 2 2 2 2 (18) ∣ ( )∣ ( ) (∣∣) (∣∣) ≤ h t x y p t ψ x ξ y , , , 1 1 1 1 (17) ∣ ( )∣ ( ) (∣∣) (∣∣) ≤ h t x y p t ψ x ξ y , , , 2 2 2 2 (18) ∣ ( )∣ ( ) (∣∣) (∣∣) ≤ h t x y p t ψ x ξ y , , , 1 1 1 1 (17) ∣ ( )∣ ( ) (∣∣) (∣∣) ≤ h t x y p t ψ x ξ y , , , 2 2 2 2 (18) (17) for all [ ] ∈ t x 0, 1 , and y are the elements in R; ( ) H4 The positive number r satisﬁes ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ⎜ ⎟ − ⎛ ⎝ + − − + + − − ⎞ ⎠ < ρ g f ϕ ϕ g f ϕ ϕ r 1 1 Λ 1 Λ Λ 1 Λ , 1 1 1 0 2 2 1 0 (19) ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ⎜ ⎟ − ⎛ ⎝ + − − + + − − ⎞ ⎠ < ρ g f ϕ ϕ g f ϕ ϕ r 1 1 Λ 1 Λ Λ 1 Λ , 1 1 1 0 2 2 1 0 (19) where (19) ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ − ⎝ − − − − ⎠ ρ ϕ ϕ ϕ ϕ 1 1 Λ 1 Λ 1 0 1 0 where where ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ = ⎧⎨⎩ + − − + − − ⎫⎬⎭ ρ ϕ ϕ ϕ ϕ ϕ ϕ ϕ ϕ max Λ 1 Λ , Λ 1 Λ , 1 0 1 0 1 0 1 0 (20) ( ) ( ) ( ) ( ) ( ) ( )‖ ‖ ( ) ( ) ( ) ( ) ( ) ( ) ( )‖ ‖ ( ) = − + − + + + − + − + + a f a a g a a b f b b g b b ψ r ξ r p γ a f a a g a a b f b b g b b ψ r ξ r p ν Λ 1 2 0, , 0, , 1 2 1, , 1, , 2 Γ 1 1 2 0, , 0, , 1 2 1, , 1, , 2 Γ 1 , 1 1 1 2 1 1 2 1 1 1 2 1 1 2 1 1 1 2 2 1 2 2 1 2 2 2 1 2 2 1 2 2 2 2 (21) 1 1 ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ = ⎧⎨⎩ + − − + − − ⎫⎬⎭ ρ ϕ ϕ ϕ ϕ ϕ ϕ ϕ ϕ max Λ 1 Λ , Λ 1 Λ , 1 0 1 0 1 0 1 0 (20) (20) ( ) ( ) ( ) ( ) ( ) ( )‖ ‖ ( ) ( ) ( ) ( ) ( ) ( ) ( )‖ ‖ ( ) = − + − + + + − + − + + a f a a g a a b f b b g b b ψ r ξ r p γ a f a a g a a b f b b g b b ψ r ξ r p ν Λ 1 2 0, , 0, , 1 2 1, , 1, , 2 Γ 1 1 2 0, , 0, , 1 2 1, , 1, , 2 Γ 1 , 1 1 1 2 1 1 2 1 1 1 2 1 1 2 1 1 1 2 2 1 2 2 1 2 2 2 1 2 2 1 2 2 2 2 (21) (21) and ‖ ‖ ‖ ‖ + < ϕ ϕ Λ 1 0 1 2, ‖ ‖ ‖ ‖ + < ϕ ϕ Λ 1 0 1 2, ∣( )∣ [ ] = ∈ f f t sup , 0, 0 t 1 0,1 1 , ∣( )∣ [ ] = ∈ f f t sup , 0, 0 t 2 0,1 2 , ∣( )∣ [ ] = ∈ g g t sup , 0, 0 t 1 0,1 1 , ∣( )∣ [ ] = ∈ g g t sup , 0, 0 . t ν t ν 2 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 (12) (12) We give the hypotheses of this study. g yp y ( ) H1 [ ] × → f f h h R R , , , : 0, 1 1 2 1 2 2 and [ ] { } × → g g R R , : 0, 1 \ 0 1 2 2 are continuous functions; ( ) H2 ϕ ϕ ϕ ϕ , , ¯ , ¯ 0 1 0 1 are four positive functions such that ( ) H2 ϕ ϕ ϕ ϕ , , ¯ , ¯ 0 1 0 1 are four positive functions such that ∣( ) ( )∣ ( )[∣ ∣ ∣ ∣] − ≤ − + − f t x y f t x y ϕ t x x y y , , , ¯, ¯ ¯ ¯ , 1 1 0 (13) (13) ong Ji et al. 6  Dehong Ji et al. 6  Dehong Ji et al. 2 2 2 2 (28) (27) Therefore, the operator T in equation (10) can be written as follows: T in equation (10) can be written as follows: Therefore, the operator T in equation (10) can be written as follows: Therefore, the operator T in equation (10) can be written as follows: Therefore, the operator T in equation (10) can be written as follows: ( )( ) ( ( )( ) ( )( )) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) = = + T x y t T x y t T x y t A x t y t B x t y t C x t y t , , , , , , , , 1 2 ( )( ) ( ( )( ) ( )( )) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) = = + T x y t T x y t T x y t A x t y t B x t y t C x t y t , , , , , , , , 1 2 y y y y y y where ( ) ( ) = = A A A B B B , , , 1 2 1 2 , and ( ) = C C C , . 1 2 In the following, we prove that the conditions in Lemma 2.2 can be satisﬁed. where ( ) ( ) = = A A A B B B , , , 1 2 1 2 , and ( ) = C C C , . 1 2 In the following, we prove that the conditions in Lemma 2.2 can be satisﬁed. First, we prove that A and C are Lipschitz on the space E. Given ( ) ( ) ∈ x y x y E , , , 1 1 2 2 , and [ ] ∈ t 0, 1 , then by ( ) H , 2 we obtain First, we prove that A and C are Lipschitz on the space E. ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ∫ ∫ = − + − + − + − − − B x t y t a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , t ν t ν 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 (25) (25) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) = = C x t y t f t x t y t C x t y t f t x t y t , , , , and , , , . 1 1 2 2 (26) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) = = C x t y t f t x t y t C x t y t f t x t y t , , , , and , , , . 1 1 2 2 (26) (26) Then, operators T1 and T2 given by equations (11) and (12) are equivalent to Then, operators T1 and T2 given by equations (11) and (12) are equivalent to Then, operators T1 and T2 given by equations (11) and (12) are equivalent to ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) = + T x t y t A x t y t B x t y t C x t y t , , , , , 1 1 1 1 (27) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) = + T x t y t A x t y t B x t y t C x t y t , , , , . Proof. Let {( ) ‖( )‖ } = ∈ ≤ S x y E x y r , : , . (22) (22) Then, S is a bounded convex closed subset of E. Deﬁne operators → A C E X , : i i and ( ) → = B S X i : 1, 2 i by ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) ( ( ) ( )) = = A x t y t g t x t y t A x t y t g t x t y t , , , , , , , , 1 1 2 2 (23) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ∫ ∫ = − + − + − + − − − B x t y t a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , t γ t γ 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 (24) (23) (24) Solutions of a system of hybrid fractional boundary value problems  7 Solutions of a system of hybrid fractional boundary value problems  7 Solutions of a system of hybrid fractional boundary value problems  7 Solutions of a system of hybrid fractional boundary value problems  7 Similarly, 1 1 We consider ( ) ( ) → ∈ C E X x y x y E : , , , , , 1 1 2 2 then which implies that A is Lipschitz on the space E with Lipschitz constant ‖ ‖ ‖ ‖ + ϕ ϕ . 1 1 We consider ( ) ( ) → ∈ C E X x y x y E : , , , , , 1 1 2 2 then which implies that A is Lipschitz on the space E with Lipschitz constant ‖ ‖ ‖ ‖ + ϕ ϕ . 1 1 We consider ( ) ( ) → ∈ C E X x y x y E : , , , , , 1 1 2 2 then ∣ ( )( ) ( )( )∣ ∣( ( ) ( )) ( ( ) ( ))∣ ( )(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ − + − C x y t C x y t f t x t y t f t x t y t ϕ t x t x t y t y t , , , , , , . 1 1 1 1 2 2 1 1 1 1 2 2 0 1 2 1 2 (31) ∣ ( )( ) ( )( )∣ ∣( ( ) ( )) ( ( ) ( ))∣ ( )(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ − + − C x y t C x y t f t x t y t f t x t y t ϕ t x t x t y t y t , , , , , , . 1 1 1 1 2 2 1 1 1 1 2 2 0 1 2 1 2 (31) (31) Given ( ) ( ) ∈ x y x y E , , , 1 1 2 2 , and [ ] ∈ t 0, 1 , then by ( ) H , 2 we obtain ∣ ( )( ) ( )( )∣ ∣( ( ) ( )) ( ( ) ( ))∣ ( )(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ − + − A x y t A x y t g t x t y t g t x t y t ϕ t x t x t y t y t , , , , , , . 1 1 1 1 2 2 1 1 1 1 2 2 1 1 2 1 2 (29) (29) Similarly, Similarly, Similarly, ∣ ( )( ) ( )( )∣ ∣( ( ) ( )) ( ( ) ( ))∣ ( )(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ − + − A x y t A x y t g t x t y t g t x t y t ϕ t x t x t y t y t , , , , , , . 2 1 1 2 2 2 2 1 1 2 2 2 1 1 2 1 2 (30) (30) From equations (29) and (30), we obtain ∣( )( ) ( )( )∣ ∣( )( ) ( )( )∣ ( ( ) ( ))(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ + − + − A x y t A x y t A A x y A A x y ϕ t ϕ t x t x t y t y t , , , , , , 1 1 2 2 1 2 1 1 1 2 2 2 1 1 1 2 1 2 From equations (29) and (30), we obtain ∣( )( ) ( )( )∣ ∣( )( ) ( )( )∣ ( ( ) ( ))(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ + − + − A x y t A x y t A A x y A A x y ϕ t ϕ t x t x t y t y t , , , , , , 1 1 2 2 1 2 1 1 1 2 2 2 1 1 1 2 1 2 for all t on the interval [ ] 0, 1 . Taking the supremum on both sides of the aforementioned formula with respect to t, we have for all t on the interval [ ] 0, 1 . Taking the supremum on both sides of the aforementioned formula with respect to t, we have ‖ ( ) ( )‖ (‖ ‖ ‖ ‖)(‖ ‖ ‖ ‖) − ≤ + − + − A x y A x y ϕ ϕ x x y y , , , 1 1 2 2 1 1 1 2 1 2 which implies that A is Lipschitz on the space E with Lipschitz constant ‖ ‖ ‖ ‖ + ϕ ϕ . 8  Dehong Ji et al. 8  Dehong Ji et al. Similarly, Similarly, ∣ ( )( ) ( )( )∣ ∣( ( ) ( )) ( ( ) ( ))∣ ( )(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ − + − C x y t C x y t f t x t y t f t x t y t ϕ t x t x t y t y t , , , , , , . 2 1 1 2 2 2 2 1 1 2 2 2 0 1 2 1 2 (32) ∣ ( )( ) ( )( )∣ ∣( ( ) ( )) ( ( ) ( ))∣ ( )(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ − + − C x y t C x y t f t x t y t f t x t y t ϕ t x t x t y t y t , , , , , , . 2 1 1 2 2 2 2 1 1 2 2 2 0 1 2 1 2 (32) (32) From equations (31) and (32), we obtain ∣( )( ) ( )( )∣ ∣( )( ) ( )( )∣ ( ( ) ( ))(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ + − + − C x y t C x y t C C x y C C x y ϕ t ϕ t x t x t y t y t , , , , , , 1 1 2 2 1 2 1 1 1 2 2 2 0 0 1 2 1 2 From equations (31) and (32), we obtain ∣( )( ) ( )( )∣ ∣( )( ) ( )( )∣ ( ( ) ( ))(∣( ) ( )∣ ∣ ( ) ( )∣) − = − ≤ + − + − C x y t C x y t C C x y C C x y ϕ t ϕ t x t x t y t y t , , , , , , 1 1 2 2 1 2 1 1 1 2 2 2 0 0 1 2 1 2 8 for all t on the interval [ ] 0, 1 . Taking the supremum on both sides of the aforementioned formula with respect to t, we obtain ‖ ( ) ( )‖ (‖ ‖ ‖ ‖)(‖ ‖ ‖ ‖) − ≤ + − + − C x y C x y ϕ ϕ x x y y , , , 1 1 2 2 0 0 1 2 1 2 which implies that C is Lipschitz on the space E with Lipschitz constant ‖ ‖ ‖ ‖ + ϕ ϕ . 0 0 Second, we prove that B is completely continuous mapping S to X. In Step 1, we show that B is a continuous operator on S. To prove this, assume that { } { } x y , n n are convergent sequences and satisfy → → x x y y , n n as →+∞ n . By the Lebesgue dominated convergence theorem, we have ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ∫ ∫ ⎜ = ⎛ ⎝ − + − + − + − ⎞ ⎠ ⎟ = − + − →∞ →∞ − − B x y a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s a f a a g a a b f b b g b b lim , lim 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d 1 2 0, , 0, , 1 2 1, , 1, , n n n n t γ n n t γ n n 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ∫ ∫ ∫ ∫ ∫ ∫ ∫ ∫ ⎜ = ⎛ ⎝ − + − + − + − ⎞ ⎠ ⎟ = − + − + − + − = − + − + − + − = − + − + − + − = →∞ →∞ − − →∞ − →∞ − − →∞ − →∞ − − B x y a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s B x lim , lim 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ lim , , d 1 Γ lim , , d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ lim , , d 1 Γ lim , , d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , n n n n t γ n n t γ n n n t γ n n n t γ n n t γ n n n t γ n n n t γ γ 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 (33) Solutions of a system of hybrid fractional boundary value problems  9 Solutions of a system of hybrid fractional boundary value problems  9 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ∫ ∫ ∫ ∫ ∫ ∫ ∫ ∫ ⎜ = ⎛ ⎝ − + − + − + − ⎞ ⎠ ⎟ = − + − + − + − = − + − + − + − = − + − + − + − = →∞ →∞ − − →∞ − →∞ − − →∞ − →∞ − − B x y a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s B x y lim , lim 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ lim , , d 1 Γ lim , , d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ lim , , d 1 Γ lim , , d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , n n n n t ν n n t ν n n n t ν n n n t ν n n t ν n n n t ν n n n t ν t ν 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ) ( ) ∫ ∫ ⎜ = ⎛ ⎝ − + − + − + − ⎞ ⎠ ⎟ = − + − →∞ →∞ − − B x y a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s a f a a g a a b f b b g b b lim , lim 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d 1 2 0, , 0, , 1 2 1, , 1, , n n n n t ν n n t ν n n 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 (34) In view of equations (33) and (34), for all [ ] ∈ t 0, 1 , the operator ( ) = B B B ,1 2 is a continuous operator. 10  Dehong Ji et al. 10  Dehong Ji et al. In view of equations (33) and (34), for all [ ] ∈ t 0, 1 , the operator ( ) = B B B ,1 2 is a continuous operator. t γ t γ t γ t γ 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 1 1 1 0 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 1 1 1 ∣ ( ( ) ( ))∣ ( ) ( ) ( ) ( ) ≤ − + − B x t y t a f a a g a a b f b b g b b , 1 2 0, , 0, , 1 2 1, , 1, , 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 (35) ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ( ) ( ) ‖ ‖ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ‖ ‖ ( ) ( ) ( ) ∫ ∫ ∫ ∫ + − + − ≤ − + − + ⎛ ⎝ ⎜ − + − ⎞ ⎠ ⎟ = − + − + + − − − − ν t s h s x s y s s ν s t h s x s y s s a f a a g a a b f b b g b b p ψ r ξ r ν t s s s t s a f a a g a a b f b b g b b p ψ r ξ r ν 1 Γ , , d 1 Γ , , d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ d d 1 2 0, , 0, , 1 2 1, , 1, , 2 Γ 1 . In Step 2, we prove that in S, the set ( ) B S is uniformly bounded. For any ( ) ∈ x y S , , in consideration of ( ) H , 3 we obtain In Step 2, we prove that in S, the set ( ) B S is uniformly bounded. For any ( ) ∈ x y S , , in consideration of ( ) H , 3 we obtain ∣ ( ( ) ( ))∣ ( ) ( ) ( ) ( ) ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ‖ ‖ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ‖ ‖ ( ) ( ) ( ) ∫ ∫ ∫ ∫ ∫ ∫ ≤ − + − + − + − ≤ − + − + − + − = − + − + ⎛ ⎝ ⎜ − + − ⎞ ⎠ ⎟ = − + − + + − − − − − − B x t y t a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s a f a a g a a b f b b g b b γ t s p s ψ r ξ r s γ s t p s ψ r ξ r s a f a a g a a b f b b g b b p ψ r ξ r γ t s s s t s a f a a g a a b f b b g b b p ψ r ξ r γ , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ d 1 Γ d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ d d 1 2 0, , 0, , 1 2 1, , 1, , 2 Γ 1 . t γ t γ t γ t γ t γ t γ 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 1 1 1 0 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 1 1 1 (35) ∣ ( ( ) ( ))∣ ( ) ( ) ( ) ( ) ≤ − + − B x t y t a f a a g a a b f b b g b b , 1 2 0, , 0, , 1 2 1, , 1, , t 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 ∣ ( ( ) ( ))∣ ( ) ( ) ( ) ( ) ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ( ) ( ) ∫ ∫ ≤ − + − + − + − ≤ − + − − − B x t y t a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s a f a a g a a b f b b g b b , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d 1 2 0, , 0, , 1 2 1, , 1, , t γ t γ t 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ‖ ‖ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ‖ ‖ ( ) ( ) ( ) ∫ ∫ ∫ ∫ ≤ − + − + − + − = − + − + ⎛ ⎝ ⎜ − + − ⎞ ⎠ ⎟ = − + − + + − − − − a f a a g a a b f b b g b b γ t s p s ψ r ξ r s γ s t p s ψ r ξ r s a f a a g a a b f b b g b b p ψ r ξ r γ t s s s t s a f a a g a a b f b b g b b p ψ r ξ r γ 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ d 1 Γ d 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ d d 1 2 0, , 0, , 1 2 1, , 1, , 2 Γ 1 . γ γ γ γ γ γ γ γ γ γ 1 1 1 2 2 1 1 2 1 2 1 1 1 1 2 1 2 1 Similarly, we obtain Similarly, we obtain ∣ ( )( ) ( )( )∣ ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ∫ ∫ ∫ ∫ − = − + − − − − − ⎛ − − − − B x y t B x y t ν t s h s x s y s s ν s t h s x s y s s ν t s h s x s y s s ν s t h s x s y s s , , 1 Γ , , d 1 Γ , , d 1 Γ , , d 1 Γ , , d t ν t ν t ν t ν t t 2 2 2 1 0 2 1 2 1 2 1 2 0 1 1 2 1 1 1 2 2 2 1 1 1 2 ∣ ( )( ) ( )( )∣ ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ‖ ‖ ( ) ( ) ( ) (( ) ( ) ) (( ) ( ) ) (( ) ( ) ) ‖ ‖ ( ) ( ) ( ) [ ( ) ( ) ] ∫ ∫ ∫ ∫ ∫ ∫ ∫ − = − + − − − − − ≤ + ⎛ ⎝ ⎜ − − − + − − − + − − − ⎞ ⎠ ⎟ = + − + − − − − − − − − − − − − − B x y t B x y t ν t s h s x s y s s ν s t h s x s y s s ν t s h s x s y s s ν s t h s x s y s s p ψ r ξ r ν t s t s s t s s t s s t s t s p ψ r ξ r ν t t t t , , 1 Γ , , d 1 Γ , , d 1 Γ , , d 1 Γ , , d Γ 1 d d d Γ 1 1 1 . 11 In the following, we show that the operator B is equicontinuous. To prove this, given ( ) ≤ ≤ ≤ ∈ t t x y S 0 1, , , 1 2 we can ﬁnd In the following, we show that the operator B is equicontinuous. To prove this, given ( ) ≤ ≤ ≤ ∈ t t x y S 0 1, , , 1 2 we can ﬁnd ∣ ( )( ) ( )( )∣ ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) (( ) ( ) )∣ ( ( ) ( ))∣ ( ) (( ) ( ) )∣ ( ( ) ( ))∣ ( ) (( ) ( ) )∣ ( ( ) ( ))∣ ‖ ‖ ( ) ( ) ( ) (( ) ( ) ) (( ) ( ) ) (( ) ( ) ) ‖ ‖ ( ) ( ) ( ) [( ( ) ) ( ) ( ) ( ) ] ‖ ‖ ( ) ( ) ( ) [ ( ) ( ) ] ∫ ∫ ∫ ∫ ∫ ∫ ∫ ∫ ∫ ∫ − = − + − − − − − ≤ − − − + − − − + − − − ≤ ⎛ ⎝ ⎜ − − − + − − − + − − − ⎞ ⎠ ⎟ = + − − − + − − − + − = + − + − − − − − − − − − − − − − − − − − − − B x y t B x y t γ t s h s x s y s s γ s t h s x s y s s γ t s h s x s y s s γ s t h s x s y s s γ t s t s h s x s y s s γ t s s t h s x s y s s γ s t s t h s x s y s s p ψ r ξ r γ t s t s s t s s t s s t s t s p ψ r ξ r γ t t t t t t t t p ψ r ξ r γ t t t t , , 1 Γ , , d 1 Γ , , d 1 Γ , , d 1 Γ , , d 1 Γ , , d 1 Γ , , d 1 Γ , , d Γ d d d Γ 1 1 1 Γ 1 1 1 . t ν t ν t ν t ν 0 1 2 1 1 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 2 2 2 0 1 1 1 2 2 1 2 2 1 2 2 2 1 2 2 1 2 2 2 2 (36) (36) Thus, ‖ ( )‖ ≤ B x y , Λ for all ( ) ∈ x y S , with Λ given in equation (21), which implies that the set ( ) B S is a uniformly bounded set in S. Solutions of a system of hybrid fractional boundary value problems  11 t γ t γ t γ t γ t γ γ t t γ γ t γ γ t γ γ t t γ γ t γ γ γ γ γ γ γ γ γ γ γ γ 1 2 1 1 0 2 1 1 1 2 1 1 0 1 1 1 1 1 1 1 0 2 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 0 2 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 2 1 2 1 1 1 1 2 1 2 1 2 2 1 1 1 1 2 2 1 1 2 2 (37) ∣ ( )( ) ( )( )∣ ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) ( ) ( ( ) ( )) ( ) (( ) ( ) )∣ ( ( ) ( ))∣ ∫ ∫ ∫ ∫ ∫ − = − + − − − − − ≤ − − − − − − − − − B x y t B x y t γ t s h s x s y s s γ s t h s x s y s s γ t s h s x s y s s γ s t h s x s y s s γ t s t s h s x s y s s , , 1 Γ , , d 1 Γ , , d 1 Γ , , d 1 Γ , , d 1 Γ , , d t γ t γ t γ t γ t γ γ 1 2 1 1 0 2 1 1 1 2 1 1 0 1 1 1 1 1 1 1 2 1 1 1 1 2 2 1 1 1 (37) ‖ ‖ ( ) ( ) ( ) [( ( ) ) ( ) ( ) ( ) ] ‖ ‖ ( ) ( ) ( ) [ ( ) ( ) ] = + − − − + − − − + − = + − + − − − p ψ r ξ r γ t t t t t t t t p ψ r ξ r γ t t t t Γ 1 1 1 Γ 1 1 1 . Take any two elements x y , from E and any two elements x y ¯, ¯ from S, which satisfy ( ) = x y , ( ) ( ) ( ) + A x y B x y C x y , , , ; using ( ) ( ) H H , 2 3 , and ( ) H , 4 we ﬁnd ∣( )∣ ∣ ( ( ) ( ))∣∣ ( ( ) ( ))∣ ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ ( ) ( ) ( ) ( ) ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ (∣( ( ) ( )) ( )∣ ∣( )∣) ( ) ( ) ( ) ( ) ∫ ∫ ⎜ ⎜ ≤ + ≤ ⎛ ⎝ − + − + − + − ⎞ ⎠ ⎟+ ≤ − + ⎛ ⎝ − + − − − x t A x t y t B x t y t C x t y t g t x t y t a f a a g a a b f b b g b b γ t s h s x s y s s γ s t h s x s y s s f t x t y t g t x t y t g t g t a f a a g a a b f b b g b b , , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , , , 0, 0 , 0, 0 1 2 0, , 0, , 1 2 1, , 1, , t γ t γ 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 0 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 (39) )∣∣ ( ( ) ( ))∣ ∣ ( ( ) ( ))∣ ))∣ ( ) ( ) ( ) ( ) ⎜ + ⎛ ⎝ − + − B x t y t C x t y t a f a a g a a b f b b g b b , , 1 2 0, , 0, , 1 2 1, , 1, , 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ (∣( ( ) ( )) ( )∣ ∣( )∣) ( ) ( ) ( ) ( ) ∫ ∫ ⎜ + − + − ⎞ ⎠ ⎟+ ≤ − + ⎛ ⎝ − + − − − γ t s h s x s y s s γ s t h s x s y s s f t x t y t g t x t y t g t g t a f a a g a a b f b b g b b 1 Γ , , d 1 Γ , , d , , , 0, 0 , 0, 0 1 2 0, , 0, , 1 2 1, , 1, , t γ t γ 0 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ ( ) ( ) ∫ ∫ + − + − ⎞ ⎠ ⎟+ ⎛ − − γ t s h s x s y s s γ s t h s x s y s s f t x t y t a f a a b f b b 1 Γ , , d 1 Γ , , d , 1 0 1 1 t γ t γ 0 1 1 1 1 1 1 ( ) ( ) (∣( ( ) ( )) ( )∣ ∣( )∣) ( ) ( ) ( ) ( ) ∫ ∫ ⎜ ⎠ ⎟ ≤ − + ⎛ ⎝ − + − γ γ g t x t y t g t g t a f a a g a a b f b b g b b Γ Γ , , 0, 0 , 0, 0 1 2 0, , 0, , 1 2 1, , 1, , t 0 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 2 (39) ‖ ‖ ( ) ( ) ( ) ( ) ( ) (∣( ( ) ( )) ( )∣ ∣( )∣) (‖ ‖(∣( )∣ ∣( )∣) ) ‖ ‖(∣( )∣ ∣( )∣) ∫ ∫ + − + − ⎞ ⎠ ⎟+ − + ≤ + + + + + − − p ψ r ξ r γ t s s s t s f t x t y t f t f t ϕ x t y t g ϕ x t y t f Γ d d , , 0, 0 , 0, 0 Λ . t ν t ν t ν t ν t ν ν t t ν ν t ν ν ν ν ν ν 2 2 2 1 0 2 1 2 1 2 1 2 0 1 1 2 1 1 1 2 2 2 2 0 2 1 1 1 2 1 1 1 1 2 1 1 1 2 2 2 2 1 2 1 2 2 1 1 1 1 2 2 (38) (38) Using equations (37) and (38), we ﬁnd that Using equations (37) and (38), we ﬁnd that Using equations (37) and (38), we ﬁnd that Using equations (37) and (38), we ﬁnd that ∣( )( ) ( )( )∣ ‖ ‖ ( ) ( ) ( ) [ ( ) ( ) ] ‖ ‖ ( ) ( ) ( ) [ ( ) ( ) ] − ≤ + − + − − − + + − + − − − B x y t B x y t p ψ r ξ r γ t t t t p ψ r ξ r ν t t t t , , Γ 1 1 1 Γ 1 1 1 . γ γ γ γ ν ν ν ν 2 1 1 1 1 2 1 2 1 2 2 2 2 1 2 1 12  Dehong Ji et al.  Dehong Ji et al. 12  Dehong Ji et al. 12 Thus, when → t t , 2 1 we have ∣( )( ) ( )( )∣ − → B x y t B x y t , , 0, 2 1 so that we can ﬁnd that B is equicontinuous; there- fore, we have the operator B that is compact according to the Arzela-Ascoli theorem. Thus, when → t t , 2 1 we have ∣( )( ) ( )( )∣ − → B x y t B x y t , , 0, 2 1 so that we can ﬁnd that B is equicontinuous; there- fore, we have the operator B that is compact according to the Arzela-Ascoli theorem. Third, we prove that conditions (c) of Lemma 2.2 holds. t γ t γ 1 1 1 0 1 1 1 1 1 1 1 1 0 1 Taking the supremum on both sides of the aforementioned formula with respect to t, ‖ ‖ (‖ ‖(‖ ‖ ‖ ‖) ) ‖ ‖(‖ ‖ ‖ ‖∣) ≤ + + + + + x ϕ x y g ϕ x y f Λ , 1 1 0 1 Taking the supremum on both sides of the aforementioned formula with respect to t, ‖ ‖ (‖ ‖(‖ ‖ ‖ ‖) ) ‖ ‖(‖ ‖ ‖ ‖∣) ≤ + + + + + x ϕ x y g ϕ x y f Λ , 1 1 0 1 this equals to this equals to ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ≤ + + + − − x ϕ ϕ y g f ϕ ϕ Λ Λ 1 Λ . 1 0 1 1 1 0 Similarly, we obtain ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ≤ + + + − − x ϕ ϕ y g f ϕ ϕ Λ Λ 1 Λ . 1 0 1 1 1 0 ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ≤ + + + − − x ϕ ϕ y g f ϕ ϕ Λ Λ 1 Λ . t ν t ν t ν t ν 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 2 2 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 2 2 2 0 1 1 1 2 2 2 1 2 0 2 (40) ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ ∫ ∫ + − + − ⎞ ⎠ ⎟+ − − ν t s h s x s y s s ν s t h s x s y s s f t x t y t 1 Γ , , d 1 Γ , , d , t ν t ν 0 1 2 1 1 2 2 ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ (∣( ( ) ( )) ( )∣ ∣( )∣) ( ) ( ) ∫ ∫ ⎜ + − + − ⎞ ⎠ ⎟+ ≤ − + ⎛ ⎝ − − − ν t s h s x s y s s ν s t h s x s y s s f t x t y t g t x t y t g t g t a f a a g a a 1 Γ , , d 1 Γ , , d , , , 0, 0 , 0, 0 1 2 0, , 0 t ν t ν 0 1 2 1 1 2 2 2 2 2 2 2 1 2 (40) (∣( ( ) ( )) ( )∣ ∣( )∣) ( ) ( ) ⎜ ≤ − + ⎛ ⎝ − g t x t y t g t g t a f a a g a a , , 0, 0 , 0, 0 1 2 0, , 0, , 2 2 2 2 2 1 2 2 1 2 ( ) ( ) ‖ ‖ ( ) ( ) ( ) ( ) ( ) ∫ ∫ + − + − + − ⎞ ⎠ ⎟ − − b f b b g b b p ψ r ξ r ν t s s s t s 1 2 1, , 1, , Γ d d t ν t ν 2 2 1 2 2 1 2 2 2 2 0 1 1 1 (‖ ‖(∣( )∣ ∣( )∣) ) ‖ ‖(∣( )∣ ∣( )∣) ≤ + + + + + f y f f ϕ x t y t g ϕ x t y t f Λ . 1 0 1 1 1 0 Similarly, we obtain ∣( )∣ ∣ ( ( ) ( ))∣∣ ( ( ) ( ))∣ ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ ( ) ( ) ( ) ( ) ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ ∫ ∫ ⎜ ≤ + ≤ ⎛ ⎝ − + − + − + − ⎞ ⎠ ⎟+ − − y t A x t y t B x t y t C x t y t g t x t y t a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s f t x t y t , , , , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , t ν t ν 2 2 2 2 2 2 1 2 2 1 2 2 2 1 2 2 1 2 0 1 2 1 1 2 2 ∣( ( ) ( ))∣ ( ) ( ) ( ) ( ) ( ) ( ) ∣ ( ( ) ( ))∣ ( ) ( ) ∣ ( ( ) ( ))∣ ∣( ( ) ( ))∣ (∣( ( ) ( )) ( )∣ ∣( )∣) ( ) ( ) ( ) ( ) ‖ ‖ ( ) ( ) ( ) ( ) ( ) (∣( ( ) ( )) ( )∣ ∣( )∣) (‖ ‖(∣( )∣ ∣( )∣) ) ‖ ‖(∣( )∣ ∣( )∣) ∫ ∫ ∫ ∫ ⎜ ⎜ ≤ ⎛ ⎝ − + − + − + − ⎞ ⎠ ⎟+ ≤ − + ⎛ ⎝ − + − + − + − ⎞ ⎠ ⎟ + − + ≤ + + + + + − − − − y y y y g t x t y t a f a a g a a b f b b g b b ν t s h s x s y s s ν s t h s x s y s s f t x t y t g t x t y t g t g t a f a a g a a b f b b g b b p ψ r ξ r ν t s s s t s f t x t y t f t f t ϕ x t y t g ϕ x t y t f , 1 2 0, , 0, , 1 2 1, , 1, , 1 Γ , , d 1 Γ , , d , , , 0, 0 , 0, 0 1 2 0, , 0, , 1 2 1, , 1, , Γ d d , , 0, 0 , 0, 0 Λ . 4 Example (45) = = = b b 0 1 1 1 2 ( ) ( ) = = x x 0 0, 1 1, ( ) ( ) = = y y 0 0, 1 1. (45) ( ) ( ) = = y y 0 0, 1 1. (45) We note that = = = = = = γ ν a a b b , 0, 0, 1, 1, 1 2 1 2 1 2 We note that = = = = = = γ ν a a b b , 0, 0, 1, 1, 1 2 1 2 1 2 ( ) (∣∣ ∣∣ ) ( ) (∣∣ ∣∣ ) = + + = + + f t x y t x y f t x y t x y , , 100 10 , , , 50 10 , 1 2 2 2 ( ) ∣∣ ∣∣ ∣∣ ∣∣ ( ) ∣∣ ∣∣ ∣∣ ∣∣ = + + + + = + + + + g t x y x x y y g t x y x x y y , , 1 1 10, , , 1 1 11, 1 2 ( ) ( ) ∣∣ ∣∣ ( ) ( ) ∣∣ ∣∣ = + = ⎛ ⎝ ⎞ ⎠ + h t x y t x t y y h t x y t t x t y y , , 10,000 sin 1 , and , , 10,000 cos 4 sin 1 . 1 2 We can show that We can show that ( ) ( ) (∣ ∣ ∣ ∣) ( ) ( ) (∣ ∣ ∣ ∣) ( ) ( ) ∣ ∣ ∣ ∣ ( ) ( ) ∣ ∣ ∣ ∣ − ≤ − + − − ≤ − + − − ≤ − + − − ≤ − + − f t x y f t x y t x x y y f t x y f t x y t x x y y g t x y g t x y x x y y g t x y g t x y x x y y , , , ¯, ¯ 100 ¯ ¯ , , , , ¯, ¯ 50 ¯ ¯ , , , , ¯, ¯ ¯ ¯ , , , , ¯, ¯ ¯ ¯ . □ Therefore, all the conditions of Lemma 2.2 are satisﬁed, so that ( )( ) ( ) ( ) ( ) = + T x y t A x y B x y C x y , , , , has a coupled ﬁxed point in S; consequently, Problems (1)–(4) have a solution deﬁned on [0,1]. □ ‖( )‖ ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ = + ≤ + + + − − + + + + − − = + − − + + − − + + − − + + − − x y x y ϕ ϕ y g f ϕ ϕ ϕ ϕ x g f ϕ ϕ ϕ ϕ y ϕ ϕ g f ϕ ϕ ϕ ϕ x ϕ ϕ g f ϕ ϕ , Λ Λ 1 Λ Λ Λ 1 Λ Λ 1 Λ Λ 1 Λ Λ 1 Λ Λ 1 Λ . 1 0 1 1 1 0 1 0 2 2 1 0 1 0 1 0 1 1 1 0 1 0 1 0 2 2 1 0 (41) efore, we have ‖( )‖ ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ = + ≤ + + + − − + + + + − − x y x y ϕ ϕ y g f ϕ ϕ ϕ ϕ x g f ϕ ϕ , Λ Λ 1 Λ Λ Λ 1 Λ 1 0 1 1 1 0 1 0 2 2 1 0 (41) (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ = + − − + + − − + + − − + + − − ϕ ϕ y ϕ ϕ g f ϕ ϕ ϕ ϕ x ϕ ϕ g f ϕ ϕ Λ 1 Λ Λ 1 Λ Λ 1 Λ Λ 1 Λ . 1 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 2 2 1 0 (41) (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ = + − − + + − − + + − − + + − − ϕ ϕ y ϕ ϕ g f ϕ ϕ ϕ ϕ x ϕ ϕ g f ϕ ϕ Λ 1 Λ Λ 1 Λ Λ 1 Λ Λ 1 Λ . 1 0 1 0 1 1 1 0 1 0 1 0 2 2 1 0 Therefore, we have Therefore, we have ‖( )‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ⎜ ⎟ ≤ − ⎛ ⎝ + − − + + − − ⎞ ⎠ < x y ρ g f ϕ ϕ g f ϕ ϕ r , 1 1 Λ 1 Λ Λ 1 Λ . 1 1 1 0 2 2 1 0 Fourth, we show that + < δM ρ 1. , ρ Since ‖ ( )‖ ( ∣( )( )∣) ( ) [ ] = = < ∈ ∈ M B S B x y t sup sup , Λ, x y S t , 0,1 using ( ) H , 4 we ﬁnd (‖ ‖ ‖ ‖) ‖ ‖ ‖ ‖ (‖ ‖ ‖ ‖) ‖ ‖ ‖ ‖ + + + ≤ + + + < ϕ ϕ M ϕ ϕ ϕ ϕ ϕ ϕ Λ 1. 1 1 0 0 1 1 0 0 Therefore, all the conditions of Lemma 2.2 are satisﬁed, so that ( )( ) ( ) ( ) ( ) = + T x y t A x y B x y C x y , , , , has a coupled ﬁxed point in S; consequently, Problems (1)–(4) have a solution deﬁned on [0,1]. □ Therefore, all the conditions of Lemma 2.2 are satisﬁed, so that ( )( ) ( ) ( ) ( ) = + T x y t A x y B x y C x y , , , , has a coupled ﬁxed point in S; consequently, Problems (1)–(4) have a solution deﬁned on [0,1]. 2 2 2 1 2 0 2 (‖ ‖(∣( )∣ ∣( )∣) ) ‖ ‖(∣( )∣ ∣( )∣) ≤ + + + + + ϕ x t y t g ϕ x t y t f Λ . 2 2 2 1 2 0 2 Thus, Thus, ‖ ‖ (‖ ‖(‖ ‖ ‖ ‖) ) ‖ ‖(‖ ‖ ‖ ‖∣) ≤ + + + + + y ϕ x y g ϕ x y f Λ , 1 2 0 2 ‖ ‖ (‖ ‖(‖ ‖ ‖ ‖) ) ‖ ‖(‖ ‖ ‖ ‖∣) ≤ + + + + + y ϕ x y g ϕ x y f Λ , 1 2 0 2 which equals to which equals to ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ≤ + + + − − y ϕ ϕ x g f ϕ ϕ Λ Λ 1 Λ , 1 0 2 2 1 0 ‖ ‖ (‖ ‖ ‖ ‖)‖ ‖ ‖ ‖ ‖ ‖ ≤ + + + − − y ϕ ϕ x g f ϕ ϕ Λ Λ 1 Λ , 1 0 2 2 1 0 so, Solutions of a system of hybrid fractional boundary value problems  13 Solutions of a system of hybrid fractional boundary value problems  13 4 Example We consider the hybrid boundary value systems with the Riesz–Caputo derivative: We consider the hybrid boundary value systems with the Riesz–Caputo derivative: ( ) (∣( )∣ ∣( )∣ ) ( ) ∣( )∣ ∣( )∣ ∣ ( ) ∣ ∣ ( ) ∣ ∣( ) ∣ ∣( ) ∣ ⎛ ⎝ ⎜⎜ − + + + + ⎞ ⎠ ⎟⎟= + + + D x t x t y t t x t y t y t 10 10 10,000 sin 1 , RC t x t x t y t y t 1 1 2 0 100 1 1 2 (42) ( ) (∣( )∣ ∣( )∣ ) ( ) ∣( )∣ ∣( )∣ ∣ ( ) ∣ ∣ ( ) ∣ ∣( ) ∣ ∣( ) ∣ ⎛ ⎝ ⎜⎜ − + + + + ⎞ ⎠ ⎟⎟= ⎛ ⎝ ⎞ ⎠ + + + D y t x t y t t t x t y t y t 10 11 10,000 cos 4 sin 1 , RC t x t x t y t y t 1 1 2 0 50 1 1 2 (43) (42) ( ) (∣( )∣ ∣( )∣ ) ( ) ∣( )∣ ∣( )∣ ∣ ( ) ∣ ∣ ( ) ∣ ∣( ) ∣ ∣( ) ∣ ⎛ ⎝ ⎜⎜ − + + + + ⎞ ⎠ ⎟⎟= ⎛ ⎝ ⎞ ⎠ + + + D y t x t y t t t x t y t y t 10 11 10,000 cos 4 sin 1 , RC t x t x t y t y t 1 1 2 0 50 1 1 2 (43) (43) ( ) ( ) = = x x 0 0, 1 1, (44) ( ) ( ) = = y y 0 0, 1 1. (45) ( ) ( ) = = x x 0 0, 1 1, (44) ( ) ( ) = = y y 0 0, 1 1. (45) We note that = = = = = = γ ν a a b b 0 0 1 1 1 ( ) ( ) = = x x 0 0, 1 1, (44) ( ) ( ) = = y y 0 0, 1 1. 1 2 1 2 1 2 We choose = r 10; by calculation = = ρ Λ 0.1168, 0.158; thus, ( ) ( ) ( ) ( ) ( ) ( ) = = = = = = f f f f g g 0, 0, 0 0, 0, 0, 0 0, 1, 1, 1 12 100 , 1, 1, 1 12 50 , 1, 1, 1 11, and 1, 1, 1 12. 1 2 1 2 1 2 We choose = r 10; by calculation = = ρ Λ 0.1168, 0.158; thus, We choose = r 10; by calculation = = ρ Λ 0.1168, 0.158; thus, We choose = r 10; by calculation = = ρ Λ 0.1168, 0.158; thus, ‖ ‖ ‖ ‖ ‖ ‖ ‖ ‖ ⎜ ⎟ − ⎛ ⎝ + − − + + − − ⎞ ⎠ = < = ρ g f ϕ ϕ g f ϕ ϕ r 1 1 Λ 1 Λ Λ 1 Λ 3.768 10 . 1 1 1 0 2 2 1 0 So far, we have proved that all conditions in Theorem 3.2 are satisﬁed, which implies that Problems (42)–(45) have a solution. So far, we have proved that all conditions in Theorem 3.2 are satisﬁed, which implies that Problems (42)–(45) have a solution. 4 Example 1 1 2 2 2 2 1 1 2 2 14 4  Dehong Ji et al. Dehong Ji et al. Hence, we obtain ( ) ( ) ( ) ( ) ( ) ( ) ( ) = = = = + = + = = φ t φ t φ t φ t γ ν , , 1, 1, Γ 1 Γ 1 Γ 0.886, t t 0 100 0 50 1 1 3 2 2 2 then ‖ ‖ = φ0 ‖ ‖ ‖ ‖ = = φ φ , , 1 1 100 0 1 50 1 , and ‖ ‖ = φ 1, 1 Hence, we obtain ( ) ( ) ( ) ( ) ( ) ( ) ( ) = = = = + = + = = φ t φ t φ t φ t γ ν , , 1, 1, Γ 1 Γ 1 Γ 0.886, t t 0 100 0 50 1 1 3 2 2 2 then ‖ ‖ = φ0 ‖ ‖ ‖ ‖ = = φ φ , , 1 1 100 0 1 50 1 , and ‖ ‖ = φ 1, 1 ∣ ( )∣ ( ) (∣∣) (∣∣) ∣ ( )∣ ( ) (∣∣) (∣∣) ≤ ≤ h t x y p t ψ x ξ y h t x y p t ψ x ξ y , , , , , , 1 1 1 1 2 2 2 2 ∣ ( )∣ ( ) (∣∣) (∣∣) ∣ ( )∣ ( ) (∣∣) (∣∣) ≤ ≤ h t x y p t ψ x ξ y h t x y p t ψ x ξ y , , , , , , 1 1 1 1 2 2 2 2 where where ( ) (∣∣) ∣∣ (∣∣) ∣∣ ( ) (∣∣) ∣∣ (∣∣) ∣∣ = = = = ⎛ ⎝ ⎞ ⎠ = = p t t ψ x x ξ y y p t t t ψ x x ξ y y 10,000 , , , 10,000 cos 4 , , , 1 1 1 2 2 2 then ‖ ‖ ‖ ‖ = = p p , 1 1 10,000 2 1 10,000 , and ( ) (∣∣) ∣∣ (∣∣) ∣∣ ( ) (∣∣) ∣∣ (∣∣) ∣∣ = = = = ⎛ ⎝ ⎞ ⎠ = = p t t ψ x x ξ y y p t t t ψ x x ξ y y 10,000 , , , 10,000 cos 4 , , , 1 1 1 2 2 2 then ‖ ‖ ‖ ‖ = = p p , 1 1 10,000 2 1 10,000 , and ∣( )∣ ∣( )∣ [ ] [ ] [ ] [ ] = = = = = = ∈ ∈ ∈ ∈ f f t t f f t t sup , 0, 0 sup 10 1 10 , sup , 0, 0 sup 5 1 5 , t t t t 1 0,1 1 0,1 2 2 0,1 2 0,1 2 ∣( )∣ ∣( )∣ [ ] [ ] [ ] [ ] = = = = = = ∈ ∈ ∈ ∈ g g t g g t sup , 0, 0 sup 10 10, sup , 0, 0 sup 11 11, t t t t 1 0,1 1 0,1 2 0,1 2 0,1 ( ) ( ) ( ) ( ) ( ) = = = = = f f f f g g 0, 0, 0 0, 0, 0, 0 0, 1, 1, 1 12 100 , 1, 1, 1 12 50 , 1, 1, 1 11, and 1 2 1 2 1 ( ) ( ) ( ) ( ) ( ) ( ) = = = = = = f f f f g g 0, 0, 0 0, 0, 0, 0 0, 1, 1, 1 12 100 , 1, 1, 1 12 50 , 1, 1, 1 11, and 1, 1, 1 12. 5 Conclusion We have presented the existence results of a coupled system of hybrid boundary value problems with the Riesz-Caputo derivative with the aid of the Dhage ﬁxed point theorem. The novelty of this kind of fractional derivative is Riesz-Caputo fractional derivative that can reﬂect both the past and the future memory eﬀects. Acknowledgements: We express our sincere thanks to the anonymous reviewers for their valuable comments and suggestions. This work was supported by the Natural Science Foundation of Tianjin (No. 19JCYBJC30700), Ministry of Education Industry-University Cooperative Education Project in 2022 (Nos. 220503009174427 and 220505265245612). Tianjin University of Technology Teaching Fund (No. KG21-04). Funding information: This study was funded by National Science Foundation of Tianjin (No. 19JCYBJC30700), the ﬁrst batch of the cooperation and education projects of the Ministry of Education in 2022 (Nos 220503009174427 and 220505265245612), and Tianjin University of Technology Teaching Fund (No. KG21-04). Author contributions: All authors accepted responsibility for the entire content of this manuscript and approved its submission. Conﬂict of interest: The authors state that there is no conﬂict of interest. Ethical approval: The conducted research is not related to either human or animal use. Ethical approval: The conducted research is not related to either human or animal use. Data availability statement: Data sharing is not applicable to this article as no datasets were generated or analyzed during this study. Data availability statement: Data sharing is not applicable to this article as no datasets were generated or analyzed during this study. Solutions of a system of hybrid fractional boundary value problems  15 15 References [1] R. Hilfer, (Ed.), Application of Fractional Calculus in Physics, World Scientiﬁc, Singapore, 2000. [1] R. Hilfer, (Ed.), Application of Fractional Calculus in Physics, World Scientiﬁc, Singapore, 2000. [2] A. A. Kilbas, H. M. Srivastava, and J. J. 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https://openalex.org/W2553345748	https://academicjournals.org/journal/ERR/article-full-text-pdf/39DDDE461677.pdf	English	null	Evaluation of fourth-grade primary school students attitudes and self-efficacy towards physical education course using socio-cultural approach	Educational research and reviews	2,016	cc-by	7,874	Vol. 11(22), pp. 2034-2047, 23 November, 2016 DOI: 10.5897/ERR2016.2962 Article Number: 39DDDE461677 ISSN 1990-3839 Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/ERR Educational Research and Reviews Vol. 11(22), pp. 2034-2047, 23 November, 2016 DOI: 10.5897/ERR2016.2962 Article Number: 39DDDE461677 ISSN 1990-3839 Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/ERR Educational Research and Reviews Full Length Research Paper Hayri Aydoğan School of Physical Education and Sports, Recep Tayyip Erdoğan Unive Received 6 August, 2016; Accepted 25 October, 2016 School of Physical Education and Sports, Recep Tayyip Erdoğan University, Rize, Turkey. Received 6 August, 2016; Accepted 25 October, 2016 Changes in the national education system in Turkey have negative effects on the development of sports. Because, as classroom teachers teach Physical Education courses themselves, instead Physical Education teachers teach the course. This was because of a recent legislation released by the Ministry of National Education related to 4+4+4 compulsory schooling and the last resort for compulsory schooling model was made as 4+4+4 on 2012 to 2013 education season. In this context, the purpose of this research is to evaluate fourth-grade primary school students’ attitudes and self-efficacy towards physical education course based on socio-cultural approach. Physical Education Predisposition Scale (BEYÖ in Turkish) was used as the data collection tool and “Explanatory Factor Analysis (EFA)”, “Cronbach Alpha Coefficient” and “Item-Total Correlation Analysis” were conducted on fourth-grade students in order to maintain validity and reliability of the data collected. In the normality hypothesis analysis of the collected data, it is thought that “Kolmogorov-Smirnov Test” along with skewness and kurtosis coefficients will contribute to existing literature. Key words: Attitude, self-efficacy, physical education, primary school. Full Length Research Paper Evaluation of fourth-grade primary school students’ attitudes and self-efficacy towards physical education course using socio-cultural approach Full Length Research Paper INTRODUCTION attitude towards an object or an idea, he drifts away from it and even takes negative actions towards it. However, attitudes might change and new attitudes might be formed over time. For that reason, researches related to attitudes might provide useful findings for physical education and sports instructors (Şişko and Demirhan, 2002). ) Another important factor which affects attitudes is self- efficacy. “Self-efficacy” is one of the major concepts in Bandura‟s social learning theory and Bandura (1997) defines self-efficacy as people's beliefs about their capabilities to produce designated levels of performance that exercise influence over events that affect their lives. Self-efficacy beliefs determine how people feel, think, motivate themselves and behave. Such beliefs produce these diverse effects through four major processes. They include cognitive, motivational, affective and selection processes. A strong sense of efficacy enhances human accomplishment and personal well-being in many ways. People with high assurance in their capabilities approach difficult tasks as challenges to be mastered rather than as threats to be avoided. Such an efficacious outlook fosters intrinsic interest and deep engrossment in activities. They set themselves challenging goals and maintain strong commitment to them. They heighten and sustain their efforts in the face of failure. They quickly recover their sense of efficacy after failures or setbacks. They attribute failure to insufficient effort or deficient knowledge and skills which are acquirable. They approach threatening situations with assurance that they can exercise control over them. Such an efficacious outlook produces personal accomplishments, reduces stress and lowers vulnerability to depression (Bandura, 1994). p y The basic resource which helps countries to develop socially, culturally and artistically is healthy manpower. In order for a country to raise a healthy generation, a modern education approach should be implemented and physical education classes should be integrated to the curriculums, which affect millions of primary and junior high school students are affected. It is of great importance to help contribute to personal development of millions of students through activities in physical education classes. Intra-curriculum and extra-curriculum physical education activities are among the most important activities that help primary and junior high school students, who have a high level of activity to use their energy in a positive way, to be stress free and socialize, adapt to rules, keep away from harmful habits, enhance their knowledge, skills and abilities (Akgün and İnan, 2010). Students generally have a receptive approach towards physical education classes. Population and sample The population of this study includes fourth-grade primary school students. The sample of the study consists of 424 fourth-grade students from the following 12 primary schools: Kurtuluş Primary School located in Rize City Center; Karasu, Ambarlık, Şehit Nedim Çalık, and Dörtyol primary schools located in the Central District; Kıbledağı Şehit Metin Çetin, Başköy, Ulucami, and Adacami primary schools from villages of Güneysu District; İMKB Primary School located in Güneysu District Center; 28 Şubat primary schools located in Off District in Trabzon Province; Zeki Bilge Primary School and Uzungöl primary schools located in Çaykara District in Trabzon. INTRODUCTION Franzoi (2003) defines the concept of attitude as “an individual‟s positive or negative evaluation of an object”. When attitudes are categorized as positive or negative; having negative beliefs about ideas or objects, rejecting or disliking them, taking negative actions towards these might be considered as negative attitudes and having positive beliefs about ideas or objects, accepting and liking them might be considered as positive attitudes (Demirhan and Altay, 2001). People have their attitudes as a result of their previous experiences, thus, attitudes are not innate behaviors (Kağıtçıbaşı, 2005). According Franzoi (2003) defines the concept of attitude as “an individual‟s positive or negative evaluation of an object”. When attitudes are categorized as positive or negative; having negative beliefs about ideas or objects, rejecting or disliking them, taking negative actions towards these might be considered as negative attitudes and having positive beliefs about ideas or objects, accepting and liking them might be considered as positive attitudes (Demirhan and Altay, 2001). People have their attitudes as a result of their previous experiences, thus, attitudes are not innate behaviors (Kağıtçıbaşı, 2005). According to Sakallı (2006), there are various factors that affect our attitudes. Family, environment and direct personal experiences are major factors among these. Kağıtçıbaşı (2005) indicated that the age of a person is also an important factor in terms of attitudes. Many attitudes are formed especially during childhood (between 6 to 12 ages) by imitating parents‟ behaviors. In the first stage of adulthood (between 21 to 30 ages), these attitudes are reinforced. If an individual forms a positive attitude towards an object or an idea, he approaches to it and supports it; however if an individual forms a negative E-mail: hayriaydogan@hotmail.com. Tel:+00905327200913. E-mail: hayriaydogan@hotmail.com. Tel:+00905327200913. Aydoğan 2035 status of children (Haskell, 1994) as well as cognitive performance (Sibley and Etnier, 2003) and attention and concentration (Hillman et al., 2005; Shephard, 1996; Taras, 2005). Students feel satisfaction when they participate in physical education activities during school. These physical education activities can greatly enhance students‟ learning experience and positively influence their physical, mental, social and interpersonal wellbeing (Huang, 2007). Physical education course aims to improve individuals‟ social competence and positive sense of self as well as basic motor skills (Göktaş and Arıkıran, 2007). In this way self-efficacy that students develop might be by means of their participation in physical education courses. INTRODUCTION The findings of this study support this hypothesis. Taşmektepli et al (2006) also had similar findings. Their interpretation of those findings was that students generally show a higher level of interest in physical education classes and they have a receptive attitude. y p ( , ) According to Seçkin and Başbay (2013), self-efficacy is an individual‟s self-evaluation of his random behaviors. Self-efficacy also affects emotional reactions and ways of thinking of individuals. People who have a high level of self-efficacy might be relatively comfortable and productive in difficult activities and tasks. Physical education is the whole of theoretical and consistent studies conducted in order to contribute to physical and body development of individuals, to prepare them for the conditions of daily life and work life and to enhance their national awareness and citizenship feelings (Yamaner, 2001). Physical education not only develops physical features, but also promotes a sense of self-respect, mutual respect, cooperation and teamwork and supports and enhances leadership abilities (Buck et al., 2004). Young children are leading increasingly sedentary lives, with physical activity frequently displaced by television viewing, Internet surfing, and video gaming (Myers et al., 1996). Much concern has surfaced for this lifestyle change as childhood obesity has risen greatly in the last 10 years (World Health Organization, 2000). Within the school context, incorporating physical activity or fitness training is a likely means to improve the physical health Data collection tools “Personal Information Form” (Annex -2) and “Physical Education 2036 Educ. Res. Rev. Predisposition Scale” (Annex-3) were used as data collection tools in this research. with and sufficient for the data collected from the scale which consists of 11 items and is designated for fourth- grade students. The obtained results are given in Table 1. According to the findings listed in the Table 1, the obtained results are perfect since KMO coefficient is 0.868. For that reason, the sample size of the research is sufficient. Another test which determines whether explanatory factor analysis is compatible with a set of data is the sphericity test developed by Bartlett to determine the homoscedasticity of the sample. Factor analysis is conducted to detect high level of correlation between reverse floating variables of regression analysis. In this context, Bartlett test helps us to determine whether there is a significant correlation between variables in the main sample (Nakip, 2006). According to the results listed in the table, it is determined that the data collected are compatible with factor analysis (B=1413. 97; p = 0.000 < 0.05). Physical education predisposition scale (PEPS) Physical Education Predisposition Scale (PEPS), which was developed by Hilland et al. (2009) and adapted to Turkish language by Öncü, Gürbüz, Küçükkılıç and Keskin (2015), was used to measure students‟ self-efficacy and attitudes towards physical education. The scale, which consists of 11 items, includes 2 factors. Factors of the scale are “Attitude” (6 items) and „Self-efficacy‟ (5 items). Negative items included in the scale are scored reversely. The scale is a 5-point Likert-type scale with the following scoring system: “I strongly disagree (1)”, “I disagree (2)”, “I neither agree nor disagree (3)”, “I agree (4)” and “I strongly agree (5)” The lowest score that can be achieved in the scale is 11 and the highest one is 55. The lowest and the highest scores of “Attitude” sub-dimension of the scale are 6 and 30, whereas the lowest and the highest scores of “Self-efficacy” sub-dimension of the scale are 5 and 25, respectively. Results of factor analysis After determining that the data collected are compatible with factor analysis, explanatory factor analysis was conducted using Principal Components Analysis and Varimax Rotation methods to determine the factor structure of the scale. Factors collected as a result of conducted explanatory factor analysis and factor weight values of items listed under these factors are listed in Table 2. It is understood that latent values of 2 factors listed in Table 2 are greater than 1. The most significant and prominent method for determining whether a factor should be included in the scale is based on the latent value of the factor (those greater than 1 are included as a factor). (Büyüköztürk, 2002). Variances of these two factors are 37.75 and 14.72% and total variance listed is 52.47%. According to these findings, it is understood that these two factors reflect 0.52% of total variance of the main structure. This value should be deemed acceptable according to Kline since it is over 40% (Ceyhan and Namlu, 2000). According to these results, it might be concluded that the scale proved valid results on fourth grade students and it has a solid factor structure. Statistical methods Explanatory Factor Analysis (EFA), “Cronbach Alpha Coefficient”, and “Item-Total Correlation Analysis” were conducted in order to confirm the validity and reliability of “ PEPS” (or BEYÖ in Turkish), which was used on fourth grade students as a data collection tool in this research. Kolmogorov-Smirnov test and coefficient of skewness and kurtosis were used in normality analysis of the collected data. Number of groups was taken into consideration for comparison of attitude and self-efficacy scores of different groups. In accordance with this, “t test for independent groups”, which is a parametric statistical method, was used for comparison of the average scores of two independent groups and “one way analysis of variance test (ANOVA)”, which is another parametric statistical method, was used for comparison of the average scores of three or more groups. If ANOVA tests proved differences, Tukey multiple comparison test (Post Hoc) was used in order to determine which group is the source of these differences. IBM SPSS package program was used for application of aforementioned statistical methods within the scope of this research. FINDINGS If internal consistency determinant Cronbach α coefficient is lower than 0.40, the scale is “unreliable”; if it is between 0.40 to 0.59, the scale has a “low level of reliability” and if it is between 0.60 to 0.79, the scale has a “high level of reliability” (Tavşancıl, 2002). In this study, internal consistency of the PEPS (BEYÖ in Turkish) scale was analyzed measuring Cronbach Alpha coefficients of the whole scale and its sub-dimensions separately. Cronbach Alpha coefficients of each factor and the whole Personal information form “Personal Information Form” developed by the researchers aims to collect data from students which constitute the sample of this research. This section includes information related to students‟ demographic and independent variables: gender, past injuries in PE classes, education level of the mother, education level of the father and location of the school. Factor analysis Kaiser-Meyer-Olkin coefficient and Bartlett Test of Sphericity were used in order to determine whether conducting an explanatory factor analysis is compatible 2037 Aydoğan Table 1. Collected data‟s compatibility with factor analysis. Table 1. Collected data‟s compatibility with factor analysis. Kaiser-Meyer-Olkin (KMO) Variance Sufficiency of the Sample 0.868 Bartlett Test Ki-Square Value 1413.97 S.d 55 P 0.000 Table 2. Factor analysis results. Scale sub-dimension Items Factor weight value Latent Value Variance (%) Cumulative Variance (%) Attitude Item 1 0.643 4.153 37.75 37.75 Item 3 0.440 Item 6 0.723 Item 8 0.614 Item 10 0.567 Item 11 0.719 Self-Efficacy Item 2 0.803 1.619 14.72 52.47 Item 4 0.758 Item 5 0.783 Item 7 0.696 Item 9 0.770 Table 2. Factor analysis results. Table 3. Internal consistency coefficients of the whole scale and all sub-dimensions. Factors Number of items Cronbach alpha coefficient (α) Factor 1- Attitude 6 0.700 Factor 2- Self-efficacy 5 0.841 Total 11 0.814 Table 3. Internal consistency coefficients of the whole scale and all sub-dimensions. from the test (Tezbaşaran, 1996). In other words, it displays whether each item of a scale samples similar behaviors or not. In this context, item-total score correlation is expected to be greater than 0.25. Significant correlation coefficients between scale item or sub-dimension scores and total scale scores are deemed as a sign of internal consistency. High correlation coefficients show the high compatibility of the respective item with measured theoretical structure. In case correlation coefficient is lesser than 0.25, it is suggested to remove these items from the scale (Karataş, 2014). scale are listed in Table 3 accordingly. According to the values listed on the table, Cronbach α reliability coefficient of PEPS (BEYÖ in Turkish) sub dimensions are 0.700 for Factor 1 and 0.841 for Factor 2, respectively. Based on this, it might be concluded that “self-efficacy” factor has an acceptable level of reliability and “attitude” factor has a good level of reliability. Cronbach α reliability coefficient of 11 items listed in the scale is 0.814. Based on this value, it might be suggested that the scale has a good level of reliability. According to the findings listed in Table 4, item-total correlation coefficients of all 11 items are greater than 0.25. Factor analysis Based on this, it might be suggested that all items have a high correlation with the whole of the scale and they move in the same direction with the fundamental structure measured by the scale. In this case, there is no Comparison of fourth grade student behaviors based on demographic variables T test was used in independent groups to determine whether attitude sub-dimension scores of fourth grade students have a significant difference based on gender. The results are presented in Table 7. Kolmogorov-Smirnov test was used to determine whether total attitude score variable obtained from the attitude sub-dimension of the scale has a normal distribution Table 6. According to Kolmogorov-Smirnov Test, total attitude scores of fourth-grades are not distributed evenly (p<0.05). In this case, normality of the data collected using the Likert scale is tested using another criterion commonly accepted in literature whose skewness and kurtosis coefficients are between -1.5 and +1.5 (Fidell et al., 2013). Based on this and taking into account skewness and kurtosis coefficients of attitude variable, it might be considered to be distributed evenly (normal). In this case, parametric methods are used to determine Based on the results listed in the Table 7 and Figure 1, fourth-grade students‟ attitudes towards physical education have a significant statistical difference (t=2.875; p<0.05). Analyzing the average scores of genders, it is understood that female students have a more positive attitude towards physical education compared to males. Item-total score correlation analysis Item-total score correlation analysis is an objective control mechanism which explains the relation between scores obtained from items and total scores obtained 2038 Educ. Res. Rev. Table 4. Item-total correlation coefficients. Table 4. Item-total correlation coefficients. Parameter Item-total correlation Cronbach alpha coef. if there are any Items Deleted Item 1 0.260 0.813 Item 2 0.498 0.783 Item 3 0.441 0.789 Item 4 0.508 0.783 Item 5 0.547 0.778 Item 6 0.328 0.800 Item 7 0.526 0.780 Item 8 0.603 0.773 Item 9 0.586 0.774 Item 10 0.507 0.782 Item 11 0.380 0.795 Table 5. Statistics related to attitude dimension. Parameter N Mean ss. Skewness Kurtosis Minimum Maximum Attitude 424 23.52 4.50 -0.519 -0.051 6 30 Table 6. K-S normality test results. Parameter Kolmogorov-Smirnov Statistics sd Sig.(p) Attitude 0.075 424 0.000 Parameter Item-total correlation Cronbach alpha coef. if there are any Items Deleted Item 1 0.260 0.813 Item 2 0.498 0.783 Item 3 0.441 0.789 Item 4 0.508 0.783 Item 5 0.547 0.778 Item 6 0.328 0.800 Item 7 0.526 0.780 Item 8 0.603 0.773 Item 9 0.586 0.774 Item 10 0.507 0.782 Item 11 0.380 0.795 Parameter Item-total correlation Cronbach alpha coef. if there are any Items Deleted Item-total correlation Cronbach alpha coef. if there are any Items Deleted Table 5. Statistics related to attitude dimension. Table 5. Statistics related to attitude dimension. Parameter N Mean ss. Skewness Kurtosis Minimum Maximum Attitude 424 23.52 4.50 -0.519 -0.051 6 30 Table 6. K-S normality test results. Parameter Kolmogorov-Smirnov Statistics sd Sig.(p) Attitude 0.075 424 0.000 Table 5. Statistics related to attitude dimension. Parameter N Mean ss. Skewness Kurtosis Minimum Maximum Attitude 424 23.52 4.50 -0.519 -0.051 6 30 Table 6. K-S normality test results. Parameter Kolmogorov-Smirnov Statistics sd Sig.(p) Attitude 0.075 424 0.000 whether attitude scores have any significant differences based on certain demographic variables. need to remove any items from the scale and it might be suggested that PEPS (BEYÖ in Turkish) maintains the number of items on fourth grade student population Table 5. need to remove any items from the scale and it might be suggested that PEPS (BEYÖ in Turkish) maintains the number of items on fourth grade student population Table 5. Table 7. (t) Test results of attitude score averages based on gender. Table 7. (t) Test results of attitude score averages based on gender. Table 7. (t) Test results of attitude score averages based on gender. Gender N Mean ss T p Female 228 24.11 4.04 2.875 0.004 Male 196 22.84 4.90 Figure 1. Average attitude scores based on gender. 24.4 24 23.5 23 22.5 22 Figure 1. Average attitude scores based on gender. Table 8. (t) Test results of attitude score averages based on past injuries in PE classes. Past injuries N Mean ss t p Yes 49 22.69 3.93 -1.365 0.173 No 375 23.63 4.56 Figure 2. Attitude score averages based on past injuries in PE classes. 24 23.5 23 22.5 22 Table 8. (t) Test results of attitude score averages based on past injuries in PE classes. Past injuries N Mean ss t p Yes 49 22.69 3.93 -1.365 0.173 No 375 23.63 4.56 Figure 2. Attitude score averages based on past injuries in PE classes. 24 23.5 23 22.5 22 Table 8. (t) Test results of attitude score averages based on past injuries in PE classes. Figure 2. Attitude score averages based on past injuries in PE classes. whether fourth grade students‟ past injuries in physical education classes cause a significant difference in their attitudes towards physical education. The results are given in Table 8 and Figure 2. According to t test results on this chart, fourth grade students‟ attitudes towards physical education classes do not differ significantly based on whether they have past injuries in PE classes (t=-1.365; p>0.05). In this case, fourth grade students‟ negative past experiences in PE classes do not affect their attitudes towards this class. determine whether education level of mothers of fourth grade students significantly affects their attitude scores towards physical education classes. The test results are as given in Table 9 and Figure 3. According to ANOVA test results on this chart, fourth grade students‟ attitudes towards physical education classes do not differ significantly based on education level of their mothers (F=2.105; p>0.05). Comparison of attitude scores based on past injuries in PE classes T test was used in independent groups to determine 2038 Aydoğan Comparison of attitude scores based on education level of mother One way variance analysis (ANOVA) was used to determine whether education level of fathers of fourth grade students significantly affects their attitude scores One way variance analysis (ANOVA) was used to 2040 Educ. Res. Rev. Table 9. Anova test related to attitude score averages based on education level of mothers. Education level of mother N Mean ss F p Primary 147 22.97 4.980 2.105 0.079 Junior high school 128 23.42 4.534 High school 93 23.65 3.917 Undergraduate 50 25.04 3.779 Post-graduate 6 24.50 3.728 Figure 3. Average attitude scores based on education level of mothers. 25.5 25 24.5 24 23.5 23 22.5 22 21.5 Table 9. Anova test related to attitude score averages based on education level of mothers. Education level of mother N Mean ss F p Primary 147 22.97 4.980 2.105 0.079 Junior high school 128 23.42 4.534 High school 93 23.65 3.917 Undergraduate 50 25.04 3.779 Post-graduate 6 24.50 3.728 Figure 3. Average attitude scores based on education level of mothers. 25.5 25 24.5 24 23.5 23 22.5 22 21.5 Table 9. Anova test related to attitude score averages based on education level of mothers. Table 9. Anova test related to attitude score averages based on education level of mothers. Education level of mother N Mean ss F p Primary 147 22.97 4.980 2.105 0.079 Junior high school 128 23.42 4.534 High school 93 23.65 3.917 Undergraduate 50 25.04 3.779 Post-graduate 6 24.50 3.728 Figure 3. Average attitude scores based on education level of mothers. 25.5 25 24.5 24 23.5 23 22.5 22 21.5 Figure 3. Average attitude scores based on education level of mothers. Table 10. Anova test related to attitude score averages based on education level of fathers Education level of father N Mean ss F p Primary 61 22.61 4.514 6.603 0.000 Junior high school 115 22.21 4.545 High school 134 23.97 3.983 Undergraduate 102 24.58 4.723 Post-graduate 12 26.67 3.473 Figure 4. Average attitude scores based on education level of fathers. 27 26.5 25 24 23 22 21 Table 10. Anova test related to attitude score averages based on education level of fathers. Education level of father N Mean ss F p Primary 61 22.61 4.514 6.603 0.000 Junior high school 115 22.21 4.545 High school 134 23.97 3.983 Undergraduate 102 24.58 4.723 Post-graduate 12 26.67 3.473 Figure 4. Average attitude scores based on education level of fathers. Comparison of attitude scores based on education level of mother 27 26.5 25 24 23 22 21 Table 10. Anova test related to attitude score averages based on education level of fathers. Figure 4. Average attitude scores based on education level of fathers. p<0.05). Tukey multiple comparison test (Post Hoc) was used in order to determine which group is the source of difference and the following results were obtained Table 11. According to the findings in this table, there are significant differences between primary-undergraduate, towards physical education classes. The test results are seen in Table 10 and Figure 4. According to ANOVA test results on this chart, fourth grade students‟ attitudes towards physical education classes differ significantly based on education level of their fathers (F=6.603; 2041 Aydoğan Table 11. Tukey multiple comparison test results based on education level of fathers. Table 11. Tukey multiple comparison test results based on education level of fathers. Table 11. Tukey multiple comparison test results based on education level of fathers. Parameter Mean difference Sig (p) Primary Junior high school 0.398 0.979 High school -1.364 0.262 Undergraduate -1.972* 0.045 Post-graduate -4.060* 0.029 Junior high school Primary -0.398 0.979 High school -1.761* 0.015 Undergraduate -2.370* 0.001 Post-graduate -4.458* 0.008 High School Primary 1.364 0.262 Junior high school 1.761* 0.015 Undergraduate -0.608 0.830 Post-graduate -2.697 0.249 Undergraduate Primary 1.972* 0.045 Junior high school 2.370* 0.001 High school 0.608 0.830 Post-graduate -2.088 0.525 Post-graduate Primary 4.060* 0.029 Junior high school 4.458* 0.008 High school 2.697 0.249 Post-graduate 2.088 0.525 Table 12. Anova test related to attitude score averages based on locations of schools. Locations of School N Mean ss F p City center 27 23.63 3.835 5.669 0.001 City center village 123 23.07 4.555 District center 173 24.51 4.706 District village 101 22.35 3.905 Table 12. Anova test related to attitude score averages based on locations of schools. primary-postgraduate, junior high school-high school, junior high school-postgraduate groups based on education level of their fathers. In addition, when analyzing the average scores of the groups, students‟ attitude scores increase as their fathers‟ education level changes from primary to post-graduate. Therefore, it might be concluded that students‟ attitudes towards physical education classes increase positively in parallel with education level of their fathers. on this chart, fourth grade students‟ attitudes towards physical education classes differ significantly based on locations of their schools (F=5.669; p<0.05). Comparison of attitude scores based on education level of mother Tukey multiple comparison tests were used to determine which group is the source of difference and following results were obtained Table 13. According to the findings in Table 14, there are significant differences between city center village-district center and district village-district center location groups. In addition, average scores of groups indicate that the attitudes of fourth grade students attending schools in district centers are more positive towards physical education classes. primary-postgraduate, junior high school-high school, junior high school-postgraduate groups based on education level of their fathers. In addition, when analyzing the average scores of the groups, students‟ attitude scores increase as their fathers‟ education level changes from primary to post-graduate. Therefore, it might be concluded that students‟ attitudes towards physical education classes increase positively in parallel with education level of their fathers. Comparison of self-efficacy levels of fourth grade students based on demographic variables One way variance analysis (ANOVA) was used to determine whether school locations of fourth grade students significantly affect attitude scores towards physical education classes. Test results are given in Table 12 and Figure 5. According to ANOVA test results Kolmogorov-Smirnov test was used to determine whether total self-efficacy score variable obtained from the self- Educ. Res. Rev. 2042 Figure 5. Average attitude scores based on locations of schools. Figure 5. Average attitude scores based on locations of schools. Table 13. Tukey multiple comparison test results based on locations of schools. Table 13. Tukey multiple comparison test results based on locations of schools. Parameter Mean difference Sig. (p) City center City center village 0.565 0.932 District center -0.879 0.773 District village 1.283 0.540 City center village City center -0.565 0.932 District center -1.444* 0.030 District village 0.719 0.622 District center City center 0.879 0.773 City center village 1.444* 0.030 District village 2.162* 0.001 District village City center -1.283 0.540 City center village -0.719 0.622 District center -2.162* 0.001 Table 14. Statistics related to self-efficacy dimension. Parameter N Mean ss. Skewness Kurtosis Minimum Maximum Self-efficacy 424 19.95 4.16 -0.833 0.511 5 25 Table 15. K-S normality test results. Parameter Kolmogorov-Smirnov Statistic sd Sig.(p) Self-efficacy 0.113 424 0.000 Table 13. Tukey multiple comparison test results based on locations of schools. Parameter Mean difference Sig. (p) City center City center village 0.565 0.932 District center -0.879 0.773 District village 1.283 0.540 City center village City center -0.565 0.932 District center -1.444* 0.030 District village 0.719 0.622 District center City center 0.879 0.773 City center village 1.444* 0.030 District village 2.162* 0.001 District village City center -1.283 0.540 City center village -0.719 0.622 District center -2.162* 0.001 Table 14. Statistics related to self-efficacy dimension. Parameter N Mean ss. Skewness Kurtosis Minimum Maximum Self-efficacy 424 19.95 4.16 -0.833 0.511 5 25 Table 15. K-S normality test results. Parameter Kolmogorov-Smirnov Statistic sd Sig.(p) Self-efficacy 0.113 424 0.000 Table 14. Statistics related to self-efficacy dimension. efficacy sub-dimension of the scale has a normal distribution Table 15. According to Kolmogorov-Smirnov Test, total self-efficacy scores of fourth grades are not distributed evenly (p<0.05). In this case, normality of the data collected using the Likert scale is tested using another criterion commonly accepted in literature whose skewness and kurtosis coefficients are between -1.5 and +1.5 (Fidell et al., 2013). Comparison of self-efficacy levels of fourth grade students based on demographic variables Based on this and taking into account skewness and kurtosis coefficients of attitude variable, it might be considered to be distributed evenly (normal). In this case, just as in attitude sub-dimension, parametric methods are used to determine whether self- 2043 Aydoğan Table 16. (t) test results of self-efficacy score averages based on gender. Table 16. (t) test results of self-efficacy score averages based on gender. Gender N Mean ss t p Female 228 19.82 3.80 -0.708 0.479 Male 196 20.11 4.56 Figure 6. Average self-efficacy scores based on gender. 20.5 20 19.5 Table 16. (t) test results of self-efficacy score averages based on gender. Gender N Mean ss t p Female 228 19.82 3.80 -0.708 0.479 Male 196 20.11 4.56 Figure 6. Average self-efficacy scores based on gender. 20.5 20 19.5 Figure 6. Average self-efficacy scores based on gender. Table 17. (t) test results of self-efficacy score averages based on past injuries in PE classes. Past injuries N Mean ss t p Yes 49 19.14 4.64 -1.45 0.194 No 375 20.06 4.09 Figure 7. Self-efficacy scores based on past injuries. 20.5 20 19.5 19 18.5 Table 17. (t) test results of self-efficacy score averages based on past injuries in PE classes. Past injuries N Mean ss t p Yes 49 19.14 4.64 -1.45 0.194 No 375 20.06 4.09 Figure 7. Self-efficacy scores based on past injuries. 20.5 20 19.5 19 18.5 Table 17. (t) test results of self-efficacy score averages based on past injuries in PE classes. Figure 7. Self-efficacy scores based on past injuries. Comparison of self-efficacy scores based on past injuries in PE classes efficacy scores have any significant differences based on certain demographic variables. efficacy scores have any significant differences based on certain demographic variables. efficacy scores have any significant differences based on certain demographic variables. Comparison of self-efficacy scores based on past injuries in PE classes T test was used in independent groups to determine whether fourth grade students‟ past injuries in physical education classes cause a significant difference in their self-efficacy perceptions of physical education. The results are given in Table 17 and Figure 7. According to t test results on this chart, fourth grade students‟ self- efficacy perceptions of physical education classes do not differ significantly based on whether they have past injuries in PE classes (t=-1.45; p>0.05). In this case, fourth grade students‟ negative past experiences in PE classes do not affect their self-efficacy perceptions of Comparison of self-efficacy scores based on gender T test was used in independent groups to determine whether self-efficacy sub-dimension scores of fourth grade students have a significant difference based on gender. The results are given in Table 16 and Figure 6. Based on the results listed on this chart, fourth grade students‟ self-efficacy perceptions of physical education classes do not have a significant statistical difference (t= - 0.708; p>0.05). 2044 Educ. Res. Rev. Table 18. Anova test related to self- efficacy score averages based on education level of mothers. Education level of mother N Mean ss F p Primary 147 19.85 4.350 0.630 0.641 Junior high school 128 20.16 3.983 High school 93 19.49 4.127 Undergraduate 50 20.46 4.282 Post-graduate 6 20.83 3.125 Figure 8. Self-efficacy scores based on education level of mothers 21 20.5 20 19.5 19 18.5 Table 18. Anova test related to self- efficacy score averages based on education level of mothers. Education level of mother N Mean ss F p Primary 147 19.85 4.350 0.630 0.641 Junior high school 128 20.16 3.983 High school 93 19.49 4.127 Undergraduate 50 20.46 4.282 Post-graduate 6 20.83 3.125 Figure 8. Self-efficacy scores based on education level of mothers 21 20.5 20 19.5 19 18.5 Table 18. Anova test related to self- efficacy score averages based on education level of mothers. Table 18. Anova test related to self- efficacy score averages based on education level of mothers. Education level of mother N Mean ss F p Primary 147 19.85 4.350 0.630 0.641 Junior high school 128 20.16 3.983 High school 93 19.49 4.127 Undergraduate 50 20.46 4.282 Post-graduate 6 20.83 3.125 Figure 8. Self-efficacy scores based on education level of mothers 21 20.5 20 19.5 19 18.5 Figure 8. Self-efficacy scores based on education level of mothers Table 19. Anova test related to self-efficacy score averages based on education level of fathers. Table 19. Anova test related to self-efficacy score averages based on education level of fathers. Education Level of Father N Mean ss F p Primary 61 19.64 4.191 2.564 0.038 Junior high school 115 19.14 4.279 High school 134 20.51 3.805 Undergraduate 102 20.08 4.407 Post-graduate 12 22.00 3.464 determine whether education level of fathers of fourth grade students significantly affects self-efficacy scores towards physical education classes. Test results are given in Table 19 and Figure 9. Comparison of self-efficacy scores based on education level of mother One way variance analysis (ANOVA) was used to determine whether education level of mothers of fourth grade students significantly affects self-efficacy scores towards physical education classes. Test results are given in Table 18 and Figure 8. According to ANOVA test results on this chart, fourth grade students‟ self-efficacy perceptions of physical education classes do not differ significantly based on education level of their mothers (F=0,630; p>0.05). Comparison of self-efficacy scores based on locations of schools One way variance analysis (ANOVA) was used to determine whether school locations of fourth grade students significantly affect self-efficacy scores towards physical education classes. Test results are given in Table 20 and Figure 10. According to ANOVA test results Comparison of self-efficacy scores based on gender According to ANOVA test results on this chart, fourth grade students‟ self-efficacy perceptions of physical education classes differ significantly based on education level of their fathers (F=2.564; p<0.05). In addition, group averages indicate that self-efficacy perceptions of students who have fathers with a highest level of education are the highest. this class. this class. Comparison of Self-Efficacy Scores Based on Education Level of Fathers One way variance analysis (ANOVA) was used to 2045 Aydoğan Figure 9. Self-efficacy scores based on education level of fathers. Figure 9. Self-efficacy scores based on education level of fathers. Figure 9. Self-efficacy scores based on education level of fathers. Table 20. Anova test related to self-efficacy score averages based on locations of schools. Locations of school N Mean ss F p City center 27 19.78 4.734 3.709 0.012 City center village 123 19.79 4.289 District center 173 20.66 3.949 District village 101 18.97 4.046 Figure 10. Average self-efficacy scores based on locations of schools. 21 20.5 20 19.5 19 18.5 18 Table 20. Anova test related to self-efficacy score averages based on locations of schools. Locations of school N Mean ss F p City center 27 19.78 4.734 3.709 0.012 City center village 123 19.79 4.289 District center 173 20.66 3.949 District village 101 18.97 4.046 Figure 10. Average self-efficacy scores based on locations of schools. 21 20.5 20 19.5 19 18.5 18 Table 20. Anova test related to self-efficacy score averages based on locations of schools. Figure 10. Average self-efficacy scores based on locations of schools. details were included in charts. Summarized interpretation of the collected data shows that students‟ attitudes and self-efficacy perceptions do not differ greatly in terms of demographic variables. Analysis of average scores indicate that female students show greater interest in physical education classes when compared to male students. According to the findings of the study, fourth-grade students‟ attitudes and self- efficacy perceptions do not differ greatly based on their past injuries in PE classes. Hence, it might be concluded that past injuries do not change fourth-grade students‟ attitudes towards physical education classes. Another finding suggests that students‟ attitudes do not differ greatly based on education level of their mothers. However, education level of fathers creates a significant difference (F=6.603; p<0.05). According to the findings of on this chart, fourth grade students‟ self-efficacy per- ceptions of physical education classes differ significantly based on locations of their schools (F=3.709; p<0.05). Tukey multiple comparison test was used to determine which group is the source of the difference and the following results were obtained Table 21. Based on the findings listed in the table, locations of schools cause a significant difference only between villages and city centers. DISCUSSION AND CONCLUSION In this study, statistical calculations related to fourth- grade primary school students‟ attitudes and self-efficacy towards physical education classes were made and the 2046 Educ. Res. Rev. Table 21. Tukey multiple comparison test results based on locations of schools. Table 21. Tukey multiple comparison test results based on locations of schools. Parameter Mean difference Sig. (p) City center City center village -0.011 1.000 District center -0.887 0.727 District village 0.807 0.803 City center village City center 0.011 1.000 District center -0.876 0.274 District village 0.818 0.452 District center City center 0.887 0.727 City center village 0.876 0.274 District village 1.694* 0.006 District village City center -0.807 0.803 City center village -0.818 0.452 District center -1.694* 0.006 significant others (Bandura, 1986; e.g., peers, family members, educators, and health professionals), and (c) persuading children and adolescents that regular PA is an important and enjoyable part of everyday life. Research findings of a positive correlation between support for PA from parents and siblings and youth PA involvement (Aarnio et al., 1997; Zakarian et al., 1994) suggest that combining social support, selfefficacy, and positive experiential components in interventions to increase PA may be promising (Robbins et al., 2004). this study, there are significant differences between primary-undergraduate and junior high school -post- graduate education level groups. In addition, when group averages are taken into consideration, as fathers‟ education level increases from primary school to under- graduate, attitude score averages generally increase. g g g y According to this, it might be suggested that the better fathers‟ education level is the more positive students‟ attitudes towards the class are. On the other hand, it might also be suggested that locations of schools create a significant difference in attitudes towards PE classes (F=5.669; p<0. 05). According to the results of this research, there are significant differences between villages in city center-district centers and villages in district centers-district centers. In a study conducted by Yıldırım and İlhan (2010), average scores of groups indicated that the attitudes of fourth grade students attending schools in district centers are more positive towards physical education classes and this is also supported by our findings. Moreover, findings of Endler et al. (2001) suggested that gender does not create significant differences in terms of overall self-efficacy. Analyzing the self-efficacy dimension of the study, it is suggested that fourth grade students‟ self-efficacy perceptions of physical education classes do not have any significant differences. REFERENCES Nakip M (2006). Marketing Research: Techniques and Applications with SPSS. Ankara: Seçkin Press Ö Akgün İB, İnan M (2010). 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Sports Exercise 28:852-859. Conflict of interests Kağıtçıbaşı Ç (2005). New Person and People. (Tenth Ed.). İstanbul: Evrim Press. Karataş Z (2014). Advanced Statistical Applications Courses in Social Services: Regression, Correlation, and Factor Analysis. 19.06.2016. Retrieved from. The author has not declared any conflict of interest. DISCUSSION AND CONCLUSION Hence, the results of our study are parallel to the findings of Endler et al. (2001). y p g ( ) According to our study, fourth- grade students‟ past negative experiences do not affect their self-efficacy perceptions of PE classes. It is also understood that self- efficacy perceptions of fourth- grade students do not differ significantly based on education level of their mothers; however, it is based on education level of their fathers. Furthermore, group averages indicate that self- efficacy perceptions of students who have fathers with a highest level of education are the highest. It is understood that self-efficacy perceptions of physical education classes do not differ significantly based on locations of schools (F=3.709; p<0.05). In conclusion, primary school students are accompanied by other branch teachers such as English teachers and religious culture and moral knowledge teachers in addition to guidance counselors after the second grade. Physical Education and Sports classes have a vital importance on children‟s personal and moral development as suggested earlier. In this context, if Physical Education specialists instruct children‟s PE classes as a requirement of a more professional approach. This will help children have a healthier education life and it will also contribute to the development of sports in the country; also, increased weekly hours of Physical Education and Sports classes will provide students with the opportunity to participate in sports activities more frequently. In a study of Lorraine B. Robbins et al. 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https://openalex.org/W4320342313	https://repositorium.sdum.uminho.pt/bitstream/1822/63570/1/PhysRevD.100.032007.pdf	English	null	Search for the electroweak diboson production in association with a high-mass dijet system in semileptonic final states in <math xmlns="http://www.w3.org/1998/Math/MathML"> <mi>p</mi> <mi>p</mi> </math> collisions at <math xmlns="http://www.w3.org/1998/Math/MathML"> <msqrt> <mi>s</mi> </msqrt> <mo>=</mo> <mn>13</mn> </math> TeV with the ATLAS detector	OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information)	2,019	cc-by	38,790	PHYSICAL REVIEW D 100, 032007 (2019) PHYSICAL REVIEW D 100, 032007 (2019) (Received 21 May 2019; published 22 August 2019) This paper reports on a search for electroweak diboson (WW=WZ=ZZ) production in association with a high-mass dijet system, using data from proton-proton collisions at a center-of-mass energy of ﬃﬃﬃs p ¼ 13 TeV. The data, corresponding to an integrated luminosity of 35.5 fb−1, were recorded with the ATLAS detector in 2015 and 2016 at the Large Hadron Collider. The search is performed in final states in which one boson decays leptonically, and the other boson decays hadronically. The hadronically decaying W=Z boson is reconstructed as either two small-radius jets or one large-radius jet using jet substructure techniques. The electroweak production of WW=WZ=ZZ in association with two jets is measured with an observed (expected) significance of 2.7 (2.5) standard deviations, and the fiducial cross section is measured to be 45.1 8.6ðstat:Þþ15.9 −14.6ðsyst:Þ fb. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. Search for electroweak diboson production in association with a high-mass dijet system in semileptonic final states in pp collisions at ﬃﬃs p = 13 TeV with the ATLAS detector G. Aad et al.* (ATLAS Collaboration) I. INTRODUCTION VVjj (V ¼ W, Z, γ), with a large separation in rapidity of jets and a large dijet invariant mass. Multiple processes can produce the same final state of two bosons and two jets. The production of VVjj at tree level has an EW contri- bution involving only electroweak-interaction vertices, and a strong contribution (QCD induced) involving two strong- interaction vertices. The EW production is further divided into two components. The first component is EW VBS production with actual scattering of the two electroweak bosons. The scattering occurs via quartic gauge vertices, or triple gauge vertices involving the s- or t-channel exchange of a Higgs boson or a W=Z boson. The second component is EW non-VBS production that has electro- weak vertices only, but where the two bosons do not scatter. The EW non-VBS component cannot be separated from the EW VBS component in a gauge invariant way [13] and contributes significantly to the total cross section. It is therefore included in the signal generation. Representative Feynman diagrams at tree level are shown in Fig. 1. Both the ATLAS and CMS Collaborations have searched for experimental evidence of VBS. So far, electroweak VVjj production is only observed in the same-sign WWjj channel [14] and WZjj channel [15] in the fully leptonic final states using data collected at a center-of-mass energy of ﬃﬃﬃs p ¼ 13 TeV. Evidence of electroweak VVjj production is also obtained in the WWjj [16–18] and Zγjj [19] channels using pp collisions at ﬃﬃﬃs p ¼ 8 TeV. Limits on fiducial cross sections of electroweak VVjj production are reported for the WZjj [20,21], ZZjj [22], Zγjj [23] and Wγjj [24] channels. Constraints on anomalous quartic gauge couplings are reported in Refs. [16–19,21,23–27]. VVjj (V ¼ W, Z, γ), with a large separation in rapidity of jets and a large dijet invariant mass. Multiple processes can produce the same final state of two bosons and two jets. The production of VVjj at tree level has an EW contri- bution involving only electroweak-interaction vertices, and a strong contribution (QCD induced) involving two strong- interaction vertices. The EW production is further divided into two components. The first component is EW VBS production with actual scattering of the two electroweak bosons. The scattering occurs via quartic gauge vertices, or triple gauge vertices involving the s- or t-channel exchange of a Higgs boson or a W=Z boson. *Full author list given at the end of the article. DOI: 10.1103/PhysRevD.100.032007 2The ATLAS experiment uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the center of the detector and the z axis along the axis of the beam pipe. The x axis points from the IP to the center of the LHC ring, and the y axis points upwards. Cylindrical coordinates ðr; ϕÞ are used in the transverse plane, ϕ being the azimuthal angle around the z axis. The pseudorapidity is defined in terms of the polar angle θ as η ¼ −ln tanðθ=2Þ. Angular distance is measured in units of ΔR ≡ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðΔηÞ2 þ ðΔϕÞ2 p . I. INTRODUCTION Representative Feynman diagrams for (a) EW VVjj production via VBS, (b) EW VVjj production via non-VBS contribution, and (c) QCD VVjj production. (c) (b) (a) (c) (b) (a) FIG. 1. Representative Feynman diagrams for (a) EW VVjj production via VBS, (b) EW VVjj production via non-VBS contribution, and (c) QCD VVjj production. final states, each using resolved or merged V →qq reconstruction techniques. Reference [26] reports a study similar to the one in this paper, albeit focused on EW production of VVjj in the WV →lνqq channel only and performed at ﬃﬃﬃs p ¼ 8 TeV. This paper presents a study of the EW production of VVjj (V ¼ W, Z) with the vector-boson pair decaying semi- leptonically. A larger data sample is used and additional diboson signal processes with similar final states are included. In order to extract the signal and to measure the cross section for the EW production of VVjj in a fiducial volume, multivariate discriminants, which combine observ- ables sensitive to the kinematics of the VBS process, are used to separate EW-induced VVjj production from QCD- induced VVjj production. This analysis measures the cross section of EW VVjj production in a region of kinematic phase space close to the acceptance of the detector. Fiducial cross sections are measured in the 0-, 1- and 2-lepton channels, where lepton refers to e and μ. Final states with V decaying into one or more τ-leptons (both leptonically and hadronically decaying τ-leptons) are included as signal, but the con- tribution of V from top quark decay is not considered as signal. Three VV semileptonic decay channels are explored: a Z boson decaying into a pair of neutrinos, Z →νν1; a W boson decaying into a charged lepton (an electron or muon, denoted by l) and a neutrino, W →lν; and a Z boson decaying into a pair of light charged leptons, Z →ll. In all cases, the other vector boson V is required to decay into a pair of quarks, V →qq, leading to ZV →ννqq, WV → lνqq and ZV →llqq final states. These processes overlap in the fiducial region of the measurement because of the geometrical acceptance of the detector for leptons and jets. I. INTRODUCTION The second component is EW non-VBS production that has electro- weak vertices only, but where the two bosons do not scatter. The EW non-VBS component cannot be separated from the EW VBS component in a gauge invariant way [13] and contributes significantly to the total cross section. It is therefore included in the signal generation. Representative Feynman diagrams at tree level are shown in Fig. 1. Both the ATLAS and CMS Collaborations have searched for experimental evidence of VBS. So far, electroweak VVjj production is only observed in the same-sign WWjj channel [14] and WZjj channel [15] in the fully leptonic final states using data collected at a center-of-mass energy of ﬃﬃﬃs p ¼ 13 TeV. Evidence of electroweak VVjj production is also obtained in the WWjj [16–18] and Zγjj [19] channels using pp collisions at ﬃﬃﬃs p ¼ 8 TeV. Limits on fiducial cross sections of electroweak VVjj production are reported for the WZjj [20,21], ZZjj [22], Zγjj [23] and Wγjj [24] channels. Constraints on anomalous quartic gauge couplings are reported in Refs. [16–19,21,23–27]. Vector-boson scattering (VBS) is a key process for probing the non-Abelian gauge structure of the electroweak (EW) sector of the Standard Model (SM), since it involves both the self-couplings of the vector bosons and their coupling with the Higgs boson. In the absence of the SM Higgs boson, the amplitudes for VBS would increase as a function of partonic center-of-mass energy and ultimately violate unitarity [1,2]. The discovery of a Higgs boson in 2012 at the LHC [3,4], with measured properties [5–8] consistent with those of the SM Higgs boson, represents a major milestone in the understanding of electroweak symmetry breaking. The study of the VBS process provides an important check of the SM by testing whether the Higgs mechanism is the sole source of electroweak symmetry breaking. Theories of new phenomena beyond the SM that alter the quartic gauge couplings [9,10], or include the presence of additional resonances [11,12], predict enhancements of VBS at high transverse momentum of the vector bosons and at high invariant mass of the diboson system. The experimental signature of VBS is characterized by the presence of a pair of vector bosons and two forward jets, © 2019 CERN, for the ATLAS Collaboration 032007-1 2470-0010=2019=100(3)=032007(36) PHYS. REV. D 100, 032007 (2019) G. AAD et al. (a) (b) (c) FIG. 1. 1To simplify the notation, antiparticles are not explicitly labeled in this paper. I. INTRODUCTION The decay channels are selected as 0-, 1- and 2-lepton final states, where the 1-lepton (2-lepton) final state receives only contribution from WV→lνqq (ZV→llqq) proc- esses, and the 0-lepton final state receives about equal contributions from WV →lνqq and ZV →ννqq processes. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … constant. Both the VBS amplitudes and non-VBS ampli- tudes of the VVjj process with one boson decaying hadronically and the other leptonically were included, using factorized on-shell decays for the gauge bosons. The NNPDF30LO [31] PDF set was used. The parton showers and hadronization were modeled with PYTHIA 8.186 [32] using the A14 set of tuned parameters (tune) for the underlying event [33]. The end cap and forward regions are instrumented with LAr calorimeters for both EM and hadronic energy measurements up to jηj ¼ 4.9. A muon spectrometer (MS) system incorporating large superconducting toroidal air-core magnets surrounds the calorimeters. Three layers of precision wire chambers provide muon tracking in the range jηj < 2.7, while dedicated fast chambers are used for triggering in the region jηj < 2.4. The trigger system is composed of two stages [29]. The first stage, implemented with custom hardware, uses information from calorimeters and muon chambers to reduce the event rate to a maximum of 100 kHz. The second stage, called the high-level trigger, reduces the data acquisition rate to about 1 kHz on average. The high-level trigger is software-based and runs reconstruction algorithms similar to those used in the offline reconstruction. The main background sources are Z and W bosons produced in association with jets (Z þ jets and W þ jets), as well as significant contributions from top quark pro- duction (both t¯t pair and single-top) and QCD-induced vector-boson pair production. The Z þ jets and W þ jets events were simulated using the SHERPA 2.2.1 [34] event generator. Matrix elements were calculated for up to two partons at NLO and up to four partons at LO using the COMIX [35] and OPENLOOPS [36] programs. QCD-induced diboson processes with one of the bosons decaying hadronically and the other leptonically were simulated using SHERPA 2.2.1. They were simulated for up to one additional parton at NLO and up to three additional partons at LO using the COMIX and OPENLOOPS programs. There is no overlap between the QCD-induced diboson samples and the EW VVjj signal samples, as the former include diagrams of Oðα4 EWα2 SÞ. For Z þ jets, W þ jets and diboson simulation, the matrix-element calculations were merged with the SHERPA parton shower using the ME þ PS@NLO prescription [37]. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … The NNPDF30NNLO [38] PDF set was used in conjunction with a dedicated parton-shower tuning developed by the SHERPA authors. For the Z þ jets and W þ jets samples, boson decays into all lepton flavors (e, μ, τ) are included. For the generation of top quark pairs, the POWHEG- BOX V2 [39–41] event generator with the CT10 [42] PDF set in the matrix-element calculations was used. Electroweak t- channel, s-channel and Wt-channel single-top-quark events were generated using the POWHEG-BOX V1 event generator [43–45]. This event generator uses the four-flavor scheme for the NLO matrix-element calculations together with the fixed four-flavor PDF set CT10f4 [42]. For all top quark processes, top quark spin correlations are preserved (for the t-channel, top quark decay is simulated using MADSPIN [46]). The parton showers, fragmentation, and underlying event were simulated using PYTHIA 6.428 [47] with the CTEQ6L1 [48] PDF set and the corresponding Perugia 2012 tune (P2012) [49]. The top quark mass was set to 172.5 GeV. The EVTGEN V1.2.0 program [50] was used to simulate the decay of bottom and charm hadrons for the POWHEG-BOX samples. A. Data The data were collected with the ATLAS detector in 2015 and 2016 from pp collisions at a center-of-mass energy of ﬃﬃﬃs p ¼ 13 TeV, corresponding to a total integrated luminos- ity of 35.5 fb−1. The recorded 2-lepton channel and 1-lepton channel events were selected with a mixture of either multiple single-electron or single-muon triggers with varying trans- verse energy ET (electron) and transverse momentum pT (muon) thresholds, and quality and isolation requirements, that depended on the LHC running conditions. The lowest ET or pT requirement without trigger prescaling was 26 GeV for both the electrons and muons. Events for the 0-lepton channel were recorded with nonprescaled missing transverse momentum (Emiss T ) triggers where the Emiss T threshold depended on the LHC running conditions. The lowest threshold used is 110 GeV. The Emiss T triggers used are fully efficient for events passing the selection described below. The Emiss T triggers are also used in the 1-lepton channel to compensate for single-muon trigger inefficiency due to the difference in acceptance between the muon tracking and triggering. Events in this analysis have all detector systems operat- ing normally. Collision vertices are formed from tracks with pT > 400 MeV, and the one with the highest P p2 T of its associated tracks is selected as the primary vertex. II. ATLAS DETECTOR The ATLAS experiment is described in Ref. [28]. ATLAS is a multipurpose detector with a forward-back- ward symmetric cylindrical geometry and a solid-angle2 coverage of nearly 4π. The inner tracking detector (ID), covering the region jηj < 2.5, consists of a silicon pixel detector, a silicon microstrip detector and a straw-tube transition-radiation tracker. The inner detector is sur- rounded by a thin superconducting solenoid providing a 2 T magnetic field, and by a finely segmented lead/ liquid-argon (LAr) electromagnetic calorimeter covering the region jηj < 3.2. A steel/scintillator-tile hadronic calo- rimeter provides coverage in the central region jηj < 1.7. p Two different reconstruction techniques for the V →qq decay are considered: resolved and merged. The resolved reconstruction attempts to identify two separate small- radius jets (small-R jet denoted by j) of hadrons from the V →qq decay, while the merged reconstruction uses jet substructure techniques to identify the V →qq decay reconstructed as a large-radius jet (large-R jet denoted by J). The latter applies when the momentum transfer in VVjj production is high, and as a consequence the qq pair from the V boson decay is collimated. In this case, hadrons from the two quarks overlap in the detector and are more efficiently reconstructed as a single large-R jet. In total, six final states are included in this study: 0-, 1- and 2-lepton 032007-2 PHYS. REV. D 100, 032007 (2019) III. DATA AND MONTE CARLO SIMULATION A. Data B. Signal and background simulation Monte Carlo (MC) simulation is used to model signal and background processes. The simulated samples are used to optimize the event selection, to develop the multivariate discriminant, and to estimate the irreducible background yields. All simulated processes are normalized using the cur- rently available state-of-the-art theoretical predictions for their cross sections. Cross sections are calculated with up to next-to-next-to-leading-order (NNLO) QCD corrections for Z þ jets and W þ jets production [51]. Cross sections for diboson production are calculated at NLO including LO contributions with two additional partons [34,52]. The t¯t The EW VVjj signal samples were generated using MADGRAPH5_AMC@NLO 2.4.3 [30] with amplitudes of Oðα6 EWα0 SÞ, where αEW (αS) is the EW (strong) coupling 032007-3 PHYS. REV. D 100, 032007 (2019) G. AAD et al. production cross section is calculated at NNLO in QCD, including resummation of next-to-next-to-leading logarith- mic (NNLL) soft-gluon terms [53,54]. The single-top production cross sections are calculated to NLO in QCD [55], including the soft-gluon resummation at NNLL [56] for the Wt process. production cross section is calculated at NNLO in QCD, including resummation of next-to-next-to-leading logarith- mic (NNLL) soft-gluon terms [53,54]. The single-top production cross sections are calculated to NLO in QCD [55], including the soft-gluon resummation at NNLL [56] for the Wt process. [64,65] but with different values of the radius parameter R. Small-R jets and large-R jets are reconstructed inde- pendently from the same energy depositions for a given event. The treatment of the resulting overlap is discussed further below. Small-R jets are reconstructed with a radius parameter of R ¼ 0.4. Energy- and η-dependent correction factors derived from MC simulations are applied to correct jets back to the particle level [66]. Pileup effects are corrected using a jet area method [67,68]. Jets are required to have pT > 20 GeV for jηj < 2.5 and pT > 30 GeV for 2.5 < jηj < 4.5. A jet vertex tagger [67] is applied to jets with pT < 60 GeV and jηj < 2.4 in order to select only jets from the hard interaction which are associated with the primary vertex, and to suppress jets from pileup inter- actions. This tagger uses information about tracks asso- ciated with the primary vertex and pileup vertices. MC events were processed with a detailed detector simulation [57] based on GEANT4 [58]. IV. OBJECT RECONSTRUCTION Small-R jets containing b-hadrons are identified using a multivariate algorithm (b-tagging) [69] which uses infor- mation such as track impact-parameter significance and the position of explicitly reconstructed secondary decay ver- tices. The chosen b-tagging algorithm has an efficiency of 70% for b-quark jets in simulated t¯t events, with a light- flavor jet rejection factor of about 380 and a c-jet rejection factor of about 12 [70]. Electrons are identified as isolated energy clusters in the electromagnetic calorimeter matched to ID tracks, and are required to have transverse energy ET > 7 GeV and pseudorapidity jηj < 2.47. A likelihood-based requirement [61] is imposed to reduce the background from nonprompt electrons or hadrons misidentified as electrons. Electrons are classified as either “loose,” “medium” or “tight” according to the likelihood-based identification criteria described in Ref. [61]. Large-R jets are reconstructed with the radius parameter increased to R ¼ 1.0. In order to mitigate the effects of pileup and soft radiation, the large-R jets are trimmed [71]. Trimming takes the original constituents of the jet and reclusters them using the kt algorithm [72] with a smaller radius parameter, Rsubjet, to produce a collection of subjets. These subjets are discarded if they carry less than a specific fraction (fcut) of the original jet pT. The trimming param- eters were optimized for W=Z boson tagging and are Rsubjet ¼ 0.2 and fcut ¼ 5%. The large-R jet four-momenta are recomputed from the remaining subjets, and the jet energies are calibrated to particle level using correction factors derived from MC simulations [73]. The mass of a large-R jet (mJ) is computed using a combination of calorimeter and tracking information [74]. Large-R jets are required to have pT > 200 GeV and jηj < 2.0. Muons are reconstructed by a combined fit to the ID and MS tracks, and are required to have pT > 7 GeV and jηj < 2.5. Muons must pass identification requirements, based on the number of hits in the ID and MS subsystems, and the significance of the difference jq=pMS −q=pIDj [62], where q is the charge and pMSðpIDÞ is the momentum of the muon measured in the MS (ID). Similarly to electrons, muons are classified as either loose, medium or tight, following the criteria in Ref. [62]. B. Signal and background simulation Additional inelastic simulated pp collisions generated with PYTHIA 8.186 using the A2 set of tuned parameters [59] and the MSTW2008LO [60] PDF set were overlaid in order to model both the in- and out-of-time effects from additional pp collisions in the same and neighboring bunch crossings (pileup). MC samples are reweighted to match the pileup conditions in the data. All simulated events are processed using the same reconstruction algorithms as the data. A. Event selection Signal events in the 0-lepton channel are typical of a hadronically decaying V boson recoiling against a large amount of missing transverse momentum stemming from either a Z →νν decay or a W →lν decay, where the lepton is outside the acceptance of the detector. An initial selection is made by requiring Emiss T > 200 GeV, and rejecting events with electrons or muons passing the loose quality requirements. The multijet background originates primarily from the presence of mismeasured jets and noncollision phenomena. It is suppressed using a requirement on the value of the track-based missing transverse momentum, pmiss T > 50 GeV. Three further angular selection criteria are: the azimuthal separation between the ⃗Emiss T and ⃗pmiss T directions satisfies Δϕð⃗Emiss T ; ⃗pmiss T Þ < π=2; the azimuthal separation between the directions of ⃗Emiss T and the nearest small-R jet satisfies min½Δϕð⃗Emiss T ; small-R jetÞ > π=6; and the azimuthal separation between the directions of ⃗Emiss T and the reconstructed hadronically decaying candidate Vhad satisfies Δϕð⃗Emiss T ; VhadÞ > π=9. The multijet background is found to be negligible after these selections. p y The missing transverse momentum vector, ⃗Emiss T , is calculated as the negative vectorial sum of the transverse momenta of calibrated electrons, muons, and small-R jets where the calibration already includes corrections for pileup. Large-R jets and track jets are not included in the ⃗Emiss T calculation in order to avoid double-counting of energy between the small-R jets and large-R jets. Energy depositions due to the underlying event and other types of soft radiation are taken into account by constructing a “soft term” from ID tracks that are associated with the primary vertex but not used in any reconstructed object [79]. The track-based missing transverse momentum vector, ⃗pmiss T , is the negative vectorial sum of the transverse momenta of all good-quality inner-detector tracks that are associated with the primary vertex. The 1-lepton channel is typical of a leptonically decaying W boson. The W →lν candidates are selected by requiring one isolated lepton (electron or muon) satisfying the tight criteria with pT > 27 GeV. Events are required to have Emiss T > 80 GeV, and must not have any additional loose leptons. A. Event selection In order to reconstruct the invariant mass of the WV system, needed later to construct the multivariate discriminant, the neutrino momentum four-vector is reconstructed by imposing a W boson mass constraint on the lepton–neutrino system. The neutrino transverse momentum components are set equal to the missing transverse momentum of the event and the unknown z-component of the momentum (pz) is obtained from the resulting quadratic equation. The pz is chosen as either the smaller, in absolute value, of the two real solutions or, if the solution is complex, its real part. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … between the muon and the jet are small; otherwise the muon is removed if the separation satisfies ΔR < 0.4. In order to prevent double-counting of energy from an electron inside a large-R jet, the large-R jet is removed if an electron and a large-R jet are separated by ΔR < 1.0. No overlap removal is applied between large-R jets or track jets and small-R jets. (V →J) in a merged selection, and those jets are referred to as Vhad jets. Event selection criteria are chosen to guarantee the statistical independence of the channels and to maximize the sensitivity of the analysis. This selection results in nine nonoverlapping distinct signal regions (SR): one for each of the three lepton channels and three types of Vhad selections (resolved, and low- and high- purity merged). Boson tagging is applied to large-R jets in order to select those consistent with V →qq decays. A pT-dependent requirement is applied to the jet substructure variable Dðβ¼1Þ 2 , which is defined as a ratio of two-point to three-point energy correlation functions [76,77] that are based on the energies and pairwise angular separations of the particles within a jet. This variable is optimized to distinguish between jets originating from a single parton and those coming from the two-body decay of a heavy particle. A detailed description of the method and its optimization can be found in Ref. [78]. Large-R jets from V →qq decays are required to have a jet mass mJ in a pT- dependent window centered around the expected value of the boson mass. The configuration of the boson tagging algorithm is called a working point, which is designed to provide constant efficiency independent of the large-R jet pT for the signals studied. Two working points are used, one with 50% efficiency and the other one with 80% efficiency, with corresponding misidentification rates for jets from multijet production of ∼2% and ∼10%, respectively. ⃗ i The event selection for all channels and background estimations is summarized in Table I. Further details are given below. IV. OBJECT RECONSTRUCTION All electrons and muons are required to be isolated by using selections based on the sum of the pT of tracks (excluding the track associated with the lepton) in a cone of pT-dependent size around their directions. The isolation selection criteria are designed to maintain a constant efficiency of 99% in the pT-η plane for reconstructed leptons from Z →ll decays. Furthermore, leptons are required to have associated tracks satisfying jd0=σd0j < 5ð3Þ and jz0 × sin θj < 0.5 mm for electrons (muons), where d0 is the transverse impact parameter relative to the beam line, σd0 is its uncertainty, and z0 is the distance between the longitudinal position of the track along the beam line at the point where d0 is measured and the longitudinal position of the primary vertex. j j Track jets have a radius parameter of R ¼ 0.2 [75]. Inner-detector tracks originating from the primary vertex, with pT>0.5GeV and selected by impact parameter requirements, are used in the track jet reconstruction. Track jets are required to satisfy pT>20GeV and jηj<2.5. The number of track jets is an input to the multivariate discriminant described later. An overlap-removal procedure is applied to the selected leptons and jets in order to prevent double-counting. The jet is removed if an electron and a small-R jet are separated by ΔR < 0.2; the electron is removed if the separation satisfies 0.2 < ΔR < 0.4. The jet is removed if a muon and a small-R jet are separated by ΔR < 0.2 and if the jet has less than three tracks or the energy and momentum differences Three types of jets are employed in the analysis. Two of them are reconstructed from three-dimensional topological clusters of energy deposits in the calorimeter [63] (small-R jets and large-R jets), and the third type from inner-detector tracks (track jets). All three use the anti-kt algorithm 032007-4 PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … V. EVENT SELECTION AND BACKGROIUND ESTIMATION j j V boson tagging, minðjmJ −mWj; jmJ −mZjÞ 64 < mjj < 106 GeV, jj pair with minðjmjj −mWj; jmjj −mZjÞ, leading jet with pT > 40 GeV j ∉Vhad, not b-tagged, ΔRðJ; jÞ > 1.4 ηtag;j1 · ηtag;j2 < 0, mtag jj > 400 GeV, pT > 30 GeV lepton must satisfy pT > 28ð20Þ GeV. Opposite charges are required for the muon pairs but not for the electron pairs, since electrons are more susceptible to charge misidentification due to the conversion of photons from bremsstrahlung, especially at high pT. The dilepton invari- ant mass is required to be consistent with that of the Z boson: 83 < mee < 99 GeV in the case of electrons and ð−0.0117 × pμμ T þ 85.63 GeVÞ < mμμ < ð0.0185 × pμμ T þ 94 GeVÞ in the case of muons. The pT-dependent require- ment on mμμ recovers the selection efficiency at high pμμ T , which would otherwise fall due to the degraded dimuon invariant mass resolution [80]. point requirement. The former is called the high-purity (HP) signal region, and the latter the low-purity (LP) signal region. Given the different but overlappping W and Z boson tagging requirements, large-R jets are required to satisfy either W or Z boson tagging. If multiple Vhad candidates are selected, the one minimizing minðjmJ − mWj; jmJ −mZjÞ is selected. The resolved selection events are required to have two small-R signal jets with a dijet invariant mass lying in the mW=Z window: 64 < mjj < 106 GeV. If multiple Vhad candidates are selected, the one minimizing minðjmjj −mWj; jmjj −mZjÞ is used. At least one of the jets forming the selected Vhad candidate must have pT > 40 GeV, in order to improve the separation between the signal and the background; otherwise the event is not selected. The merged selection is applied as the first step in identifying a Vhad candidate. If an event is not selected, then the resolved selection is used. The order is motivated by a smaller background expectation in the merged analysis. Selecting the jets that form a Vhad candidate first and then selecting the tagging-jets from the pool of remaining jets results in an analysis with a higher sensitivity compared with doing the selection in the reverse order. The Vhad candidates are selected in three different nonoverlapping channels. V. EVENT SELECTION AND BACKGROIUND ESTIMATION Events are categorized into the 0-, 1- and 2-lepton channels depending on the number of selected electrons and muons. In addition to a leptonically decaying candi- date Vlep, events in all three channels are required to contain a hadronically decaying candidate Vhad, and two additional small-R jets (referred to as tagging-jets). The Vhad candidate is reconstructed as either two small-R jets (V →jj) in a resolved selection, or one large-R jet In the 2-lepton channel, the Z →ll candidates are identified by requiring two isolated same-flavor leptons satisfying the loose criteria. The leading (subleading) 032007-5 PHYS. REV. D 100, 032007 (2019) G. AAD et al. TABLE I. Summary of the event selection in the 0-, 1- and 2-lepton channels. Selection 0-lepton 1-lepton 2-lepton Trigger Emiss T triggers Single-electron triggers Single-lepton triggers single-muon or Emiss T triggers Leptons 0 loose leptons with pT > 7 GeV 1 tight lepton with pT > 27 GeV 2 loose leptons with pT > 20 GeV 0 loose leptons with pT > 7 GeV ≥1 lepton with pT > 28 GeV Emiss T >200 GeV >80 GeV mll 83 < mee < 99 GeV ð−0.0117 × pμμ T þ 85.63 GeVÞ < mμμ < ð0.0185 × pμμ T þ 94 GeVÞ Small-R jets pT > 20 GeV if jηj < 2.5, and pT > 30 GeV if 2.5 < jηj < 4.5 Large-R jets pT > 200 GeV, jηj < 2 Vhad →J V boson tagging, minðjmJ −mWj; jmJ −mZjÞ Vhad →jj 64 < mjj < 106 GeV, jj pair with minðjmjj −mWj; jmjj −mZjÞ, leading jet with pT > 40 GeV Tagging-jets j ∉Vhad, not b-tagged, ΔRðJ; jÞ > 1.4 ηtag;j1 · ηtag;j2 < 0, mtag jj > 400 GeV, pT > 30 GeV Num. of b-jets 0 Multijet removal pmiss T > 50 GeV Δϕð⃗Emiss T ; ⃗pmiss T Þ < π=2 min½Δϕð⃗Emiss T ; small-R jetÞ > π=6 Δϕð⃗Emiss T ; VhadÞ > π=9 of the event selection in the 0-, 1- and 2-lepton channels. B. Data control regions and background estimation The dominant backgrounds for the 1-lepton channel are W þ jets and t¯t production; for the 2-lepton channel it is Z þ jets production; while in the 0-lepton channel, they all contribute significantly. Smaller background contributions for the 1-lepton channel arise from multijet background. Single-top and QCD-induced diboson production is a small background for all three lepton channels. The background contributions are estimated using a combination of MC and data-driven techniques. The shapes of kinematic variable distributions are taken from MC simulations in all cases except for the multijet background in the 1-lepton channel. p The mtag jj spectra of simulated W þ jets (Z þ jets) events are not well modeled by the MC simulation in the WCRs (ZCRs) for the three Vhad selections in the 1-lepton (2- lepton) channel. A data-driven procedure is applied to the simulated W þ jets and Z þ jets events to correct for this shape mismodeling. Reweighting factors are derived from WCRs and ZCRs as a function of mtag jj , and applied to all SRs and CRs (for 0-, 1-, and 2-lepton regions) in the MC simulation of W þ jets and Z þ jets events, respectively. The non-W þ jetsðZ þ jetsÞ contributions are subtracted from the spectra in data. Then the reweighting factors as a function of mtag jj are determined by performing a linear fit to the ratios of data to simulation in the control regions. The reweighting is done separately for the merged and resolved analyses. For W þ jets, the reweighting factor ranges from 1.016 (1.024) at mtag jj ¼ 400 GeV to 0.47 (0.53) at mtag jj ¼ 3000 GeV in the resolved (merged) analysis. For Z þ jets, the reweighting factor ranges from 1.071 (1.062) at mtag jj ¼ 400 GeV to 0.42 (0.36) at mtag jj ¼ 3000 GeV in the resolved (merged) analysis. A Z þ jets control region (ZCR) is defined for each of the three SRs in the 2-lepton channel by reversing the mJ or mjj requirement. Events in each of the CRs are selected in exactly the same way as those in their corresponding SRs except for the requirement on mJ or mjj. For the merged selection, the leading large-R jet mass is required to be outside the large-R jet mass window of the 80% working point of the W=Z boson tagging. B. Data control regions and background estimation For the resolved selection, a requirement of 50 < mjj < 64 GeV or mjj > 106 GeV is applied. These CRs are dominated by the Z þ jets contribution, with a purity higher than 95% in all regions. They are therefore used to constrain its contribution in signal regions through simultaneous fits as discussed in Sec. X. g y Additional reweighting factors are needed for the MC simulation of W þ jets and Z þ jets events in the 0-lepton channel because the phase space is so different between the 0-lepton selection and the 1- and 2-lepton selections that the reweightings described above are not applicable. These additional reweightings are derived from MC simulation as the ratio of the numbers of W þ jets (Z þ jets) events in the 1-lepton (2-lepton) and 0-lepton channels, and are applied to the MC simulation of W þ jets (Z þ jets) events in the 0-lepton channel. Good agreement between the prediction from MC simulation and the data in the VjjCR is achieved only after the two reweightings have been applied. Unless stated otherwise, the final reweighted W þ jets and Z þ jets simulated events are used everywhere in the analysis. Three W þ jets control regions (WCRs) are formed from events satisfying the 1-lepton signal region selection except for the invariant mass requirement of the Vhad candidate, similar to the ZCRs. Approximately 86% and 77% of the selected events are from W þ jets production in the merged and resolved categories of the 1-lepton channel, respec- tively. The remaining events are primarily from t¯t production. The three t¯t control regions (TopCRs) consist of events satisfying the signal region selection of the 1-lepton channel except for the b-jet requirement, which is inverted. These CRs are dominated by t¯t production, with a purity of 79% and 59% for merged and resolved categories respec- tively, and the remainder are from single-top, V þ jets or diboson production, for both the merged and the resolved event topologies. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … misidentified as leptons or real but nonprompt leptons from decays of heavy-flavor hadrons. This contribution is negligible in all regions, except for the resolved 1-lepton SR. The fake-factor background method of Ref. [81] is used to estimate the multijet background contribution in the resolved topology of the 1-lepton channel. The estimated multijet contribution is about 10% of the total background in the resolved 1-lepton SR. Finally, 1-lepton channel events are rejected if any of the small-R jets in the event is identified as a b-jet prior to the Vhad candidate and tagging-jets selection. This reduces the contributions from top quark production. V. EVENT SELECTION AND BACKGROIUND ESTIMATION After selecting the Vhad candidate, tagging-jets are selected from the remaining small-R jets that fail the b- tagging described in Sec. IV. For the merged selection, all small-R jets with ΔRðJ; jÞ < 1.4 are excluded before the tagging-jets selection. Tagging-jets are required to be in opposite hemispheres, ηtag;j1 · ηtag;j2 < 0, and the invariant massofthetwotagging-jets mustsatisfymtag jj > 400 GeV.If there is more than one pair of jets satisfying these require- ments, the one with the highest mtag jj value is chosen. In order to suppress the contribution from pileup interactions, both tagging-jets from the selected pair must have pT > 30 GeV; otherwise the event is rejected. Merged selection events are required to have at least one large-R jet. Next the boson tagging discussed in Sec. IV is applied to select the V →qq decays. Two SRs are defined, one for events passing the 50% working point of the boson tagging requirement and the other for events failing the 50%, but passing the 80% working 032007-6 PHYS. REV. D 100, 032007 (2019) VI. MULTIVARIATE ANALYSIS Variable 0-lepton 1-lepton 2-lepton mtag jj ✓ ✓ Δηtag jj ✓ ptag;j1 T ✓ ✓ ptag;j2 T ✓ ✓ ✓ Δηjj ✓ ✓ ✓ pj1 T ✓ pj2 T ✓ ✓ ✓ wj1 ✓ ✓ ✓ wj2 ✓ ✓ ✓ nj1 tracks ✓ ✓ nj2 tracks ✓ ✓ wtag;j1 ✓ ✓ ✓ wtag;j2 ✓ ✓ ✓ ntag;j1 tracks ✓ ✓ ntag;j2 tracks ✓ ✓ nj;track ✓ ✓ nj;extr ✓ Emiss T ✓ ηl ✓ ΔRðl; νÞ ✓ ζV ✓ ✓ mVV ✓ mVVjj ✓ TABLE III. Variables used for the BDT discriminant in the resolved analysis category of each lepton channel analysis. TABLE III. Variables used for the BDT discriminant in the resolved analysis category of each lepton channel analysis. TABLE III. Variables used for the BDT discriminant in the resolved analysis category of each lepton channel analysis. Variable 0-lepton 1-lepton 2-lepton mtag jj ✓ ✓ Δηtag jj ✓ ptag;j1 T ✓ ✓ ptag;j2 T ✓ ✓ ✓ Δηjj ✓ ✓ ✓ pj1 T ✓ pj2 T ✓ ✓ ✓ wj1 ✓ ✓ ✓ wj2 ✓ ✓ ✓ nj1 tracks ✓ ✓ nj2 tracks ✓ ✓ wtag;j1 ✓ ✓ ✓ wtag;j2 ✓ ✓ ✓ ntag;j1 tracks ✓ ✓ ntag;j2 tracks ✓ ✓ nj;track ✓ ✓ nj;extr ✓ Emiss T ✓ ηl ✓ ΔRðl; νÞ ✓ ζV ✓ ✓ mVV ✓ mVVjj ✓ high-purity SR and low-purity SR are merged together for the BDT training due to an insufficient number of MC events. VI. MULTIVARIATE ANALYSIS In order to make use of the complete set of simulated MC events for the BDT training and evaluation in an unbiased way, the MC events are split for training and validation into two subsamples of equal size following the procedure in Ref. [83]. The output distributions of the BDTs trained on the two subsamples are averaged for both the simulated and data events. is also used, and it is defined as ζV ¼ minðΔη−; ΔηþÞ, where Δη−¼ min½ηðVhadÞ;ηðVlepÞ−min½ηtag;j1;ηtag;j2 and Δηþ ¼ max½ηtag;j1;ηtag;j2−max½ηðVhadÞ;ηðVlepÞ. The vari- able ζV has large values when the tagging-jets have a large separation in η, and when the two boson candidates lie between the tagging-jets in η. Variables sensitive to the quark–gluon jet separation are also included, such as the width of the small-R jets (w) [84], and the number of tracks associated with the jets (ntracks). The number of track jets, nj;track, and the number of additional small-R jets other than the Vhad jets and tagging-jets, nj;extr, are also found to be useful for the BDTs. In the 1-lepton channel, the pseudor- apidity of the lepton (ηl) is also considered. The input variables used for the BDTs are chosen in order to maximize the separation between signal and background, and are summarized in Tables II and III, for the merged and resolved category, respectively. The dis- tributions of input variables of the BDTs are compared between data and simulation, and in general are found to be in good agreement. The small-R jets are labeled in decreasing pT as j1 and j2 for the jets used to reconstruct the hadronically decaying boson, and as “tag, j1” and “tag, j2” for the tagging-jets. The invariant mass and transverse momentum of the reconstructed VV (VVjj) system are denoted by mVV (mVVjj) and pVV T (pVVjj T ), respectively. Angular variables are also considered, such as the pseu- dorapidity gap between the tagging-jets (Δηtag jj ) and between the small-R Vhad jets (Δηjj), the angular separation of the lepton and neutrino from the W boson decay (ΔRðl; νÞ) in the 1-lepton channel, and the azimuthal angle between the directions of ⃗Emiss T and the large-R jet (Δϕð⃗Emiss T ; JÞ) in the merged category of the 0-lepton channel. A topological variable named boson centrality VI. MULTIVARIATE ANALYSIS A multivariate method is used to enhance the separation between the signal and background. The analysis uses the Toolkit for Multivariate Data Analysis, TMVA [82], and its implementation of the boosted decision trees (BDTs) method. BDTs are constructed, trained and evaluated in each lepton channel and analysis region separately. The BDT training is carried out using simulated signal and all background MC samples. However, the events in In the 0-lepton channel, it is not possible to define pure control regions for W þ jets, Z þ jets and t¯t processes, thus events falling into the mass sideband regions of the Vhad, similar to WCRs and ZCRs, form three different CRs (referred to as VjjCR), one for each of the correspond- ing SRs. The contribution from multijet production primarily consists of events with jets or photon conversions 032007-7 PHYS. REV. D 100, 032007 (2019) G. AAD et al. TABLE II. Variables used for the BDT discriminant in the merged analysis category of each lepton channel. Variable 0-lepton 1-lepton 2-lepton mtag jj ✓ ✓ Δηtag jj ✓ ptag;j2 T ✓ ✓ ✓ mJ ✓ Dðβ¼1Þ 2 ✓ ✓ Emiss T ✓ Δϕð⃗Emiss T ; JÞ ✓ ηl ✓ nj;track ✓ ζV ✓ ✓ mVV ✓ pVV T ✓ mVVjj ✓ pVVjj T ✓ wtag;j1 ✓ wtag;j2 ✓ TABLE II. Variables used for the BDT discriminant in the merged analysis category of each lepton channel. TABLE III. Variables used for the BDT discriminant in the resolved analysis category of each lepton channel analysis. VIII. SYSTEMATICAL UNCERTAINTIES The sources of systematic uncertainty can be divided into three categories: experimental uncertainties related to the detector or to the reconstruction algorithms, uncertainties in the estimations of background contributions, and uncer- tainties in modeling the signal. Unless stated otherwise, the uncertainties quoted below are the uncertainties in the quantities themselves, not the impact on the analysis sensitivity. The uncertainty in the integrated luminosity of the dataset is 2.1%. It is derived from the calibration of the luminosity scale using x-y beam-separation scans, follow- ing a methodology similar to that detailed in Ref. [86], and using the LUCID-2 detector for the baseline luminosity measurements [87]. This uncertainty is applied to the normalization of the signal and also to background con- tributions whose normalizations are derived from MC simulations. In addition to the luminosity uncertainty, a variation in the pileup reweighting of MC events is also included to cover the uncertainty in the ratio of the predicted to measured inelastic cross sections in Ref. [88]. The uncertainty in the integrated luminosity of the dataset is 2.1%. It is derived from the calibration of the luminosity scale using x-y beam-separation scans, follow- ing a methodology similar to that detailed in Ref. [86], and using the LUCID-2 detector for the baseline luminosity measurements [87]. This uncertainty is applied to the normalization of the signal and also to background con- tributions whose normalizations are derived from MC simulations. In addition to the luminosity uncertainty, a variation in the pileup reweighting of MC events is also included to cover the uncertainty in the ratio of the predicted to measured inelastic cross sections in Ref. [88]. The efficiencies of the lepton triggers for events with selected leptons are high, nearly 100% in the electron channel and approximately 96% in the muon channel. The corresponding uncertainties are negligible. For the selec- tion used in the 0-lepton and 1-lepton channels, the efficiency of the Emiss T trigger is also close to 100% with negligible associated uncertainty. The modeling of the electron and muon reconstruction, identification and The selection of hadronically decaying bosons and tagging-jets follows the same steps and apply the same criteria as for reconstruction level, as shown in Table IV. For the 0-, 1- and 2-lepton channels, the number of selected fiducial leptons is required to be 0, 1 and 2, respectively. Events with additional leptons for the 1- and 2-lepton channels are vetoed. VII. FIDUCIAL CROSS-SECTION DEFINITION The fiducial phase space of the measurement is defined using stable final-state particles [85]. Leptons produced in the decay of a hadron or its descendants are not considered in the charged lepton requirement of the fiducial phase space. The fiducial selection is summarized in Table IVand details are given below. 032007-8 PHYS. REV. D 100, 032007 (2019) EARCH FOR ELECTROWEAK DIBOSON PRODUCTION … TABLE IV. Fiducial phase-space definitions used for the measurement of electroweak VVjj production. Object selection Leptons pT > 7 GeV, jηj < 2.5 Small-R jets pT > 20 GeV if jηj < 2.5, and pT > 30 GeV if 2.5 < jηj < 4.5 Large-R jets pT > 200 GeV, jηj < 2.0 Event selection Leptonic V selection 0-lepton Zero leptons, pνν T > 200 GeV 1-lepton One lepton with pT > 27 GeV, pν T > 80 GeV 2-lepton Two leptons, with leading (subleading) lepton pT > 28ð20Þ GeV 83 < mll < 99 GeV Hadronic V selection Merged One large-R jet, minðjmJ −mWj; jmJ −mZjÞ 64 < mJ < 106 GeV Resolved Two small-R jets, minðjmjj −mWj; jmjj −mZjÞ pj1 T > 40 GeV, pj2 T > 20 GeV 64 < mjj < 106 GeV Tagging-jets Two small-R non-b jets, ηtag;j1 · ηtag;j2 < 0, highest mtag jj mtag jj > 400 GeV, ptag;j1;2 T > 30 GeV Number of b-jets 0-lepton 1-lepton 0 2-lepton LE IV. Fiducial phase-space definitions used for the measurement of electroweak VVjj production. Charged leptons are required to satisfy pT > 7 GeV and jηj < 2.5. Jets are clustered from all final-state particles except prompt leptons, prompt neutrinos, and prompt photons using the anti-kt algorithm. Small-R jets are required to have pT > 20 GeV for jηj < 2.5 and pT > 30 GeV for 2.5 < jηj < 4.5. Jets within ΔR ¼ 0.2 of any charged lepton (as defined above) are rejected. Jets con- taining a b-hadron, identified using “truth” information from the MC event record, are labeled as b-jets. Large-R jets are required to have pT > 200 GeV and jηj < 2.0, and the same trimming algorithm as for the reconstruction-level large-R jets is applied. No Dðβ¼1Þ 2 requirement is applied to large-R jets. VIII. SYSTEMATICAL UNCERTAINTIES The lepton pT is required to be larger than 27 GeV for the 1-lepton channel; for the 2-lepton channel, the leading (subleading) lepton pT must be larger than 28 (20) GeV, and the invariant mass of the lepton pair must lie within 83 < mll < 99 GeV. For the 0-lepton channel, the transverse momentum of the neutrino system must satisfy pνν T > 200 GeV; and for the 1-lepton channel, the events are required to have pν T > 80 GeV and contain no b-jets. The efficiencies of the lepton triggers for events with selected leptons are high, nearly 100% in the electron channel and approximately 96% in the muon channel. The corresponding uncertainties are negligible. For the selec- tion used in the 0-lepton and 1-lepton channels, the efficiency of the Emiss T trigger is also close to 100% with negligible associated uncertainty. The modeling of the electron and muon reconstruction, identification and 032007-9 PHYS. REV. D 100, 032007 (2019) G. AAD et al. 0-lepton channel only. The normalization uncertainties in the diboson background cross sections are studied with SHERPA. The uncertainty due to missing higher-order QCD contributions (QCD scale uncertainty) is estimated by varying the renormalization (μR) and factorization (μF) scales independently by a factor ranging from one-half to two with the constraint 0.5 ≤μF=μR ≤2. The PDF uncertainty corresponds to the 68% confidence-level var- iations of the nominal PDF set NNPDF30NNLO, as well as its difference from the alternative PDF sets CT10NNLO [92] and MMHT2014NNLO [93]. The overall normaliza- tion uncertainty for the diboson background is estimated to be about 30%. For single-top-quark events, a 20% nor- malization uncertainty is assigned [94]. isolation efficiencies is studied with a tag-and-probe method using Z →ll events in data and simulation at ﬃﬃﬃs p ¼ 13 TeV [61,62]. Small corrections are applied to the simulation to better model the performance seen in data. These corrections have associated uncertainties of the order of 1%. Uncertainties in the lepton energy (or momentum) scale and resolution [62,89] are also taken into account. Uncertainties in the jet energy scale and resolution for small-radius jets are estimated using MC simulation and in situ techniques [66]. For central jets (jηj < 2.0), the total uncertainty in the jet energy scale ranges from about 6% for jets with pT ¼ 25 GeV to about 2% for pT ¼ 1 TeV. VIII. SYSTEMATICAL UNCERTAINTIES There is also an uncertainty in the jet energy resolution [66], which ranges from 10% to 20% for jets with a pT of 20 GeV to less than 5% for jets with pT > 200 GeV. Uncertainties in the lepton and jet energy scales and resolutions are propagated into the uncertainty in Emiss T . Uncertainties in the energy scale and resolution of the track soft term are also propagated into the uncertainty in Emiss T [79]. For the b-tagging efficiency of small-R jets, correction factors are applied to the simulated event samples in order to compensate for differences between data and simulation. The corrections and uncertainties in the efficiency for tagging b-jets and in the rejection factor for light jets are determined from t¯t samples [90,91]. y g The uncertainty in the modeling of the final discrimi- nants, the BDT output and mtag jj , for background processes estimated using MC simulation is assessed by comparing the nominal MC samples with alternative samples. The uncertainties are of the order of 5%–30%. The mtag jj reweighting as described in Sec. V B is also included as a shape systematic uncertainty for Z þ jets and W þ jets events by taking the difference of their respective final discriminants before and after applying the reweighting. An uncertainty in the shape of the BDT or mtag jj distribution for the t¯t background is derived by comparing the POWHEG-BOX sample with the distribution obtained using MADGRAPH5_AMC@NLO 2.2.2. Additional systematic uncertainties are estimated by comparing the nominal sample showered with PYTHIA 6.428 using the P2012 tune to one showered with Herwig þ þ 2.7.1 [95] and using the UEEE5 underlying-event tune [96]. Samples of t¯t events with the factorization and renormalization scales doubled or halved are compared with the nominal samples, and the observed differences are taken as an additional uncertainty. These modeling uncertainties for the t¯t background are 5%–30%. The shape uncertainty for diboson processes is obtained by comparing MC samples generated by SHERPA and POWHEG-BOX, and it is found to be of the order of 2%–30%. The shape uncertainty for single-top-quark events is ignored due to their relatively small contribution to the total background. The uncertainties in the scale of the large-R jet pT, mass and Dðβ¼1Þ 2 are of the order of 2%–5%. They are estimated using comparisons of data and simulation in Ref. [78]. IX. STATISTICAL ANALYSIS The statistical analysis relies on the profile likelihood test statistic [97] implemented with the RooFit [98] and RooStats [99] packages. A binned likelihood function Lðμ; θÞ is constructed as a product of Poisson probabilities over all of the bins of the fit templates considered in the analysis. This function depends on the signal-strength parameter μ, a multiplicative factor applied to the theo- retical signal production cross section, and θ, a set of nuisance parameters that encodes the effects of systematic uncertainties in the signal and expected backgrounds. The binning is chosen so that the expected numbers of events ensure that the statistical uncertainty is less than 5% in most bins, while finer binning is also allowed in signal-enriched regions. The nuisance parameters are either free to float, or constrained using Gaussian or log-normal terms defined by external studies. The likelihood function for the combina- tion of the three channels is the product of the Poisson likelihoods of the individual channels. However, only one constraint term per common nuisance parameter is included in the product. In general, one SF is introduced for each background component, common to both the SRs and CRs. One common Z þ jets SF is used for both the 0-lepton and 2-lepton channels, and one common W þ jets SF is used for both the 0-lepton and 1-lepton channels. Similarly, one common t¯t SF is used for both the 0-lepton and 1-lepton channels. However, independent SFs are used for the resolved and merged categories, to take into account different MC modelings in the different phase spaces of the same background component. The test statistic qμ is defined as the profile likelihood ratio [100], qμ ¼ −2 ln Λμ with Λμ ¼ Lðμ; ˆˆθμÞ=Lðˆμ; ˆθÞ, where ˆμ and ˆθ are the values of the parameters that maximize the likelihood function (with the constraint 0 ≤ˆμ ≤μ), and ˆˆθμ are the values of the nuisance param- eters that maximize the likelihood function for a given value of μ. The best-fit signal strength ˆμ value (μobs EW VVjj) is obtained by maximizing the likelihood function with respect to all parameters. To determine whether the observed data is compatible with the background-only hypothesis, a test statistic q0 ¼ −2 ln Λ0 is used. p A simultaneous maximum-likelihood fit is performed to the observed distributions of the final discriminants, BDT outputs, in the nine SRs to extract the signal rate informa- tion. VIII. SYSTEMATICAL UNCERTAINTIES An absolute uncertainty of 2% is assigned to the large-R jet energy resolution, and relative uncertainties of 20% and 15% are assigned to the resolution of the large-R jet mass and Dðβ¼1Þ 2 , respectively. 2 p y The overall normalization of the main backgrounds (W þ jets, Z þ jets and t¯t) is determined from the corre- sponding data control regions and is left unconstrained and floating in the global likelihood fit. For W þ jets (Z þ jets) events in the 0-lepton channel, additional normalization uncertainties are considered to account for the acceptance difference between the 0-lepton channel analysis and the 1-lepton (2-lepton) channel analysis, given that there are no corresponding pure control regions of 0-lepton events and the normalization is determined mainly from control regions with 1-lepton (2-lepton) events. This addi- tional normalization uncertainty for W þ jets (Z þ jets) events is estimated using the ratio of the event yield in each signal region of the 0-lepton channel to that in the 1-lepton (2-lepton) channel, and by comparing this ratio obtained from the nominal MC samples generated by SHERPA with the ratio from alternative samples generated by MADGRAPH5_AMC@NLO. The normalization uncer- tainty is 8% (14%) for W þ jets events in the merged (resolved) signal region, and 22% (42%) for Z þ jets events in the merged (resolved) signal region. These uncertainties are applied to the W þ jets and Z þ jets events in the The following discussion describes the uncertainties in the predictions of EW VVjj signal processes. The uncer- tainties in the signal-strength measurement, discussed in Sec. X A, include contributions from both the normaliza- tion and shape; for the fiducial cross section measurement, discussed in Sec. X B, only the shape uncertainties are taken into account for the measured fiducial cross sections, and the normalization uncertainties are included for the SM predicted fiducial cross sections. Theoretical uncertainties for EW VVjj signal pro- cesses include the PDF choice, the missing higher-order corrections, and the parton-shower modeling. The signal modeling uncertainty due to PDF uncertainties is estimated by taking the uncertainty from the PDF error sets of 032007-10 PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … NNPDF23LO and adding it in quadrature to the acceptance difference obtained using alternative PDF sets: CT10 and MMHT2014LO. The PDF uncertainties are estimated to be 3%–5%. VIII. SYSTEMATICAL UNCERTAINTIES The parton-shower uncertainty, estimated by varying relevant parameters in the A14-NNPDF tune [33], ranges from 1% to 5%. The effect of the QCD scale uncertainty, of the order of 1%–3%, is estimated by varying the factorization and renormalization scales independently by a factor of 2 with the constraint 0.5 ≤μF=μR ≤2. TABLE V. The distributions used in the global likelihood fit for the signal regions and control regions for all the categories in each channel. “One bin” implies that a single bin without any shape information is used in the corresponding fit region. Discriminants Regions Merged high-purity Merged low-purity Resolved 0-lepton SR BDT BDT BDT VjjCR mtag jj mtag jj mtag jj 1-lepton SR BDT BDT BDT WCR mtag jj mtag jj mtag jj TopCR One bin One bin One bin 2-lepton SR BDT BDT BDT ZCR mtag jj mtag jj mtag jj y μF=μR The interference between EW- and QCD-induced VVjj processes is not included in the MC simulation, since the EW- and QCD-induced VVjj samples were generated separately. The interference effect is considered as an uncertainty affecting both the normalization and the shape of the EW VVjj kinematic distributions. The effect is determined using the MADGRAPH5_AMC@NLO 2.4.3 MC generator at the truth level as a function of mtag jj . A reweighting is then applied to the simulated EW VVjj samples, resulting in shape uncertainties of 5% to 10% at low and high values of the BDT score, respectively, and a similar size for the normalization uncertainties. summarized in Table V. Signal and background contribu- tions, including their shapes in the signal and control regions, are taken from MC simulations. For each source of systematic uncertainty, the correlations across bins of BDT distributions are taken into account and are fully correlated. The correlations between different regions, as well as those between signal and background, are also included. Moreover, normalization scale factors (SFs) are applied to the MC estimates of the Z þ jets, W þ jets and top quark contributions. These SFs are free parameters in the fit and are therefore constrained by the data in both the signal and control regions. The diboson contribution is constrained to the theoretical estimate within the corre- sponding uncertainties. IX. STATISTICAL ANALYSIS The signal is shown as a filled histogram malized to the signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, gend. The size of the combined statistical and systematic uncertainty for the sum of the fitted signa atched band. The middle pane shows the ratios of the observed data to the postfit signal and back shows the ratios of the postfit and pre-fit background predictions. Events / 100 GeV Postfit/Prefit [GeV] tag jj m V ζ 3 2 1 0 1 2 3 4 Events / 0.5 100 200 300 400 500 600 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 V ζ 3 − 2 − 1 − 0 1 2 3 4 Postfit/Prefit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Events / 100 GeV 1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Postfit/Prefit 0.5 1 1.5 Events / 0.5 Postfit/Prefit V ζ 3 2 1 0 1 2 3 4 Events / 0.5 20 40 60 80 100 120 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 V ζ 3 − 2 − 1 − 0 1 2 3 4 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 1 − 10 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged high-purity SR Data/Postfit 1 2 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 1 2 Events / 0.5 Postfit/Prefit FIG. 2. IX. STATISTICAL ANALYSIS [GeV] miss T E 200 300 400 500 600 700 800 Events / 100 GeV 1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Events / 100 GeV 1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Postfit/Prefit 0.5 1 1.5 V ζ 3 2 1 0 1 2 3 4 Events / 0.5 100 200 300 400 500 600 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 V ζ 3 − 2 − 1 − 0 1 2 3 4 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 1 − 10 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged high-purity SR Data/Postfit 1 2 [G V] tag 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 1 2 V ζ 3 2 1 0 1 2 3 4 Events / 0.5 20 40 60 80 100 120 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 ζ 3 − 2 − 1 − 0 1 2 3 4 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Events / 100 GeV 1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Postfit/Prefit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Events / 100 GeV 1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Events / 100 GeV 1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Postfit/Prefit 0.5 1 1.5 V ζ 3 2 1 0 1 2 3 4 Events / 0.5 100 200 300 400 500 600 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 V ζ 3 − 2 − 1 − 0 1 2 3 4 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 1 − 10 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged high-purity SR Data/Postfit 1 2 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 1 2 V ζ 3 2 1 0 1 2 3 4 Events / 0.5 20 40 60 80 100 120 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 V ζ 3 − 2 − 1 − 0 1 2 3 4 Postfit/Prefit 0.5 1 1.5 ributions for Emiss T (top left), mtag jj (top right), mVVjj (middle left), ζV (middle right), mtag jj (bottom le on (top), 1-lepton (middle) and 2-lepton (bottom) channels for the high-purity merged signal regio r the global likelihood fit are shown as filled histograms. IX. STATISTICAL ANALYSIS The three ZCRs, WCRs and TopCRs as well as the three VjjCRs are included in the fit’s likelihood calculation; the mtag jj distributions are used for ZCRs, WCRs and VjjCRs, while for the TopCRs only one bin for each of the three Vhad decay channels is used. The purpose of using mtag jj distributions for CRs is to constrain the mtag jj reweight- ing systematic uncertainties. The different regions and the corresponding discriminants entering the likelihood fit are 032007-11 PHYS. REV. D 100, 032007 (2019) G. AAD et al. IX. STATISTICAL ANALYSIS The distributions for Emiss T (top left), mtag jj (top right), mVVjj (middle left), ζV (middle right), mtag jj (bottom left), and ζV (bottom right) in the 0-lepton (top), 1-lepton (middle) and 2-lepton (bottom) channels for the high-purity merged signal region. The background contributions after the global likelihood fit are shown as filled histograms. The signal is shown as a filled histogram on top of the fitted backgrounds normalized to the signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. The size of the combined statistical and systematic uncertainty for the sum of the fitted signal and background is indicated by the hatched band. The middle pane shows the ratios of the observed data to the postfit signal and background predictions. The bottom pane shows the ratios of the postfit and pre-fit background predictions. 032007-12 032007-12 lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 10 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Events / 10 GeV 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Postfit/Prefit 0.5 1 1.5 ibutions for Emiss T (top left), mtag jj (top right), mVVjj (middle left), pj2 T (middle right), mtag jj (bottom le pton (top), 1-lepton (middle) and 2-lepton (bottom) channels for the resolved signal region the global likelihood fit are shown as filled histograms. The signal is shown as a filled histogram malized to the signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, gend. The size of the combined statistical and systematic uncertainty for the sum of the fitted signal atched band. The middle pane shows the ratios of the observed data to the postfit signal and backg shows the ratios of the postfit and prefit background predictions. 032007-12 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 10 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Events / 100 GeV 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m [GeV] miss T E [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Events / 10 GeV 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Postfit/Prefit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Events / 100 GeV 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. 032007-12 PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … [GeV] miss T E 200 300 400 500 600 700 800 Events / 100 GeV 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 10 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Events / 100 GeV 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Postfit/Prefit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Events / 10 GeV 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. 032007-12 lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Postfit/Prefit 0.5 1 1.5 Events / 100 GeV 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Resolved SR Events / 10 GeV 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Resolved SR [GeV] miss T E 200 300 400 500 600 700 800 Events / 100 GeV 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] miss T E 200 300 400 500 600 700 800 Postfit/Prefit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 10 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Events / 100 GeV 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Postfit/Prefit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Events / 10 GeV 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. 032007-12 lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] VVjj m 1000 1500 2000 2500 3000 3500 4000 4500 Postfit/Prefit 0.5 1 1.5 Events / 10 GeV [GeV] VVjj m [GeV] 2j T p [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Events / 100 GeV 10 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] tag jj m 500 1000 1500 2000 2500 3000 3500 4000 Postfit/Prefit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Events / 10 GeV 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Resolved SR Data/Postfit 0.5 1 1.5 [GeV] 2j T p 20 40 60 80 100 120 140 160 180 200 Postfit/Prefit 0.5 1 1.5 Events / 10 GeV [GeV] 2j T p 100 120 1 FIG. 3. The distributions for Emiss T (top left), mtag jj (top right), mVVjj (middle left), pj2 T (middle right), mtag jj (bottom left), and pj2 T (bottom right) in the 0-lepton (top), 1-lepton (middle) and 2-lepton (bottom) channels for the resolved signal region. The background contributions after the global likelihood fit are shown as filled histograms. The signal is shown as a filled histogram on top of the fitted backgrounds normalized to the signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. The size of the combined statistical and systematic uncertainty for the sum of the fitted signal and background is indicated by the hatched band. The middle pane shows the ratios of the observed data to the postfit signal and background predictions. The bottom pane shows the ratios of the postfit and prefit background predictions. 032007-13 032007-13 032007-13 PHYS. REV. D 100, 032007 (2019) G. AAD et al. BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (a) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged low-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (b) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 Events / 0.1 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Resolved SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 Postfit/Prefit 0.5 1 1.5 (c) Comparisons of the observed data and expected distributions of the BDT outputs of the 0-lepton channel signal regions: purity and (b) low-purity merged signal regions; (c) the resolved signal region. The background contributions after the global d fit are shown as filled histograms. The signal is shown as a filled histogram on top of the fitted backgrounds normalized to the eld extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. The overflow are included in the last bin. The middle pane shows the ratios of the observed data to the postfit signal and nd predictions. The uncertainty in the total prediction, shown as bands, combines statistical and systematic contributions. The ane shows the ratios of the postfit and prefit background predictions. 032007-13 BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (a) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Merged low-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (b) Events / 0.1 (b) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 Events / 0.1 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z +jets W Top Quarks Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 0-lep., Resolved SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 Postfit/Prefit 0.5 1 1.5 (c) (c) FIG. 4. Comparisons of the observed data and expected distributions of the BDT outputs of the 0-lepton channel signal regions: (a) high-purity and (b) low-purity merged signal regions; (c) the resolved signal region. The background contributions after the global likelihood fit are shown as filled histograms. The signal is shown as a filled histogram on top of the fitted backgrounds normalized to the signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. The entries in overflow are included in the last bin. The middle pane shows the ratios of the observed data to the postfit signal and background predictions. The uncertainty in the total prediction, shown as bands, combines statistical and systematic contributions. The bottom pane shows the ratios of the postfit and prefit background predictions. X. RESULTS contributions shown are obtained from the signal-plus- background fits described previously. A. Results for the EW VVjj production processes SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … D 100, 032007 (2019 BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 Events / 0.1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 Postfit/Prefit 0.5 1 1.5 (a) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 Events / 0.1 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged low-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 Postfit/Prefit 0.5 1 1.5 (b) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 Events / 0.1 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged low-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 Postfit/Prefit 0.5 1 1.5 (b) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 Events / 0.1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 Postfit/Prefit 0.5 1 1.5 (a) Events / 0.1 (b) (a) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 Events / 0.1 2 10 3 10 4 10 5 10 6 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 Postfit/Prefit 0.5 1 1.5 (c) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 Events / 0.1 2 10 3 10 4 10 5 10 6 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 Postfit/Prefit 0.5 1 1.5 (c) FIG. 5. Comparisons of the observed data and expected distributions of the BDT outputs of the 1-lepton channel signal regions: (a) high-purity and (b) low-purity merged signal regions; (c) the resolved signal region. The background contributions after the global likelihood fit are shown as filled histograms. The signal is shown as a filled histogram on top of the fitted backgrounds normalized to the signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. The entries in overflow are included in the last bin. The middle pane shows the ratios of the observed data to the postfit signal and background predictions. The uncertainty in the total prediction, shown as bands, combines statistical and systematic contributions. The bottom pane shows the ratios of the postfit and prefit background predictions. (c) FIG. 5. Comparisons of the observed data and expected distributions of the BDT outputs of the 1-lepton channel signal regions: (a) high-purity and (b) low-purity merged signal regions; (c) the resolved signal region. The background contributions after the global likelihood fit are shown as filled histograms. The signal is shown as a filled histogram on top of the fitted backgrounds normalized to the signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. The entries in overflow are included in the last bin. The middle pane shows the ratios of the observed data to the postfit signal and background predictions. The uncertainty in the total prediction, shown as bands, combines statistical and systematic contributions. A. Results for the EW VVjj production processes The observed distributions of the BDT outputs in SRs used in the global likelihood fit are compared with the predictions, shown in Fig. 4 for the 0-lepton channel, Fig. 5 for the 1-lepton channel, and Fig. 6 for the 2-lepton channel. The data distributions are reasonably well Figures 2 and 3 show a selection of representative postfit distributions of input variables that are most discriminating for each of the lepton channels, for the merged and resolved categories, respectively. Background and EW VVjj signal 032007-14 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … PHYS. REV. D 100, 032007 (2019) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 Events / 0.1 10 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 Postfit/Prefit 0.5 1 1.5 (a) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 Events / 0.1 2 10 3 10 4 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Merged low-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 Postfit/Prefit 0.5 1 1.5 (b) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 Events / 0.1 2 10 3 10 4 10 5 10 6 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets W Mis-id. lepton Top Quarks +jets Z Diboson Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 1-lep., Resolved SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 Postfit/Prefit 0.5 1 1.5 (c) FIG. 5. Comparisons of the observed data and expected distributions of the BDT outputs of the 1-lepton channel signal region (a) high-purity and (b) low-purity merged signal regions; (c) the resolved signal region. The background contributions after the glob likelihood fit are shown as filled histograms. The signal is shown as a filled histogram on top of the fitted backgrounds normalized to th signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. Th entries in overflow are included in the last bin. The middle pane shows the ratios of the observed data to the postfit signal an background predictions. The uncertainty in the total prediction, shown as bands, combines statistical and systematic contributions. Th bottom pane shows the ratios of the postfit and prefit background predictions. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … PHYS. REV. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … The signal is shown as a filled histogram on top of the fitted backgrounds normalized to the ld extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. The overflow are included in the last bin. The middle pane shows the ratios of the observed data to the postfit signal and nd predictions. The uncertainty in the total prediction, shown as bands, combines statistical and systematic contributions. The ane shows the ratios of the postfit and prefit background predictions. BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (a) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged low-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (b) (b) BDT 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 10 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Resolved SR Data/Postfit 0.5 1 1.5 BDT 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (c) (c) FIG. 6. Comparisons of the observed data and expected distributions of the BDT outputs of the 2-lepton channel signal regions: (a) high-purity and (b) low-purity merged signal regions; (c) the resolved signal region. The background contributions after the global likelihood fit are shown as filled histograms. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … The bottom pane shows the ratios of the postfit and prefit background predictions. for the 1-lepton channel, and Table VIII for the 2-lepton channel. The fitted value of the signal strength is reproduced by the predicted contributions in all cases, with the smallest p-value of 0.16 from the χ2 test [101] being for the mtag jj distribution in the merged high-purity ZCR. The numbers of events observed and estimated in the SRs are summarized in Table VI for the 0-lepton channel, Table VII μobs EW VVjj ¼ 1.05þ0.42 −0.40 ¼ 1.05 0.20ðstat:Þþ0.37 −0.34ðsyst:Þ: 032007-15 PHYS. REV. D 100, 032007 (2019) G. AAD et al. BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged high-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (a) BDT 1 0 8 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 1 10 2 10 3 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Merged low-purity SR Data/Postfit 0.5 1 1.5 BDT 1 − 0.8 − 0.6 − 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (b) BDT 0 4 0 2 0 0 2 0 4 0 6 0 8 Events / 0.1 10 2 10 3 10 4 10 5 10 Data = 1.05) μ ( VVjj EW 30 × VVjj EW +jets Z Diboson Top Quarks +jets W Uncertainty ATLAS -1 = 13 TeV, 35.5 fb s 2-lep., Resolved SR Data/Postfit 0.5 1 1.5 BDT 0.4 − 0.2 − 0 0.2 0.4 0.6 0.8 Postfit/Prefit 0.5 1 1.5 (c) Comparisons of the observed data and expected distributions of the BDT outputs of the 2-lepton channel signal regions: urity and (b) low-purity merged signal regions; (c) the resolved signal region. The background contributions after the global d fit are shown as filled histograms. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … TABLE VI. Numbers of events observed and predicted for signal and background processes in the 0-lepton channel signal regions, obtained from signal-plus-background fits to the signal and control regions (Sec. X). The signal yields are calculated after the fit with the observed signal strength of 1.05 applied. The uncertainties combine statistical and systematic contributions. The fit constrains the background estimate towards the observed data, which reduces the total background uncertainty by correlating those uncertainties from the individual backgrounds. Sample Resolved Merged HP Merged LP Background W þ jets 9200 1300 259 27 582 56 Z þ jets 19 000 1400 383 29 955 69 Top quarks 3280 480 277 28 276 32 Diboson 720 120 69 12 68 14 Total 32 100 2000 988 50 1881 96 Signal WðlνÞWðqq0Þ 56 22 8.0 3.2 5.4 2.2 WðlνÞZðqqÞ 12.0 4.7 2.1 0.8 1.6 0.6 ZðννÞWðqq0Þ 66 25 9.0 3.5 7.4 2.9 ZðννÞZðqqÞ 27 10 5.1 2.0 3.1 1.2 Total 161 35 24.3 5.2 17.5 3.9 SM 32 300 2000 1012 50 1898 96 Data 32 299 1002 1935 TABLE VII. Numbers of events observed and predicted for signal and background processes in the 1-lepton channel signal regions, obtained from signal-plus-background fits to the signal and control regions (Sec. X). The signal yields are calculated after the fit with the observed signal strength of 1.05 applied. The uncertainties combine statistical and systematic contributions. The fit constrains the background estimate towards the observed data, which reduces the total background uncertainty by correlating those uncertainties from the individual backgrounds. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … The signal is shown as a filled histogram on top of the fitted backgrounds normalized to the signal yield extracted from data (μ ¼ 1.05), and unstacked as an unfilled histogram, scaled by the factor indicated in the legend. The entries in overflow are included in the last bin. The middle pane shows the ratios of the observed data to the postfit signal and background predictions. The uncertainty in the total prediction, shown as bands, combines statistical and systematic contributions. The bottom pane shows the ratios of the postfit and prefit background predictions. The background-only hypothesis is excluded in data with a significance of 2.7 standard deviations, compared with 2.5 standard deviations expected. signal-strength parameter. The probability that the signal strengths measured in the three lepton channels are com- patible is 36%. Figure 7 shows the measured signal strength from the combined fit with a single signal-strength fit parameter, and from a fit where each lepton channel has its own After the global maximum-likelihood fit, the uncertain- ties described in Sec. VIII are much reduced. The effects of systematic uncertainties on the measurement after the fit are 032007-16 PHYS. REV. D 100, 032007 (2019) EARCH FOR ELECTROWEAK DIBOSON PRODUCTION … SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Sample Resolved Merged HP Merged LP Background W þ jets 69 100 1900 1201 65 2828 97 Z þ jets 2770 370 39 3 83 6 Top quarks 7100 1100 394 56 422 63 Diboson 2660 600 163 35 229 57 Multijet 13 400 1600 Total 95 100 2800 1797 93 3560 130 Signal WðlνÞWðqq0Þ 330 120 45 17 34 13 WðlνÞZðqqÞ 78 29 11 4 5 2 Total 410 130 57 18 39 13 SM 95 500 2800 1854 95 3600 130 Data 95 366 1864 3571 TABLE VIII. Numbers of events observed and predicted for signal and background processes in the 2-lepton channel signal regions, obtained from signal-plus-background fits to the signal and control regions (Sec. X). The signal yields are calculated after the fit with the observed signal strength of 1.05 applied. The uncertainties combine statistical and systematic contributions. The fit constrains the background estimate towards the observed data, which reduces the total background uncertainty by correlating those uncertainties from the individual backgrounds. Sample Resolved Merged HP Merged LP Background Z þ jets 37 090 310 331 14 775 24 Top quarks 645 99 5.8 0.9 9.9 2.7 Diboson 830 170 34.6 7.6 36.7 8.2 Total 38 570 370 371 16 821 25 Signal ZðllÞWðqq0Þ 138 53 8.6 3.3 7.0 2.7 ZðllÞZðqqÞ 46 18 4.3 1.7 2.9 1.1 Total 185 56 12.9 3.7 9.8 2.9 SM 38 760 370 384 17 831 25 Data 38 734 371 810 PHYS. REV. D 100, 032007 (2019) G. AAD et al. SM σ / σ = μ Best fit 0 2 4 6 8 10 Combination 0-lepton 1-lepton 2-lepton 1.05 0.40 − 0.42 + ( 0.34 − 0.37 + 0.20 ± ) 2.47 1.22 − 1.33 + ( 0.93 − 1.05 + 0.80 ± ) 0.33 0.52 − 0.53 + ( 0.46 − 0.47 + 0.25 ± ) 1.97 0.77 − 0.83 + ( 0.59 − 0.65 + 0.50 ± ) Tot. ( Stat. Syst. ) Tot. Stat. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … The fitted values of the signal-strength parameter μobs EW VVjj for the 0-, 1- and 2-lepton channels and their combi- nation. The individual μobs EW VVjj values for the lepton channels are obtained from a simultaneous fit with the signal-strength param- eter for each of the lepton channels floating independently. The probability that the signal strengths measured in the three lepton channels are compatible is 36%. detailed in Sec. VIII are combined into categories. Apart from the statistics of the data, the uncertainties with the largest impact on the sensitivity of EW VVjj production are from the modeling of background (Z þ jets, W þ jets and QCD-induced diboson processes), the modeling of the signal, b-tagging, and reconstruction of small-R and large-R jets. studied using the signal-strength parameter μobs EW VVjj. The relative uncertainties in the best-fit μobs EW VVjj value from the leading sources of systematic uncertainty are shown in Table IX. The individual sources of systematic uncertainty SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … ATLAS , Observed -1 =13 TeV, 35.5 fb s SM σ / σ = μ Best fit 0 2 4 6 8 10 Combination 0-lepton 1-lepton 2-lepton 1.05 0.40 − 0.42 + ( 0.34 − 0.37 + 0.20 ± ) 2.47 1.22 − 1.33 + ( 0.93 − 1.05 + 0.80 ± ) 0.33 0.52 − 0.53 + ( 0.46 − 0.47 + 0.25 ± ) 1.97 0.77 − 0.83 + ( 0.59 − 0.65 + 0.50 ± ) Tot. ( Stat. Syst. ) Tot. Stat. ATLAS , Observed -1 =13 TeV, 35.5 fb s FIG. 7. The fitted values of the signal-strength parameter μobs EW VVjj for the 0-, 1- and 2-lepton channels and their combi- nation. The individual μobs EW VVjj values for the lepton channels are obtained from a simultaneous fit with the signal-strength param- eter for each of the lepton channels floating independently. The probability that the signal strengths measured in the three lepton channels are compatible is 36%. TABLE X. Summary of predicted and measured fiducial cross sections for EW VVjj production. The three lepton channels are combined. For the measured fiducial cross sections in the merged and resolved categories, two signal-strength parameters are used in the combined fit, one for the merged category and the other one for the resolved category; while for the measured fiducial cross section in the inclusive fiducial phase space, a single signal- strength parameter is used. For the SM predicted cross section, the error is the theoretical uncertainty (theo.). For the measured cross section, the first error is the statistical uncertainty (stat.), and the second error is the systematic uncertainty (syst.). Fiducial phase space Predicted σfid;SM EW VVjj [fb] Measured σfid;obs EW VVjj [fb] Merged 11.4 0.7 (theo.) 12.7 3.8ðstat:Þþ4.8 −4.2ðsyst:Þ Resolved 31.6 1.8 (theo.) 26.5 8.2ðstat:Þþ17.4 −17.1ðsyst:Þ Inclusive 43.0 2.4 (theo.) 45.1 8.6ðstat:Þþ15.9 −14.6ðsyst:Þ FIG. 7. The fitted values of the signal-strength parameter μobs EW VVjj for the 0-, 1- and 2-lepton channels and their combi- nation. The individual μobs EW VVjj values for the lepton channels are obtained from a simultaneous fit with the signal-strength param- eter for each of the lepton channels floating independently. The probability that the signal strengths measured in the three lepton channels are compatible is 36%. FIG. 7. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … We acknowledge the support of ANPCyT, Argentina; Yerevan Physics Institute (YerPhI), Armenia; ARC, Australia; BMWFW and FWF, Austria; Azerbaijan National Academy of Sciences (ANAS), Azerbaijan; State Science and Technology Committee (SSTC), Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and Committee for Collaboration of the Czech Republic with CERN (VSC CR), Czech Republic; DNRF and Danish Natural Science Research Council (DNSRC), Denmark; IN2P3-CNRS, CEA-DRF/ IRFU, France; Shota Rustaveli National Science Foundation of Georgia (SRNSFG), Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; Research Council of Norway (RCN), Norway; MNiSW and NCN, Poland; FCT, Portugal; Ministry of National Education, Institute of Atomic Physics (MNE/IFA), Romania; Ministry of Education and Science of the Russian Federation (MES) of Russia and National Research Centre Kurchatov Institute, Russian Federation; JINR; Ministry of Education, Science and Technological Development (MESTD), Serbia; Ministry of Education, Science, Research and Sport (MSSR), Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; Swedish Research Council (SRC) and Wallenberg Foundation, Sweden; Secretariat for Education and Research (SERI), SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, individual groups and members have received support from BCKDF, also reported in Table XI. The measured values are obtained from a simultaneous fit where each lepton channel has its own signal-strength parameter, and in each lepton channel the same signal-strength parameter is applied to both the merged and resolved categories. The predictions are from MADGRAPH5_AMC@NLO 2.4.3 at LO only, and no higher order corrections are included; the theoretical uncertainties due to the PDF, missing higher-order correc- tions, and parton-shower modeling are estimated as described in Sec. VIII. The measured fiducial cross sections are generally consistent with the SM predictions. XI. CONCLUSION A measurement of VVjj (V ¼ W, Z) electroweak production using ﬃﬃﬃs p ¼ 13 TeV pp collisions at the LHC is presented. The data were collected with the ATLAS detector in 2015 and 2016 and correspond to a total integrated luminosity of 35.5 fb−1. The study explores the final states with one boson decaying leptonically, and the other boson decaying into a pair of quarks, identified either as two separate jets or as one large-radius jet. The VVjj electroweak production cross section is measured with a significance of 2.7 standard deviations over the background-only hypothesis. The expected sig- nificance is 2.5 standard deviations. The measured signal strength relative to the leading-order SM prediction is μobs EW VVjj ¼ 1.05 0.20ðstat:Þþ0.37 −0.34ðsyst:Þ. The fiducial cross section of VVjj electroweak production is measured to be σfid;obs EW VVjj ¼ 45.1 8.6ðstat:Þþ15.9 −14.6ðsyst:Þ fb. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … TABLE XI. Summary of predicted and measured fiducial cross sections for EW VVjj production in the three lepton channels. The measured values are obtained from a simultaneous fit where each lepton channel has its own signal-strength parameter, and in each lepton channel the same signal-strength parameter is applied to both the merged and resolved categories. For the SM predicted cross section, the error is the theoretical uncertainty (theo.). For the measured cross section, the first error is the statistical uncertainty (stat.), and the second error is the systematic uncertainty (syst.). Fiducial phase space Predicted σfid;SM EW VVjj [fb] Measured σfid;obs EW VVjj [fb] Merged 0-lepton 4.1 0.3 (theo.) 10.1 3.3ðstat:Þþ4.2 −3.8ðsyst:Þ 1-lepton 6.1 0.5 (theo.) 2.0 1.5ðstat:Þþ2.9 −2.8ðsyst:Þ 2-lepton 1.2 0.1 (theo.) 2.4 0.6ðstat:Þþ0.8 −0.7ðsyst:Þ Resolved 0-lepton 9.2 0.6 (theo.) 22.8 7.4ðstat:Þþ9.4 −8.5ðsyst:Þ 1-lepton 16.4 1.0 (theo.) 5.5 4.1ðstat:Þþ7.7 −7.5ðsyst:Þ 2-lepton 6.0 0.4 (theo.) 11.8 3.0ðstat:Þþ3.8 −3.5ðsyst:Þ Inclusive 0-lepton 13.3 0.8 (theo.) 32.9 10.7ðstat:Þþ13.5 −12.3ðsyst:Þ 1-lepton 22.5 1.5 (theo.) 7.5 5.6ðstat:Þþ10.5 −10.2ðsyst:Þ 2-lepton 7.2 0.4 (theo.) 14.2 3.6ðstat:Þþ4.6 −4.2ðsyst:Þ TABLE XI. Summary of predicted and measured fiducial cross sections for EW VVjj production in the three lepton channels. The measured values are obtained from a simultaneous fit where each lepton channel has its own signal-strength parameter, and in each lepton channel the same signal-strength parameter is applied to both the merged and resolved categories. For the SM predicted cross section, the error is the theoretical uncertainty (theo.). For the measured cross section, the first error is the statistical uncertainty (stat.), and the second error is the systematic uncertainty (syst.). Fiducial phase space Predicted σfid;SM EW VVjj [fb] Measured σfid;obs EW VVjj [fb] Merged 0-lepton 4.1 0.3 (theo.) 10.1 3.3ðstat:Þþ4.2 −3.8ðsyst:Þ 1-lepton 6.1 0.5 (theo.) 2.0 1.5ðstat:Þþ2.9 −2.8ðsyst:Þ 2-lepton 1.2 0.1 (theo.) 2.4 0.6ðstat:Þþ0.8 −0.7ðsyst:Þ Resolved 0-lepton 9.2 0.6 (theo.) 22.8 7.4ðstat:Þþ9.4 −8.5ðsyst:Þ 1-lepton 16.4 1.0 (theo.) 5.5 4.1ðstat:Þþ7.7 −7.5ðsyst:Þ 2-lepton 6.0 0.4 (theo.) 11.8 3.0ðstat:Þþ3.8 −3.5ðsyst:Þ Inclusive 0-lepton 13.3 0.8 (theo.) 32.9 10.7ðstat:Þþ13.5 −12.3ðsyst:Þ 1-lepton 22.5 1.5 (theo.) 7.5 5.6ðstat:Þþ10.5 −10.2ðsyst:Þ 2-lepton 7.2 0.4 (theo.) 14.2 3.6ðstat:Þþ4.6 −4.2ðsyst:Þ institutions without whom ATLAS could not be operated efficiently. B. Cross-section measurements The determination of the fiducial cross section is performed by scaling the measured signal strengths with the corresponding SM predicted fiducial cross sections, σfid;obs EW VVjj ¼ μobs EW VVjj · σfid;SM EW VVjj. It is assumed that there is no new physics that could cause sizable kinematic mod- ifications of the background and signal. Therefore, the only new physics signals that can be detected in an unbiased way are those leading to an enhanced EW VVjj signal strength in the search region of this analysis. The fiducial cross sections for EW VVjj are measured in the merged and resolved fiducial phase-space regions described in Sec. VII and inclusively. The merged HP SR and LP SR are combined to form one single merged fiducial phase-space region. The systematic uncertainties of the measured fiducial cross sections include contributions from exper- imental systematic uncertainties, theory modeling uncer- tainties in the backgrounds, theory modeling uncertainties in the shapes of signal kinematic distributions, and lumi- nosity uncertainties. The measured and SM predicted fiducial cross sections for EW VVjj processes are sum- marized in Table X, where the measured values are obtained from two different simultaneous fits. In the first fit, two signal-strength parameters are used, one for the merged category (both HP and LP), and the other one for the resolved category; while in the second fit, a single signal-strength parameter is used. The measured and SM predicted fiducial cross sections in each lepton channel are TABLE IX. The symmetrized uncertainty σμ from each source in the best-fit signal-strength parameter μobs EW VVjj. The floating normalizations include uncertainties of normalization scale fac- tors for Z þ jets, W þ jets and top quark contributions. normalizations include uncertainties of normalization scale fac- tors for Z þ jets, W þ jets and top quark contributions. Uncertainty source σμ Total uncertainty 0.41 Statistical 0.20 Systematic 0.35 Theoretical and modeling uncertainties Floating normalizations 0.09 Z þ jets 0.13 W þ jets 0.09 t¯t 0.06 Diboson 0.09 Multijet 0.04 Signal 0.07 MC statistics 0.17 Experimental uncertainties Large-R jets 0.08 Small-R jets 0.06 Leptons 0.02 Emiss T 0.04 b-tagging 0.07 Pileup 0.04 Luminosity 0.03 032007-18 PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … ACKNOWLEDGMENTS [5] ATLAS Collaboration, Measurements of the Higgs boson production and decay rates and coupling strengths using pp collision data at ﬃﬃﬃs p ¼ 7 and 8 TeV in the ATLAS experiment, Eur. Phys. J. C 76, 6 (2016). 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Arcangeletti,51 A. T. H. Arce,49 F. A. Arduh,88 J-F. Arguin,109 S. Argyropoulos,77 J.-H. Arling,46 A. J. Armbruster,36 L. J. Armitage,92 A. Armstrong,171 O. Arnaez,167 H. Arnold,120 A. Artamonov,111,a G. Artoni,135 S. Artz,99 S. Asai,163 N. Asbah,59 E. M. Asimakopoulou,172 L. Asquith,156 K. Assamagan,29 R. Astalos,28a R. J. Atkin,33a M. Atkinson,173 N. B. Atlay,151 H. Atmani,132 K. Augsten,142 G. Avolio,36 R. Avramidou,60a M. K. Ayoub,15a A. M. Azoulay,168b G. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Azuelos,109,e M. J. Baca,21 H. Bachacou,145 K. Bachas,67a,67b M. Backes,135 F. Backman,45a,45b P. Bagnaia,72a,72b M. Bahmani,84 H. Bahrasemani,152 A. J. Bailey,174 V. R. Bailey,173 J. T. Baines,144 M. Bajic,40 C. Bakalis,10 O. K. Baker,183 P. J. Bakker,120 D. Bakshi Gupta,8 S. Balaji,157 E. M. Baldin,122b,122a P. Balek,180 F. Balli,145 W. K. Balunas,135 J. Balz,99 E. Banas,84 A. Bandyopadhyay,24 Sw. Banerjee,181,f A. A. E. Bannoura,182 L. Barak,161 W. M. Barbe,38 E. L. Barberio,104 D. Barberis,55b,55a M. Barbero,101 T. Barillari,115 M-S. Barisits,36 J. Barkeloo,131 T. Barklow,153 R. Barnea,160 S. L. Barnes,60c G. Aad,101 B. Abbott,128 D. C. Abbott,102 O. Abdinov,13,a A. Abed Abud,70a,70b K. Abeling,53 D. K. Abhayasinghe,93 S. H. Abidi,167 O. S. AbouZeid,40 N. L. Abraham,156 H. Abramowicz,161 H. Abreu,160 Y. Abulaiti,6 B. S. Acharya,66a,66b,b B. Achkar,53 S. Adachi,163 L. Adam,99 C. Adam Bourdarios,132 L. Adamczyk,83a L. Adamek,167 J. Adelman,121 M. Adersberger,114 A. Adiguzel,12c,c S. Adorni,54 T. Adye,144 A. A. Affolder,146 Y. Afik,160 C. Agapopoulou,132 M. N. Agaras,38 A. Aggarwal,119 C. Agheorghiesei,27c J. A. Aguilar-Saavedra,140f,140a,d F. Ahmadov,79 X. Ai,15a G. Aielli,73a,73b S. Akatsuka,85 T. P. A. Åkesson,96 E. Akilli,54 A. V. Akimov,110 K. Al Khoury,132 G. L. Alberghi,23b,23a J. Albert,176 M. J. Alconada Verzini,88 S. Alderweireldt,119 M. Aleksa,36 I. N. Aleksandrov,79 C. Alexa,27b D. Alexandre,19 T. Alexopoulos,10 A. Alfonsi,120 M. Alhroob,128 B. Ali,142 G. Alimonti,68a J. Alison,37 S. P. Alkire,148 C. Allaire,132 B. M. M. Allbrooke,156 B. W. Allen,131 P. P. Allport,21 A. Aloisio,69a,69b A. Alonso,40 F. Alonso,88 C. Alpigiani,148 A. A. Alshehri,57 M. Alvarez Estevez,98 B. Alvarez Gonzalez,36 D. Álvarez Piqueras,174 M. G. Alviggi,69a,69b Y. Amaral Coutinho,80b A. Ambler,103 L. Ambroz,135 C. Amelung,26 D. Amidei,105 S. P. Amor Dos Santos,140a S. Amoroso,46 C. S. Amrouche,54 F. An,78 C. Anastopoulos,149 N. Andari,145 T. Andeen,11 C. F. Anders,61b J. K. Anders,20 A. Andreazza,68a,68b V. Andrei,61a C. R. Anelli,176 S. Angelidakis,38 A. Angerami,39 A. V. Anisenkov,122b,122a A. Annovi,71a C. Antel,61a M. T. Anthony,149 M. Antonelli,51 D. J. A. Antrim,171 F. Anulli,72a M. Aoki,81 J. A. Aparisi Pozo,174 L. Aperio Bella,36 G. Arabidze,106 J. P. Araque,140a V. Araujo Ferraz,80b R. Araujo Pereira,80b C. Arcangeletti,51 A. T. H. Arce,49 F. A. Arduh,88 J-F. Arguin,109 S. Argyropoulos,77 J.-H. Arling,46 A. J. Armbruster,36 L. J. Armitage,92 A. Armstrong,171 O. Arnaez,167 H. Arnold,120 A. Artamonov,111,a G. Artoni,135 S. Artz,99 S. Asai,163 N. Asbah,59 E. M. Asimakopoulou,172 L. Asquith,156 K. Assamagan,29 R. Astalos,28a R. J. Atkin,33a M. Atkinson,173 N. B. Atlay,151 H. Atmani,132 K. Augsten,142 G. Avolio,36 R. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Ciocio,18 F. Cirotto,69a,69b Z. H. Citron,180 M. Citterio,68a D. A. Ciubotaru,27b B. M. Ciungu,167 A. Clark,54 M. R. Clark,39 P. J. Clark,50 C. Clement,45a,45b Y. Coadou,101 M. Cobal,66a,66c A. Coccaro,55b J. Cochran,78 H. Cohen,161 A. E. C. Coimbra,36 L. Colasurdo,119 B. Cole,39 A. P. Colijn,120 J. Collot,58 P. Conde Muiño,140a,n E. Coniavitis,52 S. H. Connell,33b I A Connelly 57 S Constantinescu 27b F Conventi 69a,o A M Cooper Sarkar 135 F Cormier 175 K J R Cormier 167 Cole,39 A. P. Colijn,120 J. Collot,58 P. Conde Muiño,140a,n E. Coniavitis,52 S. H. Connell,33b I. A. Connelly,57 S. Constantinescu,27b F. Conventi,69a,o A. M. Cooper-Sarkar,135 F. Cormier,175 K. J. R. Cormier,167 L D Corpe 94 M Corradi 72a,72b E E Corrigan 96 F Corriveau 103,p A Cortes Gonzalez 36 M J Costa 174 F Costanza 5 Cr´ep´e-Renaudin,58 F. Crescioli,136 M. Cristinziani,24 V. Croft,120 G. Crosetti,41b,41a A. Cueto,5 T. Cuhadar Donszelmann,149 A. R. Cukierman,153 S. Czekierda,84 P. Czodrowski,36 M. J. Da Cunha Sargedas De Sousa,60b J. V. Da Fonseca Pinto,80b C. Da Via,100 W. Dabrowski,83a T. Dado,28a S. Dahbi,35e T. Dai,105 C. Dallapiccola,102 M. Dam,40 G. D’amen,23b,23a V. D’Amico,74a,74b J. Damp,99 J. R. Dandoy,137 M. F. Daneri,30 N. P. Dang,181,f N. D Dann,100 M. Danninger,175 V. Dao,36 G. Darbo,55b O. Dartsi,5 A. Dattagupta,131 T. Daubney,46 S. D’Auria,68a,68b W. Davey,24 C D id 46 T D id k 143 D R D i 49 E D 104 I D 149 K D 8 R D A di 69a M D B 120 C v , v , v , w , w , , , , S. De Castro,23b,23a S. De Cecco,72a,72b N. De Groot,119 P. de Jong,120 H. De la Torre,106 A. De Maria,15c D. De Pedis,72a 3b,23a S. De Cecco,72a,72b N. De Groot,119 P. de Jong,120 H. De la Torre,106 A. De Maria,15c D. D Salvo,72a U. De Sanctis,73a,73b M. De Santis,73a,73b A. De Santo,156 K. De Vasconcelos Corga 132 146 9 42 41b 41 J. B. De Vivie De Regie,132 C. Debenedetti,146 D. V. Dedovich,79 A. M. Deiana,42 M. Del Gaudio,41b,41a J. Del Peso,98 46 132 145 7 69 69b 54 36 ove,132 F. Deliot,145 C. M. Delitzsch,7 M. Della Pietra,69a,69b D. Della Volpe,54 A. Dell’Acqua,36 Y. Delabat Diaz, D. Delgove, F. Deliot, C. M. Delitzsch, M. Della Pietra, , D. Della Volpe, A. Dell Acqua, L. Dell’Asta,73a,73b M. Delmastro,5 C. Delporte,132 P. A. Delsart,58 D. A. DeMarco,167 S. Demers,183 M. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … A y j O. Bessidskaia Bylund,182 N. Besson,145 A. Bethani,100 S. Bethke,115 A. Betti,24 A. J. Bevan,92 J. Beyer,115 R. Bi,139 R. M. Bianchi,139 O. Biebel,114 D. Biedermann,19 R. Bielski,36 K. Bierwagen,99 N. V. Bies T. R. V. Billoud,109 M. Bindi,53 A. Bingul,12d C. Bini,72a,72b S. Biondi,23b,23a M. Birman,180 T. Bisanz,53 J. P. Biswal,161 100 48 134 25 28 46 26 57 A. Bitadze,100 C. Bittrich,48 K. Bjørke,134 K. M. Black,25 T. Blazek,28a I. Bloch,46 C. Blocker,26 A. Blue,57 U. Blumenschein,92 G. J. Bobbink,120 V. S. Bobrovnikov,122b,122a S. S. Bocchetta,96 A. Bocci,49 D. Boerner,46 D. Bogavac,14 A. Bitadze, C. Bittrich, K. Bjørke, K. M. Black, T. Blazek, I. Bloch, C. Blocker, A. Blue, U. Blumenschein,92 G. J. Bobbink,120 V. S. Bobrovnikov,122b,122a S. S. Bocchetta,96 A. Bocci,49 D. Boerner,46 D. Bogavac,14 122b 122 45 93 53 172 83 113 61b g y M. Bomben,136 M. Bona,92 J. S. Bonilla,131 M. Boonekamp,145 H. M. Borecka-Bielska,90 A. Borisov,123 G. Borissov,89 J. Bortfeldt,36 D. Bortoletto,135 V. Bortolotto,73a,73b D. Boscherini,23b M. Bosman,14 J. D. Bossio Sola,103 K. Bouaouda,35a 032007-23 032007-23 PHYS. REV. D 100, 032007 (2019) G. AAD et al. , , g , , y , , , D. Caforio,56 H. Cai,173 V. M. M. Cairo,153 O. Cakir,4a N. Calace,36 P. Calafiura,18 A. Calandri,101 G. Calderini,136 P. Calfayan,65 G. Callea,57 L. P. Caloba,80b S. Calvente Lopez,98 D. Calvet,38 S. Calvet,38 T. P. Calvet,155 M. Calvetti,71a,71b D. Caforio, H. Cai, V. M. M. Cairo, O. Cakir, N. Calace, P. Calafiura, A. Calandri, G. Calderini, P. Calfayan,65 G. Callea,57 L. P. Caloba,80b S. Calvente Lopez,98 D. Calvet,38 S. Calvet,38 T. P. Calvet,155 M. Calvetti,71a,71b P. Calfayan,65 G. Callea,57 L. P. Caloba,80b S. Calvente Lopez,98 D. Calvet,38 S. Calvet,38 T. P. Calvet,155 M. Calvetti,71a,71b R. Camacho Toro,136 S. Camarda,36 D. Camarero Munoz,98 P. Camarri,73a,73b D. Cameron,134 R. Caminal Armadans,102 C. Camincher,36 S. Campana,36 M. Campanelli,94 A. Camplani,40 A. Campoverde,151 V. Canale,69a,69b A. Canesse,103 M. Cano Bret,60c J. Cantero,129 T. Cao,161 Y. Cao,173 M. D. M. Capeans Garrido,36 M. Capua,41b,41a R. Cardarelli,73a F. C. Cardillo,149 I. Carli,143 T. Carli,36 G. Carlino,69a B. T. Carlson,139 L. Carminati,68a,68b R. M. D. Carney,45a,45b S. Caron,119 E. Carquin,147b S. Carrá,68a,68b J. W. S. Carter,167 M. P. Casado,14,k A. F. Casha,167 D. W. Casper,171 M. Cano Bret, J. Cantero, T. Cao, Y. Cao, M. D. M. Capeans Garrido, M. Capua, R. Cardarelli, F. C. Cardillo,149 I. Carli,143 T. Carli,36 G. Carlino,69a B. T. J. Boudreau,139 E. V. Bouhova-Thacker,89 D. Boumediene,38 S. K. Boutle,57 A. Boveia,126 J. Boyd,36 D. Boye,33b,j I. R. Boyko,79 A. J. Bozson,93 J. Bracinik,21 N. Brahimi,101 G. Brandt,182 O. Brandt,61a F. Braren,46 B. Brau,102 J. E. Brau,131 W. D. Breaden Madden,57 K. Brendlinger,46 L. Brenner,46 R. Brenner,172 S. Bressler,180 B. Brickwedde,99 D. L. Briglin,21 D. Britton,57 D. Britzger,115 I. Brock,24 R. Brock,106 G. Brooijmans,39 W. K. Brooks,147b E. Brost,121 J. H Broughton,21 P. A. Bruckman de Renstrom,84 D. Bruncko,28b A. Bruni,23b G. Bruni,23b L. S. Bruni,120 S. Bruno,73a,73b B. H. Brunt,32 M. Bruschi,23b N. Bruscino,139 P. Bryant,37 L. Bryngemark,96 T. Buanes,17 Q. Buat,36 P. Buchholz,151 A. G. Buckley,57 I. A. Budagov,79 M. K. Bugge,134 F. Bührer,52 O. Bulekov,112 T. J. Burch,121 S. Burdin,90 C. D. Burgard,120 A. M. Burger,129 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Carlson,139 L. Carminati,68a,68b R. M. D. Carney,45a,45b S C 119 E C i 147b S C á 68a,68b J W S C t 167 M P C d 14,k A F C h 167 D W C 171 R. Castelijn,120 F. L. Castillo,174 V. Castillo Gimenez,174 N. F. Castro,140a,140e A R. Castelijn,120 F. L. Castillo,174 V. Castillo Gimenez,174 N. F. Castro,140a,140e A. Catinaccio,36 J. R. Catmore,134 A. Cattai,36 24 29 14 68 14 1 1b 12b J. Caudron,24 V. Cavaliere,29 E. Cavallaro,14 D. Cavalli,68a M. Cavalli-Sforza,14 V. Cavasinni,71a,71b E. Celebi,12b F. Ceradini,74a,74b L. Cerda Alberich,174 A. S. Cerqueira,80a A. Cerri,156 L. Cerrito,73a,73b F. Cerutti,18 A. Cervelli,23b,23a J. Caudron, V. Cavaliere, E. Cavallaro, D. Cavalli, M. Cavalli Sforza, V. Cavasinni, E. Celebi, F. Ceradini,74a,74b L. Cerda Alberich,174 A. S. Cerqueira,80a A. Cerri,156 L. Cerrito,73a,73b F. Cerutti,18 A. Cervelli,23b,23a S. A. Cetin,12b D. Chakraborty,121 S. K. Chan,59 W. S. Chan,120 W. Y. Chan,90 J. D. Chapman,32 B. Chargeishvili,159b D. G. Charlton,21 T. P. Charman,92 C. C. Chau,34 S. Che,126 A. Chegwidden,106 S. Chekanov,6 S. V. Chekulaev,168a G. A. Chelkov,79,l M. A. Chelstowska,36 B. Chen,78 C. Chen,60a C. H. Chen,78 H. Chen,29 J. Chen,60a J. Chen,39 S. Chen,137 y p g D. G. Charlton,21 T. P. Charman,92 C. C. Chau,34 S. Che,126 A. Chegwidden,106 S. Chekanov,6 S. V. Chekulaev,168a G A Chelkov 79,l M A Chelstowska 36 B Chen 78 C Chen 60a C H Chen 78 H Chen 29 J Chen 60a J Chen 39 S Chen 137 S. J. Chen,15c X. Chen,15b,m Y. Chen,82 Y-H. Chen,46 H. C. Cheng,63a H. J. Cheng,15a,15d A. Cheplakov,79 E. Cheremushkina,123 R. Cherkaoui El Moursli,35e E. Cheu,7 K. Cheung,64 T. J. A. Cheval´erias,145 L. Chevalier,145 S. J. Chen,15c X. Chen,15b,m Y. Chen,82 Y-H. Chen,46 H. C. Cheng,63a H. J. Cheng,15a,15d A. Cheplakov,79 E Ch hki 123 R Ch k i El M li 35e E Ch 7 K Ch 64 T J A Ch l´ i 145 L Ch li 145 g V. Chiarella,51 G. Chiarelli,71a G. Chiodini,67a A. S. Chisholm,36,21 A. Chitan,27b I. Chiu,163 Y. H. Chiu,176 M. V. Chizhov,79 g V. Chiarella,51 G. Chiarelli,71a G. Chiodini,67a A. S. Chisholm,36,21 A. Chitan,27b I. Chiu,163 Y. H. Chiu,176 M. V. Chizhov,79 K. Choi,65 A. R. Chomont,72a,72b S. Chouridou,162 Y. S. Chow,120 M. C. Chu,63a J. Chudoba,141 A. J. Chuinard,103 J. J. Chwastowski,84 L. Chytka,130 K. M. Ciesla,84 D. Cinca,47 V. Cindro,91 I. A. Cioară,27b A. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … j y y y g G. Eigen,17 K. Einsweiler,18 T. Ekelof,172 M. El Kacimi,35c R. El Kosseifi,101 V. Ellajosyula,172 M. Ellert,172 F. Ellinghaus,182 A. A. Elliot,92 N. Ellis,36 J. Elmsheuser,29 M. Elsing,36 D. Emeliyanov,144 A. Emerman,39 Y. Enari,163 J. S. Ennis,178 g y M. B. Epland,49 J. Erdmann,47 A. Ereditato,20 M. Errenst,36 M. Escalier,132 C. Escobar,174 O. Estrada Pastor,174 E. Etzion,161 ,47 A. Ereditato,20 M. Errenst,36 M. Escalier,132 C. Escobar,174 O. Estrada Pastor,174 E. Etzion,161 M. B. Epland,49 J. Erdmann,47 A. Ereditato,20 M. Errenst,36 M. Escalier,132 C. Escobar,174 O. Est 8 F. Fabbri,57 L. Fabbri,23b,23a V. Fabiani,119 G. Facini,94 R. M. Faisca Rodrigues Pereira,140a H. Evans, A. Ezhilov, F. Fabbri, L. Fabbri, V. Fabiani, G. Facini, R. M. Faisca Rodrigues Pereira, R. M. Fakhrutdinov,123 S. Falciano,72a P. J. Falke,5 S. Falke,5 J. Faltova,143 Y. Fang,15a Y. Fang,15a G. Fanourakis,44 . Falciano,72a P. J. Falke,5 S. Falke,5 J. Faltova,143 Y. Fang,15a Y. Fang,15a G. Fanourakis,44 A. Farilla,74a E. M. Farina,70a,70b T. Farooque,106 S. Farrell,18 S. M. Farrington,178 P. Farthouat,36 ,68a,68b A. Farbin,8 A. Farilla,74a E. M. Farina,70a,70b T. Farooque,106 S. Farrell,18 S. M. Farrington 35 36 9 50 32 132 F. Fassi,35e P. Fassnacht,36 D. Fassouliotis,9 M. Faucci Giannelli,50 W. J. Fawcett,32 L. Fayard,132 O. L. Fedin,138,q kert,42 S. Feigl,134 L. Feligioni,101 A. Fell,149 C. Feng,60b E. J. Feng,36 M. Feng,49 M. J. Fenton W. Fedorko,175 M. Feickert,42 S. Feigl,134 L. Feligioni,101 A. Fell,149 C. Feng,60b E. J. Feng,36 W. Fedorko, M. Feickert, S. Feigl, L. Feligioni, A. Fell, C. Feng, E. J. Feng, M. Feng, M. J. Fenton, A. B. Fenyuk,123 J. Ferrando,46 A. Ferrante,173 A. Ferrari,172 P. Ferrari,120 R. Ferrari,70a D. E. Ferreira de Lima,61b A. B. Fenyuk,123 J. Ferrando,46 A. Ferrante,173 A. Ferrari,172 P. Ferrari,120 R. Ferrari,70a D. E. Ferreira de Lima,61b y A. Ferrer,174 D. Ferrere,54 C. Ferretti,105 F. Fiedler,99 A. Filipčič,91 F. Filthaut,119 K. D. Fine ,54 C. Ferretti,105 F. Fiedler,99 A. Filipčič,91 F. Filthaut,119 K. D. Finelli,25 M. C. N. Fiolhais,14 p L. Fiorini,174 F. Fischer,114 W. C. Fisher,106 I. Fleck,151 P. Fleischmann,105 R. R. M. Fletcher,137 T. Flick,182 B. M. Flierl,114 L. F. Flores,137 L. R. Flores Castillo,63a F. M. Follega,75a,75b N. Fomin,17 G. T. Forcolin,75a,75b A. Formica,145 F. A. Förster,14 A. C. Forti,100 A. G. Foster,21 M. G. Foti,135 D. Fournier,132 H. Fox,89 P. Francavilla,71a,71b S. Francescato,72a,72b L. F. Flores,137 L. R. Flores Castillo,63a F. M. Follega,75a,75b N. Fomin,17 G. T. Forcolin,75a,75b A. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Demichev,79 , g , , , , p , q , L. Dell’Asta,73a,73b M. Delmastro,5 C. Delporte,132 P. A. Delsart,58 D. A. DeMarco,167 S. Demers,183 M. Demichev,79 p ntigny,109 S. P. Denisov,123 D. Denysiuk,120 L. D’Eramo,136 D. Derendarz,84 J. E. Derkaoui,35d p Denisov,123 D. Denysiuk,120 L. D’Eramo,136 D. Derendarz,84 J. E. Derkaoui,35d F. Derue,136 P. Dervan,90 K. Desch,24 C. Deterre,46 K. Dette,167 C. Deutsch,24 M. R. Devesa,30 P. O. Deviveiros,36 A. Dewhurst,144 S. Dhaliwal,26 F. A. Di Bello,54 A. Di Ciaccio,73a,73b L. Di Ciaccio,5 W. K. Di Clemente,137 C. Di Donato,69a,69b A. Di Girolamo,36 G. Di Gregorio,71a,71b B. Di Micco,74a,74b R. Di Nardo,102 K. F. Di Petrillo,59 R. Di Sipio,167 S. Dhaliwal,26 F. A. Di Bello,54 A. Di Ciaccio,73a,73b L. Di Ciaccio,5 W. K. Di Clemente,137 C. Di Donato,69a,69b A. Di Girolamo,36 G. Di Gregorio,71a,71b B. Di Micco,74a,74b R. Di Nardo,102 K. F. Di Petrillo,59 R. Di Sipio,167 D. Di Valentino,34 C. Diaconu,101 F. A. Dias,40 T. Dias Do Vale,140a M. A. Diaz,147a J. Dickinson,18 E. B. Diehl,105 A. Di Girolamo,36 G. Di Gregorio,71a,71b B. Di Micco,74a,74b R. Di Nardo,102 K. F. Di Petrillo,59 R. Di Sipio,167 D. Di Valentino,34 C. Diaconu,101 F. A. Dias,40 T. Dias Do Vale,140a M. A. Diaz,147a J. Dickinson,18 E. B. Diehl,105 032007-24 032007-24 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Formica,145 F. A. Förster,14 A. C. Forti,100 A. G. Foster,21 M. G. Foti,135 D. Fournier,132 H. Fox,89 P. Francavilla,71a,71b S. Francescato,72a,72b M. Franchini,23b,23a S. Franchino,61a D. Francis,36 L. Franconi,20 M. Franklin,59 A. N. Fray,92 B. Freund,109 W. S. Freund,80b M. Franchini,23b,23a S. Franchino,61a D. Francis,36 L. Franconi,20 M. Franklin,59 A. N. Fray,92 B. Freund,109 W. S. Freund,80b E. M. Freundlich,47 D. C. Frizzell,128 D. Froidevaux,36 J. A. Frost,135 C. Fukunaga,164 E. Fullana Torregrosa,174 M. Franchini, S. Franchino, D. Francis, L. Franconi, M. Franklin, A. N. Fray, B. Freund, W. S. Freund, E. M. Freundlich,47 D. C. Frizzell,128 D. Froidevaux,36 J. A. Frost,135 C. Fukunaga,164 E. Fullana Torregrosa,174 55b 55 116 174 23b 23 18 83 54 115 E. M. Freundlich,47 D. C. Frizzell,128 D. Froidevaux,36 J. A. Frost,135 C. Fukunaga,164 E. Fullana Torregrosa,174 E. Fumagalli,55b,55a T. Fusayasu,116 J. Fuster,174 A. Gabrielli,23b,23a A. Gabrielli,18 G. P. Gach,83a S. Gadatsch,54 P. Gadow,115 E. Fumagalli,55b,55a T. Fusayasu,116 J. Fuster,174 A. Gabrielli,23b,23a A. Gabrielli,18 G. P. Gach,83a S. Gadatsch,54 P. Gadow,115 G. Gagliardi,55b,55a L. G. Gagnon,109 C. Galea,27b B. Galhardo,140a G. E. Gallardo,135 E. J. Gallas,135 B. J. Gallop,144 E. Fumagalli,55b,55a T. Fusayasu,116 J. Fuster,174 A. Gabrielli,23b,23a A. Gabrielli,18 G. P. Gach,83a S. Gadatsch,54 P. Gadow,115 G. Gagliardi,55b,55a L. G. Gagnon,109 C. Galea,27b B. Galhardo,140a G. E. Gallardo,135 E. J. Gallas,135 B. J. Gallop,144 P. Gallus,142 G. Galster,40 R. Gamboa Goni,92 K. K. Gan,126 S. Ganguly,180 J. Gao,60a Y. Gao,90 Y. S. Gao,31,h C. García,174 g , y , , , , , , , G. Gagliardi,55b,55a L. G. Gagnon,109 C. Galea,27b B. Galhardo,140a G. E. Gallardo,135 E. J. Gallas,135 B. J. Gallop,144 P Gallus 142 G Galster 40 R Gamboa Goni 92 K K Gan 126 S Ganguly 180 J Gao 60a Y Gao 90 Y S Gao 31,h C García 174 g y G. Gagliardi,55b,55a L. G. Gagnon,109 C. Galea,27b B. Galhardo,140a G. E. Gallardo,135 E. J. Gallas,135 B. J. Gallop,144 g p . Gamboa Goni,92 K. K. Gan,126 S. Ganguly,180 J. Gao,60a Y. Gao,90 Y. S. Gao,31,h C. García,174 2 G. Galster,40 R. Gamboa Goni,92 K. K. Gan,126 S. Ganguly,180 J. Gao,60a Y. Gao,90 Y. S. Gao,31 J. E. García Navarro,174 J. A. García Pascual,15a C. Garcia-Argos,52 M. Garcia-Sciveres,18 R. W. Gardner,37 N. Garelli,153 S. Gargiulo,52 V. Garonne,134 A. Gaudiello,55b,55a G. Gaudio,70a I. L. Gavrilenko,110 A. Gavrilyuk,111 C. Gay,175 24 10 27b 144 53 99 61a 55b J. E. J. Dietrich,19 S. Díez Cornell,46 A. Dimitrievska,18 W. Ding,15b J. Dingfelder,24 F. Dittus,36 F. Djama,101 T. Djobava,159b J. I. Djuvsland,17 M. A. B. Do Vale,80c M. Dobre,27b D. Dodsworth,26 C. Doglioni,96 J. Dolejsi,143 Z. Dolezal,143 M. Donadelli,80d J. Donini,38 A. D’onofrio,92 M. D’Onofrio,90 J. Dopke,144 A. Doria,69a M. T. Dova,88 A. T. Doyle,57 E. Drechsler,152 E. Dreyer,152 T. Dreyer,53 A. S. Drobac,170 Y. Duan,60b F. Dubinin,110 M. Dubovsky,28a A. Dubreuil,54 E. Duchovni,180 G. Duckeck,114 A. Ducourthial,136 O. A. Ducu,109 D. Duda,115 A. Dudarev,36 A. C. Dudder,99 E. M. Duffield,18 L. Duflot,132 M. Dührssen,36 C. Dülsen,182 M. Dumancic,180 A. E. Dumitriu,27b A. K. Duncan,57 M. Dunford,61a A. Duperrin,101 H. Duran Yildiz,4a M. Düren,56 A. Durglishvili,159b D. Duschinger,48 B. Dutta,46 D. Duvnjak,1 G. I. Dyckes,137 M. Dyndal,36 S. Dysch,100 B. S. Dziedzic,84 K. M. Ecker,115 R. C. Edgar,105 T. Eifert,36 G. Eigen,17 K. Einsweiler,18 T. Ekelof,172 M. El Kacimi,35c R. El Kosseifi,101 V. Ellajosyula,172 M. Ellert,172 F. Ellinghaus,182 A. A. Elliot,92 N. Ellis,36 J. Elmsheuser,29 M. Elsing,36 D. Emeliyanov,144 A. Emerman,39 Y. Enari,163 J. S. Ennis,178 M B Epland 49 J Erdmann 47 A Ereditato 20 M Errenst 36 M Escalier 132 C Escobar 174 O Estrada Pastor 174 E Et ion 161 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … García Navarro, J. A. García Pascual, C. Garcia-Argos, M. Garcia-Sciveres, R. W. Gardner, N. Garelli, S. Gargiulo,52 V. Garonne,134 A. Gaudiello,55b,55a G. Gaudio,70a I. L. Gavrilenko,110 A. Gavrilyuk,111 C. Gay,175 G. Gaycken,24 E. N. Gazis,10 A. A. Geanta,27b C. N. P. Gee,144 J. Geisen,53 M. Geisen,99 M. P. Geisler,61a C. Gemme,55b M. H. Genest,58 C. Geng,105 S. Gentile,72a,72b S. George,93 T. Geralis,44 D. Gerbaudo,14 L. O. Gerlach,53 S. Gargiulo, V. Garonne, A. Gaudiello, G. Gaudio, I. L. Gavrilenko, A. Gavrilyuk, C. Gay, G. Gaycken,24 E. N. Gazis,10 A. A. Geanta,27b C. N. P. Gee,144 J. Geisen,53 M. Geisen,99 M. P. Geisler,61a C. Gemme,55b M. H. Genest,58 C. Geng,105 S. Gentile,72a,72b S. George,93 T. Geralis,44 D. Gerbaudo,14 L. O. Gerlach,53 G. Gaycken, E. N. Gazis, A. A. Geanta, C. N. P. Gee, J. Geisen, M. Geisen, M. P. Geisler, C. Gemme, M. H. Genest,58 C. Geng,105 S. Gentile,72a,72b S. George,93 T. Geralis,44 D. Gerbaudo,14 L. O. Gerlach,53 P. Gessinger-Befurt,99 G. Gessner,47 S. Ghasemi,151 M. Ghasemi Bostanabad,176 M. Ghneimat,24 A. Ghosh,77 B. Giacobbe,23b S. Giagu,72a,72b N. Giangiacomi,23b,23a P. Giannetti,71a A. Giannini,69a,69b S. M. Gibson,93 M. Gignac,146 P. Gessinger-Befurt,99 G. Gessner,47 S. Ghasemi,151 M. Ghasemi Bostanabad,176 M. Ghneimat,24 A. Ghosh,77 B. Giacobbe,23b S. Giagu,72a,72b N. Giangiacomi,23b,23a P. Giannetti,71a A. Giannini,69a,69b S. M. Gibson,93 M. Gignac,146 D. Gillberg,34 G. Gilles,182 D. M. Gingrich,3,e M. P. Giordani,66a,66c F. M. Giorgi,23b P. F. Giraud,145 G. Giugliarelli,66a,66c 68 73 73b 162 9 14 10 113 D. Giugni,68a F. Giuli,73a,73b S. Gkaitatzis,162 I. Gkialas,9,r E. L. Gkougkousis,14 P. Gkountoumis,10 L. K. Gladilin,113 C. Glasman,98 J. Glatzer,14 P. C. F. Glaysher,46 A. Glazov,46 M. Goblirsch-Kolb,26 S. Goldfarb,104 T. Golling,54 D. Golubkov,123 A. Gomes,140a,140b R. Goncalves Gama,53 R. Gonçalo,140a,140b G. Gonella,52 L. Gonella,21 A. Gongadze,79 F. Gonnella,21 J. L. Gonski,59 S. González de la Hoz,174 S. Gonzalez-Sevilla,54 G. R. Gonzalvo Rodriguez,174 L. Goossens,36 P. A. Gorbounov,111 H. A. Gordon,29 B. Gorini,36 E. Gorini,67a,67b A. Gorišek,91 A. T. Goshaw,49 M. I. Gostkin,79 C. A. Gottardo,24 M. Gouighri,35b D. Goujdami,35c A. G. Goussiou,148 N. Govender,33b,s C. Goy,5 E. Gozani,160 , , , , g , , P. A. Gorbounov,111 H. A. Gordon,29 B. Gorini,36 E. Gorini,67a,67b A. Gorišek,91 A. T. Goshaw,49 M. I. Gostkin,79 C. A. Gottardo,24 M. Gouighri,35b D. Goujdami,35c A. G. Goussiou,148 N. Govender,33b,s C. Goy,5 E. Gozani,160 g j y I. Grabowska-Bold,83a E. C. Graham,90 J. Gramling,171 E. Gramstad,134 S. Grancagnolo,19 M. Grandi,156 V. Gratchev,138 I. Grabowska-Bold,83a E. C. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Graham,90 J. Gramling,171 E. Gramstad,134 S. Grancagnolo,19 M. Grandi,156 V. Gratchev,138 P M Gravila 27f F G Gravili 67a,67b C Gray 57 H M Gray 18 C Grefe 24 K Gregersen 96 I M Gregor 46 P Grenier 153 P. M. Gravila,27f F. G. Gravili,67a,67b C. Gray,57 H. M. Gray,18 C. Grefe,24 K. Gregersen,96 I. M. Gregor,46 P. Grenier,153 K. Grevtsov,46 N. A. Grieser,128 J. Griffiths,8 A. A. Grillo,146 K. Grimm,31,t S. Grinstein,14,u J.-F. Grivaz,132 S. Groh,99 E. Gross,180 J. Grosse-Knetter,53 Z. J. Grout,94 C. Grud,105 A. Grummer,118 L. Guan,105 W. Guan,181 J. Guenther,36 A. Guerguichon,132 F. Guescini,168a D. Guest,171 R. Gugel,52 B. Gui,126 T. Guillemin,5 S. Guindon,36 U. Gul,57 J. Guo,60c W. Guo,105 Y. Guo,60a,v Z. Guo,101 R. Gupta,46 S. Gurbuz,12c G. Gustavino,128 P. Gutierrez,128 C. Gutschow,94 C. Guyot,145 M. P. Guzik,83a C. Gwenlan,135 C. B. Gwilliam,90 A. Haas,124 C. Haber,18 H. K. Hadavand,8 N. Haddad,35e A. Hadef,60a S. Hageböck,36 M. Hagihara,169 M. Haleem,177 J. Haley,129 G. Halladjian,106 G. D. Hallewell,101 K. Hamacher,182 P. Hamal,130 K. Hamano,176 H. Hamdaoui,35e G. N. Hamity,149 K. Han,60a,w L. Han,60a S. Han,15a,15d K. Hanagaki,81,x M. Hance,146 D. M. Handl,114 B. Haney,137 R. Hankache,136 E. Hansen,96 J. B. Hansen,40 J. D. Hansen,40 M. C. Hansen,24 P. M. Gravila,27f F. G. Gravili,67a,67b C. Gray,57 H. M. Gray,18 C. Grefe,24 K. Gregersen,96 I. M. Gregor,46 P. Grenier,153 K. Grevtsov,46 N. A. Grieser,128 J. Griffiths,8 A. A. Grillo,146 K. Grimm,31,t S. Grinstein,14,u J.-F. Grivaz,132 S. Groh,99 E. Gross,180 J. Grosse-Knetter,53 Z. J. Grout,94 C. Grud,105 A. Grummer,118 L. Guan,105 W. Guan,181 J. Guenther,36 A. Guerguichon,132 F. Guescini,168a D. Guest,171 R. Gugel,52 B. Gui,126 T. Guillemin,5 S. Guindon,36 U. Gul,57 J. Guo,60c K. Grevtsov, N. A. Grieser, J. Griffiths, A. A. Grillo, K. Grimm, , S. Grinstein, , J.-F. Grivaz, S. Groh, E. Gross,180 J. Grosse-Knetter,53 Z. J. Grout,94 C. Grud,105 A. Grummer,118 L. Guan,105 W. Guan,181 J. Guenther,36 A. Guerguichon,132 F. Guescini,168a D. Guest,171 R. Gugel,52 B. Gui,126 T. Guillemin,5 S. Guindon,36 U. Gul,57 J. Guo,60c W G 105 Y G 60a v Z G 101 R G 46 S G b 12c G G i 128 P G i 128 C G h 94 C G 145 M. P. Guzik,83a C. Gwenlan,135 C. B. Gwilliam,90 A. Haas,124 C. Haber,18 H. K. Hadavand,8 N. Haddad,35e A. Hadef,60a S. Hageböck,36 M. Hagihara,169 M. Haleem,177 J. Haley,129 G. Halladjian,106 G. D. Hallewell,101 K. Hamacher,182 032007-25 032007-25 PHYS. REV. P. H. Hansen,40 E. C. Hanson,100 K. Hara,169 A. S. Hard,181 T. Harenberg,182 S. Harkusha,107 P. F. Harrison,178 N. M. Hartmann,114 Y. Hasegawa,150 A. Hasib,50 S. Hassani,145 S. Haug,20 R. Hauser,106 L. B. Havener,39 M. Havranek,142 C. M. Hawkes,21 R. J. Hawkings,36 D. Hayden,106 C. Hayes,155 R. L. Hayes,175 C. P. Hays,135 J. M. Hays,92 H. S. Hayward,90 S. J. Haywood,144 F. He,60a M. P. Heath,50 V. Hedberg,96 L. Heelan,8 S. Heer,24 K. K. Heidegger,52 J. Heilman,34 S. Heim,46 T. Heim,18 B. Heinemann,46,y J. J. Heinrich,131 L. Heinrich,36 C. Heinz,56 J. Hejbal,141 L. Helary,61b A. Held,175 S. Hellesund,134 C. M. Helling,146 S. Hellman,45a,45b C. Helsens,36 R. C. W. Henderson,89 Y. Heng,181 S. Henkelmann,175 A. M. Henriques Correia,36 G. H. Herbert,19 H. Herde,26 V. Herget,177 Y. Hernández Jim´enez,33c H. Herr,99 M. G. Herrmann,114 T. Herrmann,48 G. Herten,52 R. Hertenberger,114 L. Hervas,36 T. C. Herwig,137 G. G. Hesketh,94 N. P. Hessey,168a A. Higashida,163 S. Higashino,81 E. Higón-Rodriguez,174 K. Hildebrand,37 E. Hill,176 J. C. Hill,32 K. K. Hill,29 K. H. Hiller,46 S. J. Hillier,21 M. Hils,48 I. Hinchliffe,18 F. Hinterkeuser,24 M. Hirose,133 S. Hirose,52 D. Hirschbuehl,182 B. Hiti,91 O. Hladik,141 D. R. Hlaluku,33c X. Hoad,50 J. Hobbs,155 N. Hod,180 M. C. Hodgkinson,149 A. Hoecker,36 F. Hoenig,114 D. Hohn,52 D. Hohov,132 T. R. Holmes,37 M. Holzbock,114 L. B. A. H Hommels,32 S. Honda,169 T. Honda,81 T. M. Hong,139 A. Hönle,115 B. H. Hooberman,173 W. H. Hopkins,6 Y. Horii,117 P. Horn,48 A. J. Horton,152 L. A. Horyn,37 J-Y. Hostachy,58 A. Hostiuc,148 S. Hou,158 A. Hoummada,35a J. Howarth,100 J. Hoya,88 M. Hrabovsky,130 J. Hrdinka,76 I. Hristova,19 J. Hrivnac,132 A. Hrynevich,108 T. Hryn’ova,5 P. J. Hsu,64 S.-C. Hsu,148 Q. Hu,29 S. Hu,60c Y. Huang,15a Z. Hubacek,142 F. Hubaut,101 M. Huebner,24 F. Huegging,24 T. B. Huffman,135 M. Huhtinen,36 R. F. H. Hunter,34 P. Huo,155 A. M. Hupe,34 N. Huseynov,79,z J. Huston,106 J. Huth,59 R. Hyneman,105 S. Hyrych,28a G. Iacobucci,54 G. Iakovidis,29 I. Ibragimov,151 L. Iconomidou-Fayard,132 Z. Idrissi,35e P. I. Iengo,36 R. Ignazzi,40 O. Igonkina,120,a,aa R. Iguchi,163 T. Iizawa,54 Y. Ikegami,81 M. Ikeno,81 D. Iliadis,162 N. Ilic,119 F. Iltzsche,48 G. Introzzi,70a,70b M. Iodice,74a K. Iordanidou,39 V. Ippolito,72a,72b M. F. Isacson,172 N. Ishijima,133 M. Ishino,163 M. Ishitsuka,165 W. Islam,129 C. Issever,135 S. Istin,160 F. Ito,169 J. M. Iturbe Ponce,63a R. Iuppa,75a,75b A. Ivina,180 H. Iwasaki,81 J. M. Izen,43 V. Izzo,69a P. Jacka,141 P. Jackson,1 R. M. Jacobs,24 V. Jain,2 G. Jäkel,182 K. B. Jakobi,99 K. Jakobs,52 S. Jakobsen,76 T. Jakoubek,141 J. Jamieson,57 K. W. Janas,83a R. Jansky,54 J. Janssen,24 M. Janus,53 P. A. Janus,83a G. Jarlskog,96 N. Javadov,79,z T. Javůrek,36 M. Javurkova,52 F. Jeanneau,145 L. Jeanty,131 J. Jejelava,159a,bb A. Jelinskas,178 P. Jenni,52,cc J. Jeong,46 N. Jeong,46 S. J´ez´equel,5 H. Ji,181 J. Jia,155 H. Jiang,78 Y. Jiang,60a Z. Jiang,153,dd S. Jiggins,52 F. A. Jimenez Morales,38 J. Jimenez Pena,174 S. Jin,15c A. Jinaru,27b O. Jinnouchi,165 H. Jivan,33c P. Johansson,149 K. A. Johns,7 C. A. Johnson,65 K. Jon-And,45a,45b R. W. L. Jones,89 S. D. Jones,156 S. Jones,7 T. J. Jones,90 J. Jongmanns,61a P. M. Jorge,140a J. Jovicevic,36 X. Ju,18 J. J. Junggeburth,115 A. Juste Rozas,14,u A. Kaczmarska,84 M. Kado,72a,72b H. Kagan,126 M. Kagan,153 C. Kahra,99 179 160 96 99 123 91 163 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … D 100, 032007 (2019) G. AAD et al. T. Honda,81 T. M. Hong,139 A. Hönle,115 B. H. Hooberman,173 W. H. Hopkins,6 Y. Horii,117 P. Horn,48 A. J. Horton,152 5 5 5 J. Hrdinka,76 I. Hristova,19 J. Hrivnac,132 A. Hrynevich,108 T. Hryn’ova,5 P. J. Hsu,64 S.-C. Hsu,148 Q. Hu,29 S. Hu,60c 5 5 6 Y. Huang,15a Z. Hubacek,142 F. Hubaut,101 M. Huebner,24 F. Huegging,24 T. B. Huffman,135 M. Huhtinen,36 R. F. H. Hunter,34 P. Huo,155 A. M. Hupe,34 N. Huseynov,79,z J. Huston,106 J. Huth,59 R. Hyneman,105 S. Hyrych,28a G. Iacobucci,54 G. Iakovidis,29 I. Ibragimov,151 L. Iconomidou-Fayard,132 Z. Idrissi,35e P. I. Iengo,36 R. Ignazzi,40 O. Igonkina,120,a,aa R. Iguchi,163 T. Iizawa,54 Y. Ikegami,81 M. Ikeno,81 D. Iliadis,162 N. Ilic,119 F. Iltzsche,48 G. Introzzi,70a,70b M. Iodice,74a 39 72 72b 172 133 163 165 129 135 G. Iakovidis,29 I. Ibragimov,151 L. Iconomidou-Fayard,132 Z. Idrissi,35e P. I. Iengo,36 R. Ignazzi,40 O. Igonkina,120,a,aa R. Iguchi,163 T. Iizawa,54 Y. Ikegami,81 M. Ikeno,81 D. Iliadis,162 N. Ilic,119 F. Iltzsche,48 G. Introzzi,70a,70b M. Iodice,74a R. Iguchi,163 T. Iizawa,54 Y. Ikegami,81 M. Ikeno,81 D. Iliadis,162 N. Ilic,119 F. Iltzsche,48 G. Introzzi,70a,70b M. Iodice,74a pp j S. Istin,160 F. Ito,169 J. M. Iturbe Ponce,63a R. Iuppa,75a,75b A. Ivina,180 H. Iwasaki,81 J. M. Izen,43 V. Izzo,69a P. Jacka,141 1 24 2 182 99 52 76 141 57 P. Jackson,1 R. M. Jacobs,24 V. Jain,2 G. Jäkel,182 K. B. Jakobi,99 K. Jakobs,52 S. Jakobsen,76 T. Jakoubek,141 J. Jamieson,57 K. W. Janas,83a R. Jansky,54 J. Janssen,24 M. Janus,53 P. A. Janus,83a G. Jarlskog,96 N. Javadov,79,z T. Javůrek,36 M. Javurkova,52 F. Jeanneau,145 L. Jeanty,131 J. Jejelava,159a,bb A. Jelinskas,178 P. Jenni,52,cc J. Jeong,46 N. Jeong,46 S. J´ez´equel,5 H. Ji,181 J. Jia,155 H. Jiang,78 Y. Jiang,60a Z. Jiang,153,dd S. Jiggins,52 F. A. Jimenez Morales,38 J. Jimenez Pena,174 S. Jin,15c A. Jinaru,27b O. Jinnouchi,165 H. Jivan,33c P. Johansson,149 K K. Jon-And,45a,45b R. W. L. Jones,89 S. D. Jones,156 S. Jones,7 T. J. Jones,90 J. Jongmanns,61a P. M. Jorge,140a J. Jovicevic,36 K. Jon-And,45a,45b R. W. L. Jones,89 S. D. Jones,156 S. Jones,7 T. J. Jones,90 J. Jongmanns,61a P. X. Ju,18 J. J. Junggeburth,115 A. Juste Rozas,14,u A. Kaczmarska,84 M. Kado,72a,72b H. Kagan,126 M. Kagan,153 C. Kahra,99 T. Kaji,179 E. Kajomovitz,160 C. W. Kalderon,96 A. Kaluza,99 A. Kamenshchikov,123 L. Kanjir,91 Y. Kano,163 V. A. Kantserov,112 J. Kanzaki,81 L. S. Kaplan,181 D. Kar,33c M. J. Kareem,168b E. Karentzos,10 S. N. Karpov,79 79 89 123 181 126 45 45b 163 X. Ju,18 J. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … J. Junggeburth,115 A. Juste Rozas,14,u A. Kaczmarska,84 M. Kado,72a,72b H. Kagan,126 M. Kagan,153 C. Kahra,99 T. Kaji,179 E. Kajomovitz,160 C. W. Kalderon,96 A. Kaluza,99 A. Kamenshchikov,123 L. Kanjir,91 Y. Kano,163 V. A. Kantserov,112 J. Kanzaki,81 L. S. Kaplan,181 D. Kar,33c M. J. Kareem,168b E. Karentzos,10 S. N. Karpov,79 Z. M. Karpova,79 V. Kartvelishvili,89 A. N. Karyukhin,123 L. Kashif,181 R. D. Kass,126 A. Kastanas,45a,45b Y. Kataoka,163 C. Kato,60d,60c J. Katzy,46 K. Kawade,82 K. Kawagoe,87 T. Kawaguchi,117 T. Kawamoto,163 G. Kawamura,53 E. F. Kay,176 122b 122 176 42 34 96 156 21 21 j j j V. A. Kantserov,112 J. Kanzaki,81 L. S. Kaplan,181 D. Kar,33c M. J. Kareem,168b E. Karentzos,10 S. N. Karpov,79 Z. M. Karpova,79 V. Kartvelishvili,89 A. N. Karyukhin,123 L. Kashif,181 R. D. Kass,126 A. Kastanas,45a,45b Y. Kataoka,163 C. Kato,60d,60c J. Katzy,46 K. Kawade,82 K. Kawagoe,87 T. Kawaguchi,117 T. Kawamoto,163 G. Kawamura,53 E. F. Kay,176 , , p , , , , p , Z. M. Karpova,79 V. Kartvelishvili,89 A. N. Karyukhin,123 L. Kashif,181 R. D. Kass,126 A. Kastanas,45a,45b Y. Kataoka,163 C. Kato,60d,60c J. Katzy,46 K. Kawade,82 K. Kawagoe,87 T. Kawaguchi,117 T. Kawamoto,163 G. Kawamura,53 E. F. Kay,176 V. F. Kazanin,122b,122a R. Keeler,176 R. Kehoe,42 J. S. Keller,34 E. Kellermann,96 D. Kelsey,156 J. J. Kempster,21 J. Kendrick,21 D. Kobayashi,87 T. Kobayashi,163 M. Kobel,48 M. Kocian,153 P. Kodys,143 P. T. Koenig,24 T. Koffas,34 N. M. Köhler,115 T. Koi,153 M. Kolb,61b I. Koletsou,5 T. Komarek,130 T. Kondo,81 N. Kondrashova,60c K. Köneke,52 A. C. König,119 g T. Kono,125 R. Konoplich,124,ee V. Konstantinides,94 N. Konstantinidis,94 B. Konya,96 R. Kopeliansky,65 S. Koperny,83a K. Korcyl,84 K. Kordas,162 G. Koren,161 A. Korn,94 I. Korolkov,14 E. V. Korolkova,149 N. Korotkova,113 O. Kortner,115 S. Kortner,115 T. Kosek,143 V. V. Kostyukhin,24 A. Kotwal,49 A. Koulouris,10 A. Kourkoumeli-Charalampidi,70a,70b C. Kourkoumelis,9 E. Kourlitis,149 V. Kouskoura,29 A. B. Kowalewska,84 R. Kowalewski,176 C. Kozakai,163 W. Kozanecki,145 A. S. Kozhin,123 V. A. Kramarenko,113 G. Kramberger,91 D. Krasnopevtsev,60a M. W. Krasny,136 A. Krasznahorkay,36 D. Krauss,115 J. A. Kremer,83a J. Kretzschmar,90 P. Krieger,167 F. Krieter,114 A. Krishnan,61b K. Krizka,18 K. Kroeninger,47 H. Kroha,115 J. Kroll,141 J. Kroll,137 J. Krstic,16 U. Kruchonak,79 H. Krüger,24 N. Krumnack,78 M. C. Kruse,49 T. Kubota,104 S. Kuday,4b J. T. Kuechler,46 S. Kuehn,36 A. Kugel,61a T. Kuhl,46 V. Kukhtin,79 R. Kukla,101 032007-26 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … D. Lellouch,180,a K. J. C. Leney,42 T. Lenz,24 B. Lenzi,36 R. Leone,7 S. Leone,71a C. Leonidopoulos,50 A. Leopold,136 G. Lerner,156 C. Leroy,109 R. Les,167 C. G. Lester,32 M. Levchenko,138 J. Levêque,5 D. Levin,105 L. J. Levinson,180 G. Lerner, C. Leroy, R. Les, C. G. Lester, M. Levchenko, J. Levêque, D. Levin, L. J. Levinson, D. J. Lewis,21 B. Li,15b B. Li,105 C-Q. Li,60a F. Li,60c H. Li,60a H. Li,60b J. Li,60c K. Li,153 L. Li,60c M. Li,15a Q. Li,15a,15d Q. Y. Li,60a S. Li,60d,60c X. Li,46 Y. Li,46 Z. Li,60b Z. Liang,15a B. Liberti,73a A. Liblong,167 K. Lie,63c S. Liem,120 C. Y. Lin,32 K. Lin,106 T. H. Lin,99 R. A. Linck,65 J. H. Lindon,21 A. L. Lionti,54 E. Lipeles,137 A. Lipniacka,17 M. Lisovyi,61b 173 hh 175 146 8 78 ii 6 29 105 60 K. Lin,106 T. H. Lin,99 R. A. Linck,65 J. H. Lindon,21 A. L. Lionti,54 E. Lipeles,137 A. Lipniacka,17 M. Lisovyi,61b 173 hh 175 146 8 78 ii 6 29 105 60 J. K. K. Liu,135 K. Liu,136 M. Liu,60a P. Liu,18 Y. Liu,15a,15d Y. L. Liu,105 Y. W. Liu,60a M. Livan,70a,70b A. Lleres,58 J. Llorente Merino,15a S. L. Lloyd,92 C. Y. Lo,63b F. Lo Sterzo,42 E. M. Lobodzinska,46 P. Loch,7 S. Loffredo,73a,73b T. Lohse,19 K. Lohwasser,149 M. Lokajicek,141 J. D. Long,173 R. E. Long,89 L. Longo,36 K. A. Looper,126 J. A. Lopez,147b y T. Lohse,19 K. Lohwasser,149 M. Lokajicek,141 J. D. Long,173 R. E. Long,89 L. Longo,36 K. A. Looper,126 J. A. Lopez,147b p p X. Lou,15a A. Lounis,132 J. Love,6 P. A. Love,89 J. J. Lozano Bahilo,174 M. Lu,60a Y. J. Lu,64 H. J. Lubatti,148 C. Luci,72a,72b A Lucotte 58 C Luedtke 52 F Luehring 65 I Luise 136 L Luminari 72a B Lund-Jensen 154 M S Lutz 102 D Lynn 29 R. Lysak,141 E. Lytken,96 F. Lyu,15a V. Lyubushkin,79 T. Lyubushkina,79 H. Ma,29 L. L. Ma,60b Y. Ma,60b G. Maccarrone,51 g y J. Maeda,82 K. Maekawa,163 S. Maeland,17 T. Maeno,29 M. Maerker,48 A. S. Maevskiy,113 V. Magerl,52 N. Magini,78 D. J. Mahon,39 C. Maidantchik,80b T. Maier,114 A. Maio,140a,140b,140d O. Majersky,28a S. Majewski,131 Y. Makida,81 132 136 84 138 58 77 6 32 y g g D. J. Mahon,39 C. Maidantchik,80b T. Maier,114 A. Maio,140a,140b,140d O. Majersky,28a S. Majewski,131 Y. Makida,81 N. Makovec,132 B. Malaescu,136 Pa. Malecki,84 V. P. Maleev,138 F. Malek,58 U. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Mehlhase,114 A Mehta 90 T Meideck 58 B Meirose 43 D Melini 174 B R Mellado Garcia 33c J D Mellenthin 53 M Melo 28a F Meloni 46 C. J. McNicol,178 R. A. McPherson,176,p J. E. Mdhluli,33c Z. A. Meadows,102 S. Meehan,148 T. Megy,52 S. Mehlhase,114 A. Mehta,90 T. Meideck,58 B. Meirose,43 D. Melini,174 B. R. Mellado Garcia,33c J. D. Mellenthin,53 M. Melo,28a F. Meloni,46 A. Mehta,90 T. Meideck,58 B. Meirose,43 D. Melini,174 B. R. Mellado Garcia,33c J. D. Mellenthin A. Melzer,24 S. B. Menary,100 E. D. Mendes Gouveia,140a,140e L. Meng,36 X. T. Meng,105 S. Menke,115 E. Meoni,41b,41a 19 139 20 54 69 69b 68 113 110 A. Melzer,24 S. B. Menary,100 E. D. Mendes Gouveia,140a,140e L. Meng,36 X. T. Meng,105 S. Menke,115 E. Meoni,41b,41a S. Mergelmeyer,19 S. A. M. Merkt,139 C. Merlassino,20 P. Mermod,54 L. Merola,69a,69b C. Meroni,68a O. Meshkov,113,110 J K R M h ki 151 A M i 72a 72b J M t lf 6 A S M t 171 C M 65 J M 160 J P M 145 S. Mergelmeyer,19 S. A. M. Merkt,139 C. Merlassino,20 P. Mermod,54 L. Merola,69a,69b C. Meroni,68a O. Meshkov,113,110 J. K. R. Meshreki,151 A. Messina,72a,72b J. Metcalfe,6 A. S. Mete,171 C. Meyer,65 J. Meyer,160 J-P. Meyer,145 H. Meyer Zu Theenhausen, F. Miano, R. P. Middleton, L. Mijović, G. Mikenberg, M. Mikestikova, M. Mikuž,91 H. Mildner,149 M. Milesi,104 A. Milic,167 D. A. Millar,92 D. W. Miller,37 A. Milov,180 D. A. Milstead,45a,45b y , , , j , g, , M. Mikuž,91 H. Mildner,149 M. Milesi,104 A. Milic,167 D. A. Millar,92 D. W. Miller,37 A. Milov,180 D. A. Milstead,45a,45b R A Mi 153 dd A A Mi k 123 M Miñ M 174 I A Mi h ili 159b A I Mi 124 B Mi d 83a M Mi 79 Y. Minegishi,163 Y. Ming,181 L. M. Mir,14 A. Mirto,67a,67b K. P. Mistry,137 T. Mitani,179 J. Mitrevski,114 V. A. Mitsou,174 M. Mittal,60c A. Miucci,20 P. S. Miyagawa,149 A. Mizukami,81 J. U. Mjörnmark,96 T. Mkrtchyan,184 M. Mlynarikova,143 T. Moa,45a,45b K. Mochizuki,109 P. Mogg,52 S. Mohapatra,39 R. Moles-Valls,24 M. C. Mondragon,106 K. Mönig,46 J. Monk,40 E M i 101 A M lb 152 J M j B li 36 M M ll 94 F M i lli 88 S M i 68a N M 132 y g j y y T. Moa,45a,45b K. Mochizuki,109 P. Mogg,52 S. Mohapatra,39 R. Moles-Valls,24 M. C. Mondragon,106 K. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Mallik,77 D. Malon,6 C. Malone,32 N. Makovec,132 B. Malaescu,136 Pa. Malecki,84 V. P. Maleev,138 F. Malek,58 U. Mallik,77 D. Malon,6 C. Malone,32 J. Manjarres Ramos,48 K. H. Mankinen,96 A. Mann,114 A. Manousos,76 B. Mansoulie,145 I. Manthos,162 S. Manzoni,120 A M i 162 G M 30 L M h 135 G M hi i 136 M M i k 141 C M 96 C A M i T b 36 M. Marjanovic,38 Z. Marshall,18 M. U. F Martensson,172 S. Marti-Garcia,174 C. B. Martin,126 T. A. Martin,178 V. J. Martin,50 B. Martin dit Latour,17 L. Martinelli,74a,74b M. Martinez,14,u V. I. Martinez Outschoorn,102 S. Martin-Haugh,144 M. Marjanovic,38 Z. Marshall,18 M. U. F Martensson,172 S. Marti-Garcia,174 C. B. Martin,126 T. A. Martin,178 V. J. Martin,50 B. Martin dit Latour,17 L. Martinelli,74a,74b M. Martinez,14,u V. I. Martinez Outschoorn,102 S. Martin-Haugh,144 V. S. Martoiu,27b A. C. Martyniuk,94 A. Marzin,36 S. R. Maschek,115 L. Masetti,99 T. Mashimo,163 R. Mashinistov,110 J. Masik,100 A. L. Maslennikov,122b,122a L. H. Mason,104 L. Massa,73a,73b P. Massarotti,69a,69b P. Mastrandrea,71a,71b B. Martin dit Latour, L. Martinelli, , M. Martinez, , V. I. Martinez Outschoorn, S. Martin-Haugh, V. S. Martoiu,27b A. C. Martyniuk,94 A. Marzin,36 S. R. Maschek,115 L. Masetti,99 T. Mashimo,163 R. Mashinistov,110 J. Masik,100 A. L. Maslennikov,122b,122a L. H. Mason,104 L. Massa,73a,73b P. Massarotti,69a,69b P. Mastrandrea,71a,71b A. Mastroberardino,41b,41a T. Masubuchi,163 A. Matic,114 P. Mättig,24 J. Maurer,27b B. Maček,91 D. A. Maximov,122b,122a 158 162 146 105 115 94 18 R. Mazini,158 I. Maznas,162 S. M. Mazza,146 S. P. Mc Kee,105 T. G. McCarthy,115 L. I. McClymont,94 W. P. McCormack,18 E. F. McDonald,104 J. A. Mcfayden,36 M. A. McKay,42 K. D. McLean,176 S. J. McMahon,144 P. C. McNamara,104 C. J. McNicol,178 R. A. McPherson,176,p J. E. Mdhluli,33c Z. A. Meadows,102 S. Meehan,148 T. Megy,52 S. Mehlhase,114 R. Mazini, I. Maznas, S. M. Mazza, S. P. Mc Kee, T. G. McCarthy, L. I. McClymont, W. P. McCormack, E. F. McDonald,104 J. A. Mcfayden,36 M. A. McKay,42 K. D. McLean,176 S. J. McMahon,144 P. C. McNamara,104 i l 178 h 176 p dhl li 33c d 102 h 148 52 hlh 114 E. F. McDonald,104 J. A. Mcfayden,36 M. A. McKay,42 K. D. McLean,176 S. J. McMahon,144 P. C. McNamara,104 C. J. McNicol,178 R. A. McPherson,176,p J. E. Mdhluli,33c Z. A. Meadows,102 S. Meehan,148 T. Megy,52 S. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Palacino,65 S. Palazzo,50 S. Palestini,36 M. Palka,83b D. Pallin,38 I. Panagoulias,10 C. E. Pandini,36 J. G. Panduro Vazquez,93 P. Pani,46 G. Panizzo,66a,66c L. Paolozzi,54 C. Papadatos,109 K. Papageorgiou,9,r A. Paramonov,6 D. Paredes Hernandez,63b S. R. Paredes Saenz,135 B. Parida,166 T. H. Park,167 I. Panagoulias, C. E. Pandini, J. G. Panduro Vazquez, P. Pani, G. Panizzo, L. Paolozzi, C. Papadatos, K. Papageorgiou,9,r A. Paramonov,6 D. Paredes Hernandez,63b S. R. Paredes Saenz,135 B. Parida,166 T. H. Park,167 A. J. Parker,89 M. A. Parker,32 F. Parodi,55b,55a E. W. P. Parrish,121 J. A. Parsons,39 U. Parzefall,52 L. Pascual Dominguez,136 V. R. Pascuzzi,167 J. M. P. Pasner,146 E. Pasqualucci,72a S. Passaggio,55b F. Pastore,93 P. Pasuwan,45a,45b S. Pataraia,99 J. R. Pater,100 A. Pathak,181 T. Pauly,36 B. Pearson,115 M. Pedersen,134 L. Pedraza Diaz,119 R. Pedro,140a T. Peiffer,53 S. V. Peleganchuk,122b,122a O. Penc,141 H. Peng,60a B. S. Peralva,80a M. M. Perego,132 A. P. Pereira Peixoto,140a 29 19 68 68b 36 69 69b 46 100 36 J. R. Pater, A. Pathak, T. Pauly, B. Pearson, M. Pedersen, L. Pedraza Diaz, R. Pedro, T. Peiffer, S. V. Peleganchuk,122b,122a O. Penc,141 H. Peng,60a B. S. Peralva,80a M. M. Perego,132 A. P. Pereira Peixoto,140a D V P lit 29 F P i 19 L P i i 68a 68b H P 36 S P ll 69a 69b K P t 46 R F Y P t 100 B A P t 36 T. C. Petersen,40 E. Petit,101 A. Petridis,1 C. Petridou,162 P. Petroff,132 M. Petrov,135 F. Petrucci,74a,74b M. Pettee,183 N. E. Pettersson,102 K. Petukhova,143 A. Peyaud,145 R. Pezoa,147b L. Pezzotti,70a,70b T. Pham,104 F. H. Phillips,106 P. W. Phillips,144 M. W. Phipps,173 G. Piacquadio,155 E. Pianori,18 A. Picazio,102 R. H. Pickles,100 R. Piegaia,30 D Pietreanu 27b J E Pilcher 37 A D Pilkington 100 M Pinamonti 73a,73b J L Pinfold 3 M Pitt 180 L Pizzimento 73a,73b , , y , , , , p , P. W. Phillips,144 M. W. Phipps,173 G. Piacquadio,155 E. Pianori,18 A. Picazio,102 R. H. Pickles,100 R. Piegaia,30 D Pietreanu 27b J E Pilcher 37 A D Pilkington 100 M Pinamonti 73a,73b J L Pinfold 3 M Pitt 180 L Pizzimento 73a,73b p pp q D. Pietreanu,27b J. E. Pilcher,37 A. D. Pilkington,100 M. Pinamonti,73a,73b J. L. Pinfold,3 M. Pitt, M.-A. Pleier,29 V. Pleskot,143 E. Plotnikova,79 D. Pluth,78 P. Podberezko,122b,122a R. Poettgen,96 R. Poggi,54 L. Poggioli,132 6 5 I. Pogrebnyak,106 D. Pohl,24 I. Pokharel,53 G. Polesello,70a A. D. Moreno,22 M. Moreno Llácer,36 C. Moreno Martinez,14 P. Morettini,55b M. Morgenstern,120 S. Morgenstern,48 D. Mori,152 M. Morii,59 M. Morinaga,179 V. Morisbak,134 A. K. Morley,36 G. Mornacchi,36 A. P. Morris,94 L. Morvaj,155 P. Moschovakos,36 B. Moser,120 M. Mosidze,159b T. Moskalets,145 H. J. Moss,149 J. Moss,31,jj K. Motohashi,165 E. Mountricha,36 E. J. W. Moyse,102 S. Muanza,101 J. Mueller,139 R. S. P. Mueller,114 D. Muenstermann,89 G. A. Mullier,96 J L M M ti 14 F J M S h 100 P M i 28b W J M 178 144 A M 68a 68b M M ški j 18 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Mönig,46 J. Monk,40 032007-27 PHYS. REV. D 100, 032007 (2019) G. AAD et al. I. Naryshkin,138 T. Naumann,46 G. Navarro,22 H. A. Neal,105,a P. Y. Nechaeva,110 F. Nechansky,46 T. J. Neep,21 A. Negri,70a,70b M. Negrini,23b C. Nellist,53 M. E. Nelson,135 S. Nemecek,141 P. Nemethy,124 M. Nessi,36,ll M. S. Neubauer,173 y p ni,23b C. Nellist,53 M. E. Nelson,135 S. Nemecek,141 P. Nemethy,124 M. Nessi,36,ll M. S. Neubauer, g g y M. Neumann,182 P. R. Newman,21 T. Y. Ng,63c Y. S. Ng,19 Y. W. Y. Ng,171 H. D. N. Nguyen,101 T. Nguyen Manh,109 E. Nibigira,38 R. B. Nickerson,135 R. Nicolaidou,145 D. S. Nielsen,40 J. Nielsen,146 N. Nikiforou,11 V. Nikolaenko,123,kk g g y M. Neumann,182 P. R. Newman,21 T. Y. Ng,63c Y. S. Ng,19 Y. W. Y. Ng,171 H. D. N. Nguyen,101 T. Nguyen Manh,109 38 135 145 40 146 11 123 kk I. Nikolic-Audit,136 K. Nikolopoulos,21 P. Nilsson,29 H. R. Nindhito,54 Y. Ninomiya,81 A. Nisati,72a N. Nishu,60c R. Nisius,115 I. Nitsche,47 T. Nitta,179 T. Nobe,163 Y. Noguchi,85 M. Nomachi,133 I. Nomidis,136 M. A. Nomura,29 M. Nordberg,36 N. Norjoharuddeen,135 T. Novak,91 O. Novgorodova,48 R. Novotny,142 L. Nozka,130 K. Ntekas,171 E. Nurse,94 34 115 136 82 39 147 26 87 S. Odaka,81 S. Oerdek,53 A. Ogrodnik,83a A. Oh,100 S. H. Oh,49 C. C. Ohm,154 H. Oide,55b,55a M. L. Ojeda,167 H. Okawa,169 Y. Okazaki,85 Y. Okumura,163 T. Okuyama,81 A. Olariu,27b L. F. Oleiro Seabra,140a S. A. Olivares Pino,147a S. Odaka,81 S. Oerdek,53 A. Ogrodnik,83a A. Oh,100 S. H. Oh,49 C. C. Ohm,154 H. Oide,55b,55a M. L. Ojeda,167 H. Okawa,169 y D. Oliveira Damazio,29 J. L. Oliver,1 M. J. R. Olsson,171 A. Olszewski,84 J. Olszowska,84 D. C. O’Neil,152 A. Onofre,140a,140e E. Onyisi,11 H. Oppen,134 M. J. Oreglia,37 G. E. Orellana,88 D. Orestano,74a,74b N. Orlando,14 R. S Onogi,117 P. U. E. Onyisi,11 H. Oppen,134 M. J. Oreglia,37 G. E. Orellana,88 D. Orestano,74a,74b N 5 60 30 8 35d 134 V. O’Shea,57 R. Ospanov,60a G. Otero y Garzon,30 H. Otono,87 M. Ouchrif,35d F. Ould-Saada,134 A. Ouraou,145 Q. Ouyang,15a M. Owen,57 R. E. Owen,21 V. E. Ozcan,12c N. Ozturk,8 J. Pacalt,130 H. A. Pacey,32 K. Pachal,49 A. Pacheco Pages,14 y g C. Padilla Aranda,14 S. Pagan Griso,18 M. Paganini,183 G. Palacino,65 S. Palazzo,50 S. Palestini,36 M. Palka,83b D. Pallin,38 li 10 C di i 36 G d 93 i 46 G i 66a 66c l i 54 C d 109 C. Padilla Aranda,14 S. Pagan Griso,18 M. Paganini,183 G. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Poley,18 A. Policicchio,72a,72b R. Polifka,143 A. Polini,23b C. S. Pollard,46 V. Polychronakos,29 D. Ponomarenko,112 L. Pontecorvo,36 S. Popa,27a G. A. Popeneciu,27d I. Pogrebnyak, D. Pohl, I. Pokharel, G. Polesello, A. Poley, A. Policicchio, R. Polifka, A. Polini, C. S. Pollard,46 V. Polychronakos,29 D. Ponomarenko,112 L. Pontecorvo,36 S. Popa,27a G. A. Popeneciu,27d 58 142 46 79 32 11 96 36 D. M. Portillo Quintero,58 S. Pospisil,142 K. Potamianos,46 I. N. Potrap,79 C. J. Potter,32 H. Potti,11 T. Poulsen,96 J. Poveda,36 T. D. Powell,149 G. Pownall,46 M. E. Pozo Astigarraga,36 P. Pralavorio,101 S. Prell,78 D. Price,100 M. Primavera,67a S. Prince,103 M. L. Proffitt,148 N. Proklova,112 K. Prokofiev,63c F. Prokoshin,147b S. Protopopescu,29 J. Proudfoot,6 M. Przybycien,83a D. Pudzha,138 A. Puri,173 P. Puzo,132 J. Qian,105 Y. Qin,100 A. Quadt,53 M. Queitsch-Maitland,46 A. Qureshi,1 P. Rados,104 F. Ragusa,68a,68b G. Rahal,97 J. A. Raine,54 S. Rajagopalan,29 A. Ramirez Morales,92 K. Ran,15a,15d 132 5 68 68b 46 114 99 149 180 M. Przybycien, D. Pudzha, A. Puri, P. Puzo, J. Qian, Y. Qin, A. Quadt, M. Queitsch-Maitland, A. Qureshi,1 P. Rados,104 F. Ragusa,68a,68b G. Rahal,97 J. A. Raine,54 S. Rajagopalan,29 A. Ramirez Morales,92 K. Ran,15a,15d Raspopov,5 M. G. Ratti,68a,68b D. M. Rauch,46 F. Rauscher,114 S. Rave,99 B. Ravina,149 I. Ravin p p J. H. Rawling,100 M. Raymond,36 A. L. Read,134 N. P. Readioff,58 M. Reale,67a,67b D. M. Rebuzzi,70a,70b A. Redelbach,177 J. H. Rawling,100 M. Raymond,36 A. L. Read,134 N. P. Readioff,58 M. Reale,67a,67b D. M. Rebu J. H. Rawling,100 M. Raymond,36 A. L. Read,134 N. P. Readioff,58 M. Reale,67a,67b D. M. Rebuzzi,70a,70b A. Redelbach,177 G. Redlinger,29 K. Reeves,43 L. Rehnisch,19 J. Reichert,137 D. Reikher,161 A. Reiss,99 A. Rej,151 C. Rembser,36 M. Renda,27b M. Rescigno,72a S. Resconi,68a E. D. Resseguie,137 S. Rettie,175 E. Reynolds,21 O. L. Rezanova,122b,122a P. Reznicek,143 E. Ricci,75a,75b R. Richter,115 S. Richter,46 E. Richter-Was,83b O. Ricken,24 M. Ridel,136 P. Rieck,115 C. J. Riegel,182 O. Rifki,46 M. Rijssenbeek,155 A. Rimoldi,70a,70b M. Rimoldi,20 L. Rinaldi,23b G. Ripellino,154 B. Ristić,89 E. Ritsch,36 I. Riu,14 J. C. Rivera Vergara,147a F. Rizatdinova,129 E. Rizvi,92 C. Rizzi,36 R. T. Roberts,100 S. H. Robertson,103,p M. Robin,46 M. Rescigno,72a S. Resconi,68a E. D. Resseguie,137 S. Rettie,175 E. Reynolds,21 O. L. Rezanova,122b,122a P. Reznicek,143 E. Ricci,75a,75b R. Richter,115 S. Richter,46 E. Richter-Was,83b O. Ricken,24 M. Ridel,136 P. Rieck,115 C. J. Riegel,182 g O. Rifki,46 M. Rijssenbeek,155 A. Rimoldi,70a,70b M. Rimoldi,20 L. Rinaldi,23b G. Ripellino,154 B. Ristić,89 E. Ritsch,36 I. Riu,14 J. C. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Rivera Vergara,147a F. Rizatdinova,129 E. Rizvi,92 C. Rizzi,36 R. T. Roberts,100 S. H. Robertson,103,p M. Robin,46 032007-28 PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … D. Salihagic,115,a A. Salnikov,153 J. Salt,174 D. Salvatore,41b,41a F. Salvatore,156 A. Salvucci,63a,63b,63c A. Salzburger,36 J. Samarati,36 D. Sammel,52 D. Sampsonidis,162 D. Sampsonidou,162 J. Sánchez,174 A. Sanchez Pineda,66a,66c H. Sandaker,134 C. O. Sander,46 I. G. Sanderswood,89 M. Sandhoff,182 C. Sandoval,22 D. P. C. Sankey,144 M. Sannino,55b,55a Y. Sano,117 , , p , p , , , , C. O. Sander,46 I. G. Sanderswood,89 M. Sandhoff,182 C. Sandoval,22 D. P. C. Sankey,144 M. Sannino,55b,55a Y. Sano,117 A. Sansoni, C. Santoni, H. Santos, S. N. Santpur, A. Santra, A. Sapronov, J. G. Saraiva, O. Sasaki, K. Sato,169 E. Sauvan,5 P. Savard,167,e N. Savic,115 R. Sawada,163 C. Sawyer,144 L. Sawyer,95,nn C. Sbarra,23b A. Sbrizzi,23a l 94 h h id 148 h h 115 h h 114 h f 37 h f 137 h ff 99 K. Sato,169 E. Sauvan,5 P. Savard,167,e N. Savic,115 R. Sawada,163 C. Sawyer,144 L. Sawyer,95,nn C. Sbarra,23b A. Sbrizzi,23a T. Scanlon,94 J. Schaarschmidt,148 P. Schacht,115 B. M. Schachtner,114 D. Schaefer,37 L. Schaefer,137 J. Schaeffer,99 T. Scanlon, J. Schaarschmidt, P. Schacht, B. M. Schachtner, D. Schaefer, L. Schaefer, J. Schaeffer, S. Schaepe,36 U. Schäfer,99 A. C. Schaffer,132 D. Schaile,114 R. D. Schamberger,155 N. Scharmberg,100 V. A. Schegelsky,138 D. Scheirich,143 F. Schenck,19 M. Schernau,171 C. Schiavi,55b,55a S. Schier,146 L. K. Schildgen,24 Z. M. Schillaci,26 E. J. Schioppa,36 M. Schioppa,41b,41a K. E. Schleicher,52 S. Schlenker,36 K. R. Schmidt-Sommerfeld,115 K. Schmieden,36 L. Serkin,66a,66b M. Sessa,60a H. Severini,128 F. Sforza,170 A. Sfyrla,54 E. Shabalina,53 J. D. Shahinian,146 N. W. Shaikh,45a,45b D. Shaked Renous,180 L. Y. Shan,15a R. Shang,173 J. T. Shank,25 M. Shapiro,18 A. Sharma,135 A. S. Sharma,1 P. B. Shatalov,111 K. Shaw,156 S. M. Shaw,100 A. Shcherbakova,138 Y. Shen,128 N. Sherafati,34 A. D. Sherman,25 P. Sherwood,94 L. Shi,158,oo S. Shimizu,81 C. O. Shimmin,183 Y. Shimogama,179 M. Shimojima,116 I. P. J. Shipsey,135 S. Shirabe,87 M. Shiyakova,79,pp J. Shlomi,180 A. Shmeleva,110 M. J. Shochet,37 J. Shojaii,104 D. R. Shope,128 S. Shrestha,126 E. Shulga,180 P. Sicho,141 A. M. Sickles,173 P. E. Sidebo,154 E. Sideras Haddad,33c O. Sidiropoulou,36 A. Sidoti,23b,23a F. Siegert,48 Dj. Sijacki,16 M. Silva Jr.,181 M. V. Silva Oliveira,80a S. B. Silverstein,45a S. Simion,132 E. Simioni,99 D. Shaked Renous,180 L. Y. Shan,15a R. Shang,173 J. T. Shank,25 M. Shapiro,18 A. Sharma,135 A. S. Sharma,1 P. B. Shatalov,111 K. Shaw,156 S. M. Shaw,100 A. Shcherbakova,138 Y. Shen,128 N. Sherafati,34 A. D. Sherman,25 P. Sherwood,94 L. Shi,158,oo S. Shimizu,81 C. O. D. Robinson,32 J. E. M. Robinson,46 A. Robson,57 E. Rocco,99 C. Roda,71a,71b S. Rodriguez Bosca,174 A. Rodriguez Perez,14 D. Rodriguez Rodriguez,174 A. M. Rodríguez Vera,168b S. Roe,36 O. Røhne,134 R. Röhrig,115 C. P. A. Roland,65 J. Roloff,59 A. Romaniouk,112 M. Romano,23b,23a N. Rompotis,90 M. Ronzani,124 L. Roos,136 S. Rosati,72a K. Rosbach,52 N-A. Rosien,53 G. Rosin,102 B. J. Rosser,137 E. Rossi,46 E. Rossi,74a,74b E. Rossi,69a,69b L. P. Rossi,55b L. Rossini,68a,68b R. Rosten,14 M. Rotaru,27b J. Rothberg,148 D. Rousseau,132 G. Rovelli,70a,70b D. Roy,33c A. Rozanov,101 Y. Rozen,160 X. Ruan,33c F. Rubbo,153 F. Rühr,52 A. Ruiz-Martinez,174 A. Rummler,36 Z. Rurikova,52 N. A. Rusakovich,79 H. L. Russell,103 L. Rustige,38,47 J. P. Rutherfoord,7 E. M. Rüttinger,46,mm Y. F. Ryabov,138 M. Rybar,39 G. Rybkin,132 A. Ryzhov,123 G. F. Rzehorz,53 P. Sabatini,53 G. Sabato,120 S. Sacerdoti,132 H. F-W. Sadrozinski,146 R. Sadykov,79 F. Safai Tehrani,72a B. Safarzadeh Samani,156 P. Saha,121 S. Saha,103 M. Sahinsoy,61a A. Sahu,182 M. Saimpert,46 M. Saito,163 T. Saito,163 H. Sakamoto,163 A. Sakharov,124,ee D. Salamani,54 G. Salamanna,74a,74b J. E. Salazar Loyola,147b P. H. Sales De Bruin,172 D. Salihagic,115,a A. Salnikov,153 J. Salt,174 D. Salvatore,41b,41a F. Salvatore,156 A. Salvucci,63a,63b,63c A. Salzburger,36 J. Samarati,36 D. Sammel,52 D. Sampsonidis,162 D. Sampsonidou,162 J. Sánchez,174 A. Sanchez Pineda,66a,66c H. Sandaker,134 C. O. Sander,46 I. G. Sanderswood,89 M. Sandhoff,182 C. Sandoval,22 D. P. C. Sankey,144 M. Sannino,55b,55a Y. Sano,117 A. Sansoni,51 C. Santoni,38 H. Santos,140a,140b S. N. Santpur,18 A. Santra,174 A. Sapronov,79 J. G. Saraiva,140a,140d O. Sasaki,81 K. Sato,169 E. Sauvan,5 P. Savard,167,e N. Savic,115 R. Sawada,163 C. Sawyer,144 L. Sawyer,95,nn C. Sbarra,23b A. Sbrizzi,23a T. Scanlon,94 J. Schaarschmidt,148 P. Schacht,115 B. M. Schachtner,114 D. Schaefer,37 L. Schaefer,137 J. Schaeffer,99 S. Schaepe,36 U. Schäfer,99 A. C. Schaffer,132 D. Schaile,114 R. D. Schamberger,155 N. Scharmberg,100 V. A. Schegelsky,138 D. Scheirich,143 F. Schenck,19 M. Schernau,171 C. Schiavi,55b,55a S. Schier,146 L. K. Schildgen,24 Z. M. Schillaci,26 E. J. Schioppa,36 M. Schioppa,41b,41a K. E. Schleicher,52 S. Schlenker,36 K. R. Schmidt-Sommerfeld,115 K. Schmieden,36 C. Schmitt,99 S. Schmitt,46 S. Schmitz,99 J. C. Schmoeckel,46 U. Schnoor,52 L. Schoeffel,145 A. Schoening,61b E. Schopf,135 M. Schott,99 J. F. P. Schouwenberg,119 J. Schovancova,36 S. Schramm,54 F. Schroeder,182 A. Schulte,99 H-C. Schultz-Coulon,61a M. Schumacher,52 B. A. Schumm,146 Ph. Schune,145 A. Schwartzman,153 T. A. Schwarz,105 Ph. Schwemling,145 R. Schwienhorst,106 A. Sciandra,146 G. Sciolla,26 M. Scodeggio,46 M. Scornajenghi,41b,41a F. Scuri,71a F. Scutti,104 L. M. Scyboz,115 C. D. Sebastiani,72a,72b P. Seema,19 S. C. Seidel,118 A. Seiden,146 T. Seiss,37 J. M. Seixas,80b G. Sekhniaidze,69a K. Sekhon,105 S. J. Sekula,42 N. Semprini-Cesari,23b,23a S. Sen,49 S. Senkin,38 C. Serfon,76 L. Serin,132 L. Serkin,66a,66b M. Sessa,60a H. Severini,128 F. Sforza,170 A. Sfyrla,54 E. Shabalina,53 J. D. Shahinian,146 N. W. Shaikh,45a,45b D. Shaked Renous,180 L. Y. Shan,15a R. Shang,173 J. T. Shank,25 M. Shapiro,18 A. Sharma,135 A. S. Sharma,1 P B Sh t l 111 K Sh 156 S M Sh 100 A Sh h b k 138 Y Sh 128 N Sh f ti 34 A D Sh 25 T. Sykora,143 D. Ta,99 K. Tackmann,46,ss J. Taenzer,161 A. Taffard,171 R. Tafirout,168a E. Tahirovic,92 H. Takai,29 R. Takashima,86 K. Takeda,82 T. Takeshita,150 E. P. Takeva,50 Y. Takubo,81 M. Talby,101 A. A. Talyshev,122b,122a N. M. Tamir,161 J. Tanaka,163 M. Tanaka,165 R. Tanaka,132 B. B. Tannenwald,126 S. Tapia Araya,173 S. Tapprogge,99 A. Tarek Abouelfadl Mohamed,136 S. Tarem,160 G. Tarna,27b,tt G. F. Tartarelli,68a P. Tas,143 M. Tasevsky,141 T. Tashiro,85 E. Tassi,41b,41a A. Tavares Delgado,140a,140b Y. Tayalati,35e A. J. Taylor,50 G. N. Taylor,104 W. Taylor,168b A. S. Tee,89 R. Teixeira De Lima,153 P. Teixeira-Dias,93 H. Ten Kate,36 J. J. Teoh,120 S. Terada,81 K. Terashi,163 J. Terron,98 S. Terzo,14 M. Testa,51 R. J. Teuscher,167,p S. J. Thais,183 T. Theveneaux-Pelzer,46 F. Thiele,40 D. W. Thomas,93 J. O. Thomas,42 J. P. Thomas,21 A. S. Thompson,57 P. D. Thompson,21 L. A. Thomsen,183 E. Thomson,137 Y. Tian,39 R. E. Ticse Torres,53 V. O. Tikhomirov,110,uu Yu. A. Tikhonov,122b,122a S. Timoshenko,112 P. Tipton,183 S. Tisserant,101 K. Todome,23b,23a S. Todorova-Nova,5 S. Todt,48 J. Tojo,87 S. Tokár,28a K. Tokushuku,81 E. Tolley,126 K. G. Tomiwa,33c M. Tomoto,117 L. Tompkins,153,dd K. Toms,118 B. Tong,59 P. Tornambe,102 E. Torrence,131 H. Torres,48 E. Torró Pastor,148 C. Tosciri,135 J. Toth,101,vv D. R. Tovey,149 C. J. Treado,124 T. Trefzger,177 F. Tresoldi,156 A. Tricoli,29 I. M. Trigger,168a S. Trincaz-Duvoid,136 W. Trischuk,167 B. Trocm´e,58 A. Trofymov,132 C. Troncon,68a M. Trovatelli,176 F. Trovato,156 L. Truong,33b M. Trzebinski,84 A. Trzupek,84 F. Tsai,46 J. C-L. Tseng,135 P. V. Tsiareshka,107,ff A. Tsirigotis,162 N. Tsirintanis,9 V. Tsiskaridze,155 E. G. Tskhadadze,159a M. Tsopoulou,162 I. I. Tsukerman,111 V. Tsulaia,18 S. Tsuno,81 D. Tsybychev,155 Y. Tu,63b A. Tudorache,27b V. Tudorache,27b T. T. Tulbure,27a A. N. Tuna,59 S. Turchikhin,79 D. Turgeman,180 I. Turk Cakir,4b,ww R. J. Turner,21 R. T. Turra,68a P. M. Tuts,39 S. Tzamarias,162 E. Tzovara,99 G. Ucchielli,47 K. Uchida,163 I. Ueda,81 M. Ughetto,45a,45b F. Ukegawa,169 G. Unal,36 A. Undrus,29 G. Unel,171 F. C. Ungaro,104 Y. Unno,81 K. Uno,163 J. Urban,28b P. Urquijo,104 G. Usai,8 J. Usui,81 L. Vacavant,101 V. Vacek,142 B. Vachon,103 K. O. H. Vadla,134 A Vaidya 94 C Valderanis 114 E Valdes Santurio 45a,45b M Valente 54 S Valentinetti 23b,23a A Valero 174 L Val´ery 46 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Shimmin,183 Y. Shimogama,179 M. Shimojima,116 I. P. J. Shipsey,135 S. Shirabe,87 M. Shiyakova,79,pp J. Shlomi,180 A. Shmeleva,110 M. J. Shochet,37 J. Shojaii,104 D. R. Shope,128 S. Shrestha,126 E. Shulga,180 P. Sicho,141 A. M. Sickles,173 P. E. Sidebo,154 E. Sideras Haddad,33c O. Sidiropoulou,36 A. Sidoti,23b,23a R. Simoniello,99 P. Sinervo,167 N. B. Sinev,131 M. Sioli,23b,23a I. Siral,105 S. Yu. Sivoklokov,113 J. Sjölin,45a,45b E. Skorda,96 , , , , , , , , S. Yu. Smirnov,112 Y. Smirnov,112 L. N. Smirnova,113,qq O. Smirnova,96 J. W. Smith,53 M. Smizanska,89 K. Smolek,142 84 110 120 131 29 176 p 171 161 P. Steinberg,29 A. L. Steinhebel,131 B. Stelzer,152 H. J. Stelzer,139 O. Stelzer-Chilton,168a H. Stenzel,56 T. J. Stevenson,156 032007-29 PHYS. REV. D 100, 032007 (2019) G. AAD et al. D. Turgeman,180 I. Turk Cakir,4b,ww R. J. Turner,21 R. T. Turra,68a P. M. Tuts,39 S. Tzamarias,162 E. Tzovara,99 G. Ucchielli,47 K. Uchida,163 I. Ueda,81 M. Ughetto,45a,45b F. Ukegawa,169 G. Unal,36 A. Undrus,29 G. Unel,171 K. Uno,163 J. Urban,28b P. Urquijo,104 G. Usai,8 J. Usui,81 L. Vacavant,101 V. Vacek,142 B. Vachon,103 K. O. H. Vadla,134 A. Vaidya,94 C. Valderanis,114 E. Valdes Santurio,45a,45b M. Valente,54 S. Valentinetti,23b,23a A. Valero,174 L. Val´ery,46 R. A. Vallance,21 A. Vallier,36 J. A. Valls Ferrer,174 T. R. Van Daalen,14 P. Van Gemmeren,6 I. Van Vulpen,120 M V di 73a 73b W V d lli 36 A V i hi 166 D V i l 72a 72b R V i 72a E W V 7 C V i 55b 55a T V l 42 y , , , , , , y, R. A. Vallance,21 A. Vallier,36 J. A. Valls Ferrer,174 T. R. Van Daalen,14 P. Van Gemmeren,6 I. Van Vulpen,120 M Vanadia 73a,73b W Vandelli 36 A Vaniachine 166 D Vannicola 72a,72b R Vari 72a E W Varnes 7 C Varni 55b,55a T Varol 42 R. A. Vallance,21 A. Vallier,36 J. A. Valls Ferrer,174 T. R. Van Daalen,14 P. Van Gemmeren,6 I. Van Vulpen,120 M. Vanadia,73a,73b W. Vandelli,36 A. Vaniachine,166 D. Vannicola,72a,72b R. Vari,72a E. W. Varnes,7 C. Varni,55b,55a T. Varol,42 132 157 27b 176 183 38 14 D. Varouchas,132 K. E. Varvell,157 M. E. Vasile,27b G. A. Vasquez,176 J. G. Vasquez,183 F. Vazeille,38 D. Vazquez Furelos,14 T V S h d 36 J V h 53 V V hi 74a 74b M J V 120 L M V l 167 F V l 140a 140c S V i 72a D. Varouchas,132 K. , g, g , , , , , g , S. Wenig,36 N. Wermes,24 M. D. Werner,78 P. Werner,36 M. Wessels,61a T. D. Weston,20 K. Whalen,131 N. L. Whallon,148 A. M. Wharton,89 A. S. White,105 A. White,8 M. J. White,1 D. Whiteson,171 B. W. Whitmore,89 F. J. Wickens,144 W. Wiedenmann,181 M. Wielers,144 N. Wieseotte,99 C. Wiglesworth,40 L. A. M. Wiik-Fuchs,52 F. Wilk,100 H. G. Wilkens,36 L. J. Wilkins,93 H. H. Williams,137 S. Williams,32 C. Willis,106 S. Willocq,102 J. A. Wilson,21 I. Wingerter-Seez,5 E. Winkels,156 F. Winklmeier,131 O. J. Winston,156 B. T. Winter,52 M. Wittgen,153 M. Wobisch,95 A. Wolf,99 T. M. H. Wolf,120 R. Wolff,101 R. W. Wölker,135 J. Wollrath,52 M. W. Wolter,84 H. Wolters,140a,140c V. W. S. Wong,175 N. L. Woods,146 S. D. Worm,21 B. K. Wosiek,84 K. W. Woźniak,84 K. Wraight,57 S. L. Wu,181 X. Wu,54 Y. Wu,60a T. R. Wyatt,100 B. M. Wynne,50 S. Xella,40 Z. Xi,105 L. Xia,178 D. Xu,15a H. Xu,60a,tt L. Xu,29 T. Xu,145 W. Xu,105 Z. Xu,60b Z. Xu,153 B. Yabsley,157 S. Yacoob,33a K. Yajima,133 D. P. Yallup,94 D. Yamaguchi,165 Y. Yamaguchi,165 A. Yamamoto,81 T. Yamanaka,163 F. Yamane,82 M. Yamatani,163 T. Yamazaki,163 Y. Yamazaki,82 Z. Yan,25 H. J. Yang,60c,60d H. T. Yang,18 S. Yang,77 X. Yang,60b,58 Y. Yang,163 Z. Yang,17 W-M. Yao,18 Y. C. Yap,46 Y. Yasu,81 E. Yatsenko,60c,60d J. Ye,42 S. Ye,29 I. Yeletskikh,79 M. R. Yexley,89 E. Yigitbasi,25 E. Yildirim,99 K. Yorita,179 K. Yoshihara,137 C. J. S. Young,36 C. Young,153 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … J. Yu,78 R. Yuan,60b X. Yue,61a S. P. Y. Yuen,24 B. Zabinski,84 G. Zacharis,10 E. Zaffaroni,54 J. Zahreddine,136 R. Zaidan,14 A. M. Zaitsev,123,kk T. Zakareishvili,159b N. Zakharchuk,34 S. Zambito,59 D. Zanzi,36 D. R. Zaripovas,57 S. V. Zeißner,47 C. Zeitnitz,182 G. Zemaityte,135 J. C. Zeng,173 O. Zenin,123 D. Zerwas,132 M. Zgubič,135 D. F. Zhang,15b F. Zhang,181 G. Zhang,60a G. Zhang,15b H. Zhang,15c J. Zhang,6 L. Zhang,15c L. Zhang,60a M. Zhang,173 R. Zhang,60a R. Zhang,24 X. Zhang,60b Y. Zhang,15a,15d Z. Zhang,63a Z. Zhang,132 P. Zhao,49 Y. Zhao,60b Z. Zhao,60a A. Zhemchugov,79 Z. Zheng,105 D. Zhong,173 B. Zhou,105 C. Zhou,181 M. S. Zhou,15a,15d M. Zhou,155 N. Zhou,60c Y. Zhou,7 C. G. Zhu,60b H. L. Zhu,60a H. Zhu,15a J. Zhu,105 Y. Zhu,60a X. Zhuang,15a K. Zhukov,110 V. Zhulanov,122b,122a D. Zieminska,65 N. I. Zimine,79 S. Zimmermann,52 Z. Zinonos,115 M. Ziolkowski,151 G. Zobernig,181 A. Zoccoli,23b,23a K. Zoch,53 T. G. Zorbas,149 37 36 R. Zou,37 and L. Zwalinski36 R. Zou,37 and L. Zwalinski36 SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … E. Varvell,157 M. E. Vasile,27b G. A. Vasquez,176 J. G. Vasquez,183 F. Vazeille,38 D. Vazquez Furelos,14 T. Vazquez Schroeder,36 J. Veatch,53 V. Vecchio,74a,74b M. J. Veen,120 L. M. Veloce,167 F. Veloso,140a,140c S. Veneziano,72a q A. Ventura,67a,67b N. Venturi,36 A. Verbytskyi,115 V. Vercesi,70a M. Verducci,74a,74b C. M. Vergel Infante,78 C. Vergis,24 uri,36 A. Verbytskyi,115 V. Vercesi,70a M. Verducci,74a,74b C. M. Vergel Infante,78 C. Vergis,24 120 120 152 147b 54 W. Verkerke,120 A. T. Vermeulen,120 J. C. Vermeulen,120 M. C. Vetterli,152,e N. Viaux Maira,147b M. Vicente Barreto Pinto,54 T. Vickey,149 O. E. Vickey Boeriu,149 G. H. A. Viehhauser,135 L. Vigani,135 M. Villa,23b,23a M. Villaplana Perez,68a,68b T. Vickey,149 O. E. Vickey Boeriu,149 G. H. A. Viehhauser,135 L. Vigani,135 M. Villa,23b,23a M. Villaplana Perez,68a,68b P. Vokac,142 S. E. von Buddenbrock,33c E. Von Toerne,24 V. Vorobel,143 K. Vorobev,112 M. Vos,174 J. H. Vossebeld,90 N. Vranjes,16 M. Vranjes Milosavljevic,16 V. Vrba,142 M. Vreeswijk,120 T. Šfiligoj,91 R. Vuillermet,36 I. Vukotic,37 T. Ženiš,28a L. Živković,16 P. Wagner,24 W. Wagner,182 J. Wagner-Kuhr,114 H. Wahlberg,88 K. Wakamiya,82 032007-30 PHYS. REV. D 100, 032007 (2019) EARCH FOR ELECTROWEAK DIBOSON PRODUCTION … (ATLAS Collaboration) 1Department of Physics, University of Adelaide, Adelaide, Australia 2Physics Department, SUNY Albany, Albany, New York, USA 3Department of Physics, University of Alberta, Edmonton, Alberta, Canada 4aDepartment of Physics, Ankara University, Ankara, Turkey 4bIstanbul Aydin University, Istanbul, Turkey 4cDivision of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5LAPP, Universit´e Grenoble Alpes, Universit´e Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 6High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA 7Department of Physics, University of Arizona, Tucson, Arizona, USA 8Department of Physics, University of Texas at Arlington, Arlington, Texas, USA 9Physics Department, National and Kapodistrian University of Athens, Athens, Greece 10Physics Department, National Technical University of Athens, Zografou, Greece 11Department of Physics, University of Texas at Austin, Austin, Texas, USA 12aBahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 12bIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 12cDepartment of Physics, Bogazici University, Istanbul, Turkey 12dDepartment of Physics Engineering, Gaziantep University, Gaziantep, Turkey 13Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14Institut de Física d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, Barcelona, Spain 15aInstitute of High Energy Physics, Chinese Academy of Sciences, Beijing, China 15bPhysics Department, Tsinghua University, Beijing, China 15cDepartment of Physics, Nanjing University, Nanjing, China 15dUniversity of Chinese Academy of Science (UCAS), Beijing, China 16Institute of Physics, University of Belgrade, Belgrade, Serbia 17Department for Physics and Technology, University of Bergen, Bergen, Norway 18Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, California, USA 19Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany 20Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 21School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 22Facultad de Ciencias y Centro de Investigaciónes, Universidad Antonio Nariño, Bogota, Colombia 23aINFN Bologna and Universita’ di Bologna, Dipartimento di Fisica, Italy 23bINFN Sezione di Bologna, Italy 24Physikalisches Institut, Universität Bonn, Bonn, Germany 25Department of Physics, Boston University, Boston, Massachusetts, USA 26Department of Physics, Brandeis University, Waltham, Massachusetts, USA 27aTransilvania University of Brasov, Brasov, Romania 27bHoria Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 27cDepartment of Physics, Alexandru Ioan Cuza University of Iasi, Iasi, Romania 27dNational Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca, Romania 27eUniversity Politehnica Bucharest, Bucharest, Romania 27fWest University in Timisoara, Timisoara, Romania 28aFaculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic Berkeley, California, USA 19Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany 20Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland l of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 21School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 22 22Facultad de Ciencias y Centro de Investigaciónes, Universidad Antonio Nariño, Bogota, Colombia 23aINFN Bologna and Universita’ di Bologna, Dipartimento di Fisica, Italy 23b ad de Ciencias y Centro de Investigaciónes, Universidad Antonio Nariño, Bogota, Colombia 23aINFN Bologna and Universita’ di Bologna, Dipartimento di Fisica, Italy 23b Physics Department, Cluj-Napoca, Romania Physics Department, Cluj-Napoca, Romania 27eUniversity Politehnica Bucharest, Bucharest, Romania 27f 27eUniversity Politehnica Bucharest, Bucharest, Romania 27f 27fWest University in Timisoara, Timisoara, Romania 28aFaculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic 23bINFN Sezione di Bologna, Italy 24Physikalisches Institut, Universität Bonn, Bonn, Germany 25Department of Physics, Boston University, Boston, Massachusetts, USA 6 25Department of Physics, Boston University, Boston, Massachusetts, USA 6 26Department of Physics, Brandeis University, Waltham, Massachusetts, USA 27 26Department of Physics, Brandeis University, Waltham, Massachusetts, USA 27 Transilvania University of Brasov, Brasov, Romania 27aTransilvania University of Brasov, Brasov, Romania b 27bHoria Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 27 27cDepartment of Physics, Alexandru Ioan Cuza University of Iasi, Iasi, Romania 27d 27dNational Institute for Research and Development of Isotopic and Molecular Technologies, 55bINFN Sezione di Genova, Italy 60cSchool of Physics and Astronomy, Shanghai Jiao Tong University, KLPPAC-MoE, SKLPPC, Shanghai, China 60d Tsung Dao Lee Institute, Shanghai, China 61aKirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 61bPhysikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 62Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 63aDepartment of Physics, Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China 63bDepartment of Physics, University of Hong Kong, Hong Kong, China 63cDepartment of Physics and Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 64Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 65Department of Physics, Indiana University, Bloomington, Indiana, USA 64Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 65 65Department of Physics, Indiana University, Bloomington, Indiana, USA 032007-31 032007-31 28bDepartment of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 29Physics Department, Brookhaven National Laboratory, Upton, New York, USA 30Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 31California State University, California, USA 32Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 33aDepartment of Physics, University of Cape Town, Cape Town, South Africa 33bDepartment of Mechanical Engineering Science, University of Johannesburg, Johannesburg, South Africa 33cSchool of Physics, University of the Witwatersrand, Johannesburg, South Africa 34Department of Physics, Carleton University, Ottawa, Ontario, Canada 35aFacult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies—Universit´e Hassan II, Casablanca, Morocco 35bFacult´e des Sciences, Universit´e Ibn-Tofail, K´enitra, Morocco 35cFacult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech, Morocco 35dFacult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda, Morocco 35eFacult´e des sciences, Universit´e Mohammed V, Rabat, Morocco 36CERN, Geneva, Switzerland 37Enrico Fermi Institute, University of Chicago, Chicago, Illinois, USA 38LPC, Universit´e Clermont Auvergne, CNRS/IN2P3, Clermont-Ferrand, France 39Nevis Laboratory, Columbia University, Irvington, New York, USA 40Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 41aDipartimento di Fisica, Universit`a della Calabria, Rende, Italy 41bINFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy 42Physics Department, Southern Methodist University, Dallas, Texas, USA 43Physics Department, University of Texas at Dallas, Richardson, Texas, USA 44National Centre for Scientific Research “Demokritos”, Agia Paraskevi, Greece 45aDepartment of Physics, Stockholm University, Sweden 45bOskar Klein Centre, Stockholm, Sweden 46Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 47Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 48Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 49Department of Physics, Duke University, Durham, North Carolina, USA 50SUPA—School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 51INFN e Laboratori Nazionali di Frascati, Frascati, Italy 52Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 53II. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany 54D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerland 55aDipartimento di Fisica, Universit`a di Genova, Genova, Italy 55bINFN Sezione di Genova, Italy 56II. 032007-31 Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 57SUPA—School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 58LPSC, Universit´e Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 59Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA 60aDepartment of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology of China, Hefei, China 60bInstitute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao, China 60cSchool of Physics and Astronomy, Shanghai Jiao Tong University, KLPPAC-MoE, SKLPPC, Shanghai, China 60dTsung-Dao Lee Institute, Shanghai, China 61aKirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 61bPhysikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 62Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 63aDepartment of Physics, Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China 63bDepartment of Physics, University of Hong Kong, Hong Kong, China 63cDepartment of Physics and Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 64Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 65Department of Physics Indiana University Bloomington Indiana USA et al. PHYS. REV. D 100, 032007 (2 PHYS. REV. D 100, 032007 (2019) G. AAD et al. 28bDepartment of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic Fukuoka, Japan p 88Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La P 89Ph i D t t L t U i it L t U it d Ki d p 88Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentin 89 89Physics Department, Lancaster University, Lancaster, United Kingdom 90 90Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 91Department of Experimental Particle Physics, Jožef Stefan Institute and Department of Physics, University of Ljubljana, Ljubljana, Slovenia 92 92School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 93Department of Physics, Royal Holloway University of London, Egham, United Kingdom 94 92School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 93Department of Physics, Royal Holloway University of London, Egham, United Kingdom 94Department of Physics and Astronomy, University College London, London, United Kingdom 95Louisiana Tech University, Ruston, Louisiana, USA 94Department of Physics and Astronomy, University College London, London, United Kingdom 95Louisiana Tech University, Ruston, Louisiana, USA 95Louisiana Tech University, Ruston, Louisiana, USA 96 96Fysiska institutionen, Lunds universitet, Lund, Sweden 97 ysiska institutionen, Lunds universitet, Lund, Swede 97Centre de Calcul de l’Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P tut National de Physique Nucl´eaire et de Physique 032007-32 032007-32 PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … 66aINFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy 66b 66aINFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy g 66bICTP, Trieste, Italy y 66cDipartimento Politecnico di Ingegneria e Architettura, Universit`a di Udine, Udine, Italy 67a y 67bDipartimento di Matematica e Fisica, Universit`a del Salento, Lecce, Italy 68 68aINFN Sezione di Milano, Italy 68bDipartimento di Fisica, Universit`a di Milano, Milano, Italy 69 p y 69bDipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 0 p y 69bDipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 70 70aINFN Sezione di Pavia, Italy 70bDipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 71 70bDipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 71 y 71bDipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 71bDipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 72 y 72bDipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 73 72bDipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 73 73aINFN Sezione di Roma Tor Vergata, Italy 73aINFN Sezione di Roma Tor Vergata, Italy 73bDipartimento di Fisica, Universit`a di Roma Tor Vergata, Roma, Italy 74 73bDipartimento di Fisica, Universit`a di Roma Tor Vergata, Roma, Italy 74 74bDipartimento di Matematica e Fisica, Universit`a Roma Tre, Roma, Italy 75 75aINFN-TIFPA, Italy y 75bUniversit`a degli Studi di Trento, Trento, Italy 76Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 77 77University of Iowa, Iowa City, Iowa, USA 78 78Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA 79 79Joint Institute for Nuclear Research, Dubna, Russia f , , 80aDepartamento de Engenharia El´etrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora Brazil partamento de Engenharia El´etrica, Universidade Federal de Juiz de Fora (UFJF), Villeurbanne, France 98Departamento de Física Teorica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, Spa 99 99Institut für Physik, Universität Mainz, Mainz, Germany 100 100School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 101CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France 102Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA 103Department of Physics, McGill University, Montreal, Quebec, Canada 104School of Physics, University of Melbourne, Victoria, Australia 102Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA 103 102Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA 103Department of Physics McGill University Montreal Quebec Canada Department of Physics, University of Massachusetts, Amherst, Massachusetts, US 103Department of Physics, McGill University, Montreal, Quebec, Canada 104 104School of Physics, University of Melbourne, Victoria, Australia 105Department of Physics, University of Michigan, Ann Arbor, Michigan, USA Juiz de Fora, Brazil 80bUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil 80cUniversidade Federal de São João del Rei (UFSJ), São João del Rei, Brazil 80dInstituto de Física, Universidade de São Paulo, São Paulo, Brazil 81KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 82 80bUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil 80cU i id d F d l d Sã J ã d l R i (UFSJ) Sã J ã d l R i B il te School of Science, Kobe University, Kobe, Japan 83aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow Poland Krakow, Poland 83bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 84 84Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 85 85Faculty of Science, Kyoto University, Kyoto, Japan 86 86Kyoto University of Education, Kyoto, Japan 86Kyoto University of Education, Kyoto, Japan 87Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, PNPI, St. Petersburg, Russia 139Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA 140aLaboratório de Instrumentação e Física Experimental de Partículas—LIP, Portugal 140b ç p , g 140bDepartamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal 140cDepartamento de Física, Universidade de Coimbra, Coimbra, Portugal 140dCentro de Física Nuclear da Universidade de Lisboa, Lisboa, Portugal 140eDepartamento de Física, Universidade do Minho, Braga, Portugal 140fUniversidad de Granada, Granada (Spain), Spain 140gDep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 141Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 142Czech Technical University in Prague, Prague, Czech Republic 143Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 144Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 145IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France 146Santa Cruz Institute for Particle Physics University of California Santa Cruz ç p g 140bDepartamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal 140cDepartamento de Física, Universidade de Coimbra, Coimbra, Portugal 140dCentro de Física Nuclear da Universidade de Lisboa, Lisboa, Portugal 140eDepartamento de Física, Universidade do Minho, Braga, Portugal 140fUniversidad de Granada, Granada (Spain), Spain 140gDep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 141Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 142Czech Technical University in Prague, Prague, Czech Republic 143Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 144Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 145IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France 146Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA 147aDepartamento de Física, Pontificia Universidad Católica de Chile, Santiago, Chile 147bDepartamento de Física, Universidad T´ecnica Federico Santa María, Valparaíso, Chile 148Department of Physics, University of Washington, Seattle, Washington, USA 149Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 150Department of Physics, Shinshu University, Nagano, Japan ç p g 140bDepartamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal 140cDepartamento de Física, Universidade de Coimbra, Coimbra, Portugal 140dCentro de Física Nuclear da Universidade de Lisboa, Lisboa, Portugal 140eDepartamento de Física, Universidade do Minho, Braga, Portugal 140fUniversidad de Granada, Granada (Spain), Spain 140gDep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 141Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic Universidade Nova de Lisboa, Caparica, Portuga Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 142Czech Technical University in Prague, Prague, Czech Republic 143 143Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 144Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 145 145IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France 146 146Santa Cruz Institute for Particle Physics, University of California Santa Cruz Moscow, Russia 114Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 115 Fakultät für Physik, Ludwig-Maximilians-Universi 5 anck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 116 115Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 116 116Nagasaki Institute of Applied Science, Nagasaki, Japan g f pp g p 117Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Jap 118 raduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan partment of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA stitute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, 118Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, US 119Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhe 032007-33 032007-33 G. AAD et al. PHYS. REV. D 100, 032007 (2019) 106Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA 107B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 108Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus 109Group of Particle Physics, University of Montreal, Montreal, Quebec, Canada 110P N Lebedev Physical Institute of the Russian Academy of Sciences Moscow Russia Institute for Theoretical and Experimental Physics of the National Research Centre Kurchatov Institute, University of Oklahoma, Norman, Oklahoma, USA 129Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 130 130Palacký University, RCPTM, Joint Laboratory of Optics, Olomouc, Czech Republic 131 131Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA 132 133Graduate School of Science, Osaka University, Osaka, Japan 134 134Department of Physics, University of Oslo, Oslo, Norway 135Department of Physics, Oxford University, Oxford, United Kingdom p f y , f y, f , g 136LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France 137Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA 138Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, 137Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA 138Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA 138Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, 138Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, Moscow, Russia , 112National Research Nuclear University MEPhI, Moscow, Russia National Research Nuclear University MEPhI, Mosc 113D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, 113D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow Russia 13D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia Santa Cruz, California, USA 147aDepartamento de Física, Pontificia Universidad Católica de Chile, Santiago, Chile 147bDepartamento de Física, Universidad T´ecnica Federico Santa María, Valparaíso, Chile 148Department of Physics, University of Washington, Seattle, Washington, USA 149Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 150Department of Physics, Shinshu University, Nagano, Japan 148Department of Physics, University of Washington, Seattle, Washington, USA 149Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 150Department of Physics, Shinshu University, Nagano, Japan Nijmegen, Netherlands j g 120Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlan 121 f f y y f 121Department of Physics, Northern Illinois University, DeKalb, Illinois, USA 121Department of Physics, Northern Illinois University, DeKalb, Illinois, USA 122 122bNovosibirsk State University Novosibirsk, Russia 123Institute for High Energy Physics of the National Research Centre Kurchatov Institute, Protvino, R 124 124Department of Physics, New York University, New York, New York, USA 125 125Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan 126 125Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan 126 26The Ohio State University, Columbus, Ohio, USA 127Faculty of Science, Okayama University, Okayama, Japan 128 culty of Science, Okayama University, Okayama, Ja 128Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA 032007-34 032007-34 PHYS. REV. D 100, 032007 (2019) SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … f eAlso at TRIUMF, Vancouver, British Columbia, Canada. f fAlso at Department of Physics and Astronomy, University of Louisville, Louisville, Kentucky, USA gAlso at Physics Department, An-Najah National University, Nablus, Palestine. h hAlso at Department of Physics, California State University, Fresno, California, USA. i iAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. j jAlso at Physics Dept, University of South Africa, Pretoria, South Africa. k lAlso at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. lAlso at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State mAlso at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China. nAlso at Departamento de Física, Instituto Superior T´ecnico, Universidade de Lisboa, Lisboa, Portuga oAlso at Universita di Napoli Parthenope, Napoli, Italy. oAlso at Universita di Napoli Parthenope, Napoli, Italy. pAlso at Institute of Particle Physics (IPP), Canada y qAlso at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia. qAlso at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia. qAlso at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia. rAlso at Department of Financial and Management Engineering, University of the Aegean, Chios, Gr p g g g, y g , , Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa. sAlso at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, t tAlso at Department of Physics, California State University, East Bay, California, USA. tAlso at Department of Physics, California State University, East Bay, California, USA. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Javakhishvili Tbilisi State University, Tbilisi, Georgia 159bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 160Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 161Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 162Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 163International Center for Elementary Particle Physics and Department of Physics, University of Tokyo, Tokyo, Japan 164Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 165Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 166Tomsk State University, Tomsk, Russia 167Department of Physics, University of Toronto, Toronto, Ontario, Canada 168aTRIUMF, Vancouver, British Columbia, Canada 168bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada 169Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 170Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 171Department of Physics and Astronomy, University of California Irvine, Irvine, California, USA 172Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 173Department of Physics, University of Illinois, Urbana, Illinois, USA 174Instituto de Física Corpuscular (IFIC), Centro Mixto Universidad de Valencia—CSIC, Valencia, Spain 175Department of Physics, University of British Columbia, Vancouver, British Columbia, Canada 176Department of Physics and Astronomy, University of Victoria, Victoria, British Columbia, Canada 177Fakultät für Physik und Astronomie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany 178Department of Physics, University of Warwick, Coventry, United Kingdom 179Waseda University, Tokyo, Japan 180Department of Particle Physics, Weizmann Institute of Science, Rehovot, Israel 181Department of Physics, University of Wisconsin, Madison, Wisconsin, USA 182Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 183Department of Physics, Yale University, New Haven, Connecticut, USA 184Yerevan Physics Institute, Yerevan, Armenia 8aTRIUMF, Vancouver, British Columbia, Canada 79Waseda University, Tokyo, Japan 180Department of Particle Physics, Weizmann Institute of Science, Rehovot, Israel 181 181Department of Physics, University of Wisconsin, Madison, Wisconsin, USA 182 182Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany Yale University, New Haven, Connecticut, USA of Physics, Yale University, New Haven, Connec 84 ment of Physics, Yale University, New Haven, Connecticut, USA 184 4Yerevan Physics Institute, Yerevan, Armenia aDeceased. b bAlso at Department of Physics, King’s College London, London, United Kingdom. c cAlso at Istanbul University, Dept. of Physics, Istanbul, Turkey. d cAlso at Istanbul University, Dept. of Physics, Istanbul, Turkey. d dAlso at Instituto de Fisica Teorica, IFT-UAM/CSIC, Madrid, Spai eAlso at TRIUMF, Vancouver, British Columbia, Canada. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … EARCH FOR ELECTROWEAK DIBOSON PRODUCTION … 151Department Physik, Universität Siegen, Siegen, Germany 152Department of Physics, Simon Fraser University, Burnaby, British Columbia, Canada 153SLAC National Accelerator Laboratory, Stanford, California, USA 154Physics Department, Royal Institute of Technology, Stockholm, Sweden 155Departments of Physics and Astronomy, Stony Brook University, Stony Brook, New York, USA 156Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 157School of Physics, University of Sydney, Sydney, Australia 158Institute of Physics, Academia Sinica, Taipei, Taiwan 159aE. Andronikashvili Institute of Physics, Iv. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Javakhishvili Tbilisi State University, Tbilisi, Georgia 159bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 160Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 161Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 162Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 163International Center for Elementary Particle Physics and Department of Physics, University of Tokyo, Tokyo, Japan 164Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 165Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 166Tomsk State University, Tomsk, Russia 167Department of Physics, University of Toronto, Toronto, Ontario, Canada 168aTRIUMF, Vancouver, British Columbia, Canada 168bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada 169Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 170Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 171Department of Physics and Astronomy, University of California Irvine, Irvine, California, USA 172Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 173Department of Physics, University of Illinois, Urbana, Illinois, USA 174Instituto de Física Corpuscular (IFIC), Centro Mixto Universidad de Valencia—CSIC, Valencia, Spain 175Department of Physics, University of British Columbia, Vancouver, British Columbia, Canada 176Department of Physics and Astronomy, University of Victoria, Victoria, British Columbia, Canada 177Fakultät für Physik und Astronomie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany 178Department of Physics, University of Warwick, Coventry, United Kingdom 179Waseda University, Tokyo, Japan 180Department of Particle Physics, Weizmann Institute of Science, Rehovot, Israel 181Department of Physics, University of Wisconsin, Madison, Wisconsin, USA 182Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 183Department of Physics, Yale University, New Haven, Connecticut, USA 184Yerevan Physics Institute, Yerevan, Armenia 151Department Physik, Universität Siegen, Siegen, Germany 152Department of Physics, Simon Fraser University, Burnaby, British Columbia, Canada 153SLAC National Accelerator Laboratory, Stanford, California, USA 154Physics Department, Royal Institute of Technology, Stockholm, Sweden 155Departments of Physics and Astronomy, Stony Brook University, Stony Brook, New York, USA 156Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 157School of Physics, University of Sydney, Sydney, Australia 158Institute of Physics, Academia Sinica, Taipei, Taiwan 159aE. Andronikashvili Institute of Physics, Iv. SEARCH FOR ELECTROWEAK DIBOSON PRODUCTION … Javakhishvili Tbilisi State University, Tbilisi, Georgia 159bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 160Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 161Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 162Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 163International Center for Elementary Particle Physics and Department of Physics, University of Tokyo, Tokyo, Japan 164Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 165Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 166Tomsk State University, Tomsk, Russia 167Department of Physics, University of Toronto, Toronto, Ontario, Canada 168aTRIUMF, Vancouver, British Columbia, Canada 168bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada 169Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 170Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 171Department of Physics and Astronomy, University of California Irvine, Irvine, California, USA 172Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 173Department of Physics, University of Illinois, Urbana, Illinois, USA 174Instituto de Física Corpuscular (IFIC), Centro Mixto Universidad de Valencia—CSIC, Valencia, Spain 175Department of Physics, University of British Columbia, Vancouver, British Columbia, Canada 176Department of Physics and Astronomy, University of Victoria, Victoria, British Columbia, Canada 177Fakultät für Physik und Astronomie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany 178Department of Physics, University of Warwick, Coventry, United Kingdom 179Waseda University, Tokyo, Japan 180Department of Particle Physics, Weizmann Institute of Science, Rehovot, Israel 181Department of Physics, University of Wisconsin, Madison, Wisconsin, USA 182Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 183Department of Physics, Yale University, New Haven, Connecticut, USA 184Yerevan Physics Institute, Yerevan, Armenia 151Department Physik, Universität Siegen, Siegen, Germany 152Department of Physics, Simon Fraser University, Burnaby, British Columbia, Canada 153SLAC National Accelerator Laboratory, Stanford, California, USA 154Physics Department, Royal Institute of Technology, Stockholm, Sweden 155Departments of Physics and Astronomy, Stony Brook University, Stony Brook, New York, USA 156Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 157School of Physics, University of Sydney, Sydney, Australia 158Institute of Physics, Academia Sinica, Taipei, Taiwan 159aE. Andronikashvili Institute of Physics, Iv. 032007-35 032007-35 PHYS. REV. D 100, 032007 (2019) G. AAD et al. uAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. p vAlso at Department of Physics, University of Michigan, Ann Arbor, Michigan, USA. vAlso at Department of Physics, University of Michigan, Ann Arbor, Michigan, USA. p y y g g wAlso at LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France. xAlso at Graduate School of Science, Osaka University, Osaka, Japan. xAlso at Graduate School of Science, Osaka University, Osaka, Japan. Also at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany. yAlso at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany. zAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. aa zAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. aaAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmege Netherlands. aaAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. bb bbAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. bbAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. bbAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. ccAlso at CERN, Geneva, Switzerland. dd ddAlso at Department of Physics, Stanford University, Stanford, California, USA. ddAlso at Department of Physics, Stanford University, Stanford, California, USA. so at Department of Physics, Stanford University, S eeAlso at Manhattan College, New York, New York, USA. eeAlso at Manhattan College, New York, New York, USA. ff ffAlso at Joint Institute for Nuclear Research, Dubna, Russia. ffAlso at Joint Institute for Nuclear Research, Dubna, Russia. gAlso at Hellenic Open University, Patras, Greece. h p y hhAlso at The City College of New York, New York, New York, USA. ii hhAlso at The City College of New York, New York, New York, USA. ii hAlso at The City College of New York, New York ii iiAlso at Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China. jj iiAlso at Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, Ch jj jjAlso at Department of Physics, California State University, Sacramento, USA. kk jjAlso at Department of Physics, California State University, Sacramento, USA. kk kkAlso at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. 032007-35 ll kkAlso at Moscow Institute of Physics and Technology State University, Dolgopru ll llAlso at D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Swit llAlso at D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerlan Department of Physics and Astronomy, University o nnAlso at Louisiana Tech University, Ruston, Louisiana, USA. nnAlso at Louisiana Tech University, Ruston, Louisiana, USA. y ooAlso at School of Physics, Sun Yat-sen University, Guangzhou, China. ooAlso at School of Physics, Sun Yat-sen University, Guangzhou, China. r Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Sofia, Bulg Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Scie ppAlso at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Aca at Institute for Nuclear Research and Nuclear Energ qqAlso at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Russia. at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Russia. qqAlso at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Russia. rrAlso at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United Arab rrAlso at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, U p pp y y, y j , j , ssAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. p pp y y y j j ssAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. tt ttAlso at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France. ttAlso at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France uuAlso at National Research Nuclear University MEPhI, Moscow, Russia. uuAlso at National Research Nuclear University MEPhI, Moscow, Russia. Also at National Research Nuclear University MEPhI, Moscow, Russia. vvAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. y e for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. vvAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, H vvAlso at Institute for Particle and Nuclear Physics, Wigner Research Ce wwAlso at Giresun University, Faculty of Engineering, Giresun, Turkey. wwAlso at Giresun University, Faculty of Engineering, Giresun, Turkey. xxAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. xxAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. 032007-36
https://openalex.org/W3119226716	https://zenodo.org/record/4023338/files/FinalPaper_Thomas_R%C3%A9my.pdf	English	null	A High Frequency Self-Reconfigurable Battery for Arbitrary Waveform Generation	World electric vehicle journal	2,021	cc-by	8,517	Summary This article presents an innovative self-reconfigurable battery (SRB) architecture, which is able to generate directly at its output any waveform signals. Thanks to the specific characteristic of the proposed system, it is even possible to dispense with any AC charger. Although the individual control of each cell in the battery pack to perform an efficient active cell balancing has been already studied in the literature, the system presented in this article is the first of its kind. This article describes a real prototype of a high frequency SRB of 128 cells and demonstrates that it can be charged without any dedicated charger directly on the electrical grid, by generating a sinusoidal waveform voltage, while perfectly balancing the cells in real time. Keywords: Battery Management System (BMS), Self-Reconfigurable Battery (SRB), multilevel converter, high efficiency conversion, active balancing. A High Frequency Self-Reconfigurable Battery for Arbitrary Waveform Generation R. Thomas1, G. Despesse1, S. Bacquet1, E. Fernandez1, Y. Lopez1, P. Ramahefa-andry1, L. Cassarino1 1CEA, Minatec Campus, Univ. Grenoble Alpes, F-38000 Grenoble, France, remy.thomas@cea.fr 1CEA, Minatec Campus, Univ. Grenoble Alpes, F-38000 Grenoble, France, remy.thomas@cea.fr 33nd Electric Vehicle Symposium (EVS33) Portland, Oregon, June 14 - 17, 2020 33nd Electric Vehicle Symposium (EVS33) Portland, Oregon, June 14 - 17, 2020 A High Frequency Self-Reconfigurable Battery for Arbitrary Waveform Generation R. Thomas1, G. Despesse1, S. Bacquet1, E. Fernandez1, Y. Lopez1, P. Ramahefa-andry1, L. Cassarino1 1CEA, Minatec Campus, Univ. Grenoble Alpes, F-38000 Grenoble, France, remy.thomas@cea.fr 1 Introduction Lithium-ion batteries are widely seen as a promising solution for both power generation, as in electric vehicles, and energy storage, to cope with the variable and intermittent renewable energy. Despite providing great performance through high energy density and long service life, lithium-ion batteries require an accurate monitoring to ensure the user’s safety. Indeed, high temperature, overcharge and over-discharge damage the cell and endanger the user. Therefore, all battery pack includes a Battery Management System (BMS) to stop charging and discharging the battery as soon as one cell exits its safe operating area, as described in [1]. This represents a critical limitation as unbalancing State of Health (SoH) can cause some weak cells to reach end of charge and discharge much sooner than others [2]. According to [3], 350 cycles separate the end of life of the best and worst cells of a same battery pack. For technical, economic and environmental considerations, balancing strategies [4] were developed to equalize the state of charge of cells. Typical balancing used in most of manufactured Li-ion battery systems consists in dissipating some energy of the fully charged cells to achieve the charge of the other cells. This solution compensates only a difference in coulometric efficiency between cells and not a difference in capacity. It is always the weakest cell that limits the EVS33 EVS33 1 1 capacity of the full battery pack and which ends up being the most exploited. Then the weakest cell becomes the most aging, which increases capacity differences over the time. capacity of the full battery pack and which ends up being the most exploited. Then the weakest cell becomes the most aging, which increases capacity differences over the time. In order to use the full capacity of all cells of a battery pack, more advanced balancing strategies are applied such as active balancing, which use power electronic components to exchange energy from the strongest cells to the weakest ones. Other strategies use Self Reconfigurable Battery architectures (SRB) enabling to manage the average current drawn on each cell to provide the output power [5]. Some studies address the efficiency improvement of adjusting the output voltage by such battery reconfiguration compare to the use of an external DC/DC converter [6]. Others have proposed to generate a staircase shape sinusoidal waveform in order to replace the AC/DC battery charger [7] as well as the DC/AC motor inverter [8]. 1 Introduction This is an attractive prospect as it is well known that a great amount of energy is lost in AC-DC inverters [9]. Staircase shape waveform are usually generated from multilevel converters using carrier phase shift Pulse Width Modulation (PWM) or carrier cascaded PWM [10]. Modular Multilevel Converter (MMC) with Battery Energy Storage System (MMC-BESS) have been proposed as a new three-phases topology using SRB for the traction drive [11] as well as for stationary batteries for fast charge EV charging station [12] [13]. In those studies, MMC- BESS are used in single star topology with cascaded H-Bridge, or whether in dual-star topology with only half- bridge chopper switches and twice the required number of cells. Both incorporate one to several cells in series per adjustable series level in the MMC. Due to the high number of levels, the generated three-phase AC signal offers a low total harmonic distortion (THD) on the output voltage and current. This considerably reduces the need for output power filtering and shows direct benefits on the motor efficiency [14]. Among the SRBs capable of generating sinusoidal waveforms, few offer direct on-grid recharging and all use cascaded carrier PWM control schemes. These, when embedded, are implemented on DSP and FPGA targets, and refreshed at only few KHz. Moreover, sudden fluctuations in network voltage require immediate adjustment of the output voltage, which can be difficult to achieve with such control schemes. In addition, the occurrence of cell faults requires the ability to overcome failures to avoid unexpected carrier offset under various modulation indices [15]. This paper demonstrates the feasibility of embedding cell-switching strategies in a microcontroller with the use of field buses, low voltage MOSFET as well as a single current sensor. The aim is to obtain a cost efficient SRB able, on the one hand, to charge itself directly on the power grid while reducing the requirement of passive filtering components, on the other hand to generate a perfect output waveform signal. All this while allowing the exclusion of defective cells and performing real-time cells balancing. For this purpose, this document is composed as follows: a first section introduces SRB fundamentals as well as those more specific of the proposed system. A second section describes the hardware architecture implemented. Then, a third section describes the software advanced functions. 1 Introduction A following section presents the experimental results, such as SoC balancing, THD and efficiency of the HF SRB, through the implementation of a real demonstrator of 128 cells. Finally, a last section concludes on the performances demonstrated by the results obtained and develops the perspectives that follow. 2 SRB fundamentals Figure 1: Staircase shape sine waveform generated by N-level SRB Level 1 N … Level n+1 Level n Level n-1 … (H-Bridge toggle @ T/2 and T) Level 1 t U T T/2 0 1 cell n-1 cells n cells n+1 cells N cells 𝑇𝑖𝑚𝑒𝑆𝑡𝑒𝑝௠௜௡= 𝑇 𝑁௠௔௫∗2 ∗𝜋 𝑁௠௔௫= (𝑈௥௠௦∗ 2) + 𝑡𝑜𝑙𝑒𝑟𝑎𝑛𝑐𝑒 % 𝑈𝑙𝑒𝑣𝑒𝑙୫୧୬ Ulevel Ulevel 2 delay Control time step Control time step 𝑇𝑖𝑚𝑒𝑆𝑡𝑒𝑝௠௜௡ Level 1 Figure 1: Staircase shape sine waveform generated by N-level SRB When taking EU electrical network voltage as an example, which is 230 VRMS +/- 10% with a frequency of 50 Hz, and using cells which the specified end of discharge threshold is 2800 mV, Nmax reaches 128 and the related minimum time step is then about 25 µs, which corresponds to an equivalent switching frequency of 40 kHz. This frequency can be reduced if more than one level can be switched at each control time step. Note that the battery pack could include additional cells that could be used to provide the maximum voltage output in case of cell failures; therefore, the number of cells could be greater than Nmax without affecting TimeStepmin. Recharging an SRB directly on the electrical network consists in tracking the electrical network voltage and applying a slightly smaller voltage at the SRB output. The small voltage difference 𝛿𝑉 is applied to the internal impedance of the SRB 𝑍ௌோ஻. This induces a current exchange approximately proportional to that voltage difference which can reaches 0.5 A/V for a SRB of 128 levels of single 18650 NMC cells as Sony VTC6. Therefore, generating a waveform for charging purpose imply a tightly synchronization with the electrical network voltage waveform to avoid any excessive current exchange due to control time delay, as detailed in section 4.2. The periodic aspect of the electrical network voltage facilitates this control. However, it can present stochastic deformations, which is why a power filtering stage is usually required in existing SRBs to filter out these unpredictable disturbances. The control strategy implemented in the proposed system is then quite different than carrier cascaded PWM in order to be embedded on microcontrollers. Here, a nearest level control (NLC) is performed. The number of serial levels to activate is then determined in real-time by rounding up to the nearest integer the real value given by a closed-loop control. 2 SRB fundamentals The key idea of SRB is to introduce individual control of each cell in the battery pack thanks to dedicated switches placed in series and in parallel with each cell. With such management, it becomes possible to disconnect only the weakest or damaged cells and provide the required power with the remaining ones. Moreover, bypassing switches offer the capability to adjust the number N of cells placed in serial and thus to adjust the output voltage of the battery. The generation of an AC waveform voltage from a SRB rely on the sequential superimposition of each cell voltages resulting in a staircase shape signal as shown in Fig. 1. A cell insertion increases the output voltage of one cell voltage, we name that voltage increment a “Level”, “Level 1” being the first step, and “Level n”, the nth step. A level is not attached to a particular cell, any cell of the battery pack can ensure that level. As shown on Fig.1, each level performs four switching operation within a sinusoidal period. Therefore, in order to generate a 50 Hz sinusoidal waveform, the switching frequency of a level is 200 Hz. However, in order to generate an accurate waveform in its steepest part, it is necessary to respect a minimum time TimeStepmin between two level EVS33 2 2 changes. In the case of a sine wave, the min time step must be less than or equal to the period of the fundamental harmonic of the voltage over more than six times the number of levels Nmax. changes. In the case of a sine wave, the min time step must be less than or equal to the period of the fundamental harmonic of the voltage over more than six times the number of levels Nmax. 2 SRB fundamentals The use of NLC for SRB driving have been compared with carrier cascaded PWM in [16]. It is shown that PWM control patterns tend to have a better THD when a low number of serial levels is used, while NLC induces less loss due to the reduced amount of commutations. This study has been done on a SRB of 9 levels of unit blocks having 5 cells in series, therefore the NLC THD results should be better with higher number of levels. Figure 2: On the fly cells switch principle Waveforms generated with our system are similar to those from carrier cascaded PWM but the management of electrical grid disturbances as well as cells faults are now possible within a half cycle of the sinusoidal voltage without signal perturbation. A control loop updates the reference waveform in real time, which allows a forthwith Cell A Cell A 2 Cell C Cell C Cell C 6 5 4 3 Cell A “ON” Cell C “ON” Cell B “ON” (Swap 1) Cell B “OFF” Cell G “ON” U t New cell ranking (Cell B fault) (Swap 2) Cell G “OFF”, Cell J “ON” Cell C “OFF” Cell D “OFF” Cell A “OFF” Cell J “OFF” Cell D “ON” 4th level 3rd level 2nd level 1st level t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 Cell D Cell G 1 Cell A Cell B Cell D Cell G Cell order Cell J Cell G Cell J Available cells number Cell J Cell D X t < t4 X t4< t < t6 X t > t6 Figure 2: On the fly cells switch principle Cell A “ON” Cell C “ON” Cell B “ON” (Swap 1) Cell B “OFF” Cell G “ON” U t New cell ranking (Cell B fault) (Swap 2) Cell G “OFF”, Cell J “ON” Cell C “OFF” Cell D “OFF” Cell A “OFF” Cell J “OFF” Cell D “ON” 4th level 3rd level 2nd level 1st level t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 level Figure 2: On the fly cells switch principle Figure 2: On the fly cells switch principle Waveforms generated with our system are similar to those from carrier cascaded PWM but the management of electrical grid disturbances as well as cells faults are now possible within a half cycle of the sinusoidal voltage without signal perturbation. 2 SRB fundamentals A control loop updates the reference waveform in real time, which allows a forthwith Waveforms generated with our system are similar to those from carrier cascaded PWM but the management of electrical grid disturbances as well as cells faults are now possible within a half cycle of the sinusoidal voltage without signal perturbation. A control loop updates the reference waveform in real time, which allows a forthwith EVS33 3 management of the electrical network voltage perturbations. The First In First Out (FIFO) principle is applied to connect and disconnect the cells. The selection of the cell to be connected or disconnected is performed on the fly in a priority order provided by a balancing algorithm. The failing cells are handled forthwith without disturbing the generated waveform thanks to a cell swap operation. The cell use priority order is given by a ranking X(t), which is updated over the time from the availability of the cells and by the balancing algorithm. As illustrated in Fig. 2, the first four levels are connected from time t1 to t4 according to the reference signal. At time t5, a fault occurs on cell B, which is associated with the third level. This faulty cell is disconnected and instantly removed from the list of available cells list in the ranking X(t). This ranking gives cell G as the next available cell to be connected in replacement of the faulty cell. At time t6, the cell ranking X(t) is updated by the balancing algorithm. Cell G, now with rank five, is immediately replaced by cell J which has now rank four. Then cell J is disconnected at t7 when only three levels are required in order to follow the theoretical waveform. At time t8, only two levels are required and cell C is disconnected as requested by the ranking X(t). At time t9, only one level is required and the cell D is disconnected. At time t10, the zero-crossing point of the voltage is reached. The cell A is disconnected and then all cells are bypassed. The negative part of the waveform is generated thanks to inverter H-Bridges distributed on each module containing 4 cells (see Fig. 3) and cells are connected successively in the order given by the cell ranking X(t) (cell A first). The following sections show how the CEA has implemented the innovations presented above. 3.1 Master/Slave organization Distributed systems have been commonly used in classical BMS implementation to address large cable bundles issues. They are based on a central unit and multiple remote units where remote units are arranged against groups of cells. SRB requires the use of switches whose number and control complexity increases with the number of cells used as well as the number of degrees of freedom it integrates (serial, parallel, serial and parallel, etc...). Some works address the complexity of a centralized control of all switches by using neural network and reinforcing learning to optimize the control strategy [17]. Others propose the use of different levels of abstraction distributed on different processing units to reduce this complexity [18]. Others even propose to distribute the control decision at different levels [19] [20]. EVS33 4 Figure 3: HF SRB hardware architecture We propose to exploit the concept of distributing switches and their physical drivers over the battery pack while maintaining centralized control of the overall cell layout and monitoring. For this purpose, the SRB is divided into remote modules named “Slave” and a central module named “Master”. As argued in section 3.4, each Slave is composed of four serial cells connected through half-bridge chopper circuits, an H-Bridge to reverse the polarity of the module, and a local controller. Then Slaves are chained to reach the required output voltage. An External data bus Voltage/current requirements Slave 2 Neutral (com) Iout Vout Insulated internal data bus Half-bridge Chopper circuits C1 C3 C2 C4 Inverter H-bridge Switch drivers Local Controller U, T° sensors Master Controller U, I, T° sensors Voltages/current management Cell selector ensuring BMS Iout > 0 L1 SW1 F1 L2 SW2 F2 VN VG VN 100 µH 1 mH Slave 1 Slave N Master … Figure 3: HF SRB hardware architecture External data bus Voltage/current requirements Slave 2 Neutral (com) Iout Vout Insulated internal data bus Half-bridge Chopper circuits C1 C3 C2 C4 Inverter H-bridge Switch drivers Local Controller U, T° sensors Master Controller U, I, T° sensors Voltages/current management Cell selector ensuring BMS Iout > 0 L1 SW1 F1 L2 SW2 F2 VN VG VN 100 µH 1 mH Slave 1 Slave N Master … Local Controller Figure 3: HF SRB hardware architecture We propose to exploit the concept of distributing switches and their physical drivers over the battery pack while maintaining centralized control of the overall cell layout and monitoring. 3.1 Master/Slave organization For this purpose, the SRB is divided into remote modules named “Slave” and a central module named “Master”. As argued in section 3.4, each Slave is composed of four serial cells connected through half-bridge chopper circuits, an H-Bridge to reverse the polarity of the module, and a local controller. Then Slaves are chained to reach the required output voltage. An We propose to exploit the concept of distributing switches and their physical drivers over the battery pack while maintaining centralized control of the overall cell layout and monitoring. For this purpose, the SRB is divided into remote modules named “Slave” and a central module named “Master”. As argued in section 3.4, each Slave is composed of four serial cells connected through half-bridge chopper circuits, an H-Bridge to reverse the polarity of the module, and a local controller. Then Slaves are chained to reach the required output voltage. An EVS33 EVS33 4 4 overview of the overall hardware architecture is given in Fig 3. 𝑉௢௨௧ is the output-to-com voltage of the HF SRB, 𝑉ே is the line-to-neutral voltage into which the AC charger voltage comes in, and 𝑉௚ is the generated AC voltage for the batteries discharging. The Master module is in charge of managing all slave modules thanks to a master controller. It estimates the state of each cell and manages the safety according to data collected through the remote modules (mainly cells’ voltage and temperature). The selection of the cells to be inserted in the power path is performed in the master controller while Slaves ensures switches actuation and cells parameters measurement. However, [19] advises to perform the cell selection in a distributed way, otherwise the control latency will increase significantly with the number of serial levels used in a SRB. In section 4.1 we will show how to deal with this issue and what is the impact of increasing the number of levels over the driving latency with the proposed solution. The centralized management of the SRB allows to use very low-cost local controllers. The use of a shared communication bus is required in order to reduce the number of wires in the system. These must be carefully designed to withstand disturbances related to switching, as shown in the section 3.2. 3.2 Communication buses The splitting of the SRB into a distributed system requires the use of a reliable communication bus for safety reasons. The generation of arbitrary waveforms implies real-time constraints on the transmitted commands. The CAN bus is reliable but not suitable for transmitting commands at high frequency. The reliability of a faster real- time communication bus like USART can be enhanced by the use of a differential transmission lines as well as additional software mechanisms such as cyclic redundancy check (CRC). Switch command messages are less critical than cells voltage and cells temperature data. Indeed, the safety of the switch control is ensured at the level of the local controller while the real time control loop performed at the master can compensate a wrong number of activated serial levels as soon as the next control time period occurs. We propose to separate real-time data from critical data by using a combination of CAN and RS485 busses. In this way, real-time data can be transmitted at higher frequency while critical data can be safely transmitted at low frequency with a lower bandwidth. 3.3 SRB control strategy Iteratively changing the number of serially active levels allows the master controller to communicate individually and asynchronously with each local controller in order to locally modify the configuration of the cells. Thus, each local controller is only called when one of its cells is concerned by the refresh of the serial arrangement. The control frequency is limited by the master controller since the local controllers are called asynchronously at low frequency. While this sequential control has the potential to allow high command frequencies, only a few serial levels can be controlled at a time, as the length of the control message is limited by the length of the control period. A higher control frequency could be achieved by distributing the control of the slaves over several buses. An alternative could be to transmit the required number of active levels in series to all the local controllers in a single message sent over the real-time communication bus. The rank of each local controller could be transmitted in the background at low frequency on the CAN communication bus. This way, the full voltage amplitude of the SRB can be reached forthwith in one step. In counterpart, all local controllers are called up at each control command, even if this is unnecessary. The local controllers have to be as inexpensive and low power as possible due to the large amount used in the SRB. Therefore, the use of low power microcontrollers limits the call up frequency rate. For direct charging of the SRB on the electrical grid, a moderate rise time is acceptable if the control frequency is sufficient. The rest of this study is therefore based on asynchronous iterative control of the local controllers performed at 20 kHz with a maximum of three slave calls per control period, hence between 1 and 12 levels for each control periods. This reduces the computing power required by the local controller sufficiently to allow the use of microcontrollers operating at frequencies as low as 48 MHz, such as the STM32F091. EVS33 5 5 3.5 Master As for the case of central units of classical BMS, the master controller centralizes all cell voltage and temperature measurements transmitted by the local controllers. It is interfaced with the outside of the HF SRB, which provides operating mode instructions (charge/discharge mode, 48 VDC, 230 VAC, etc...) and to which it transmits status information such as SoC, SoP and SoH battery states thanks to implemented estimators. It is also the place where all the tasks that cannot be distributed to the local controllers are carried out, such as measuring the battery pack current, managing the main power relays or monitoring their upstream and downstream voltages. More specifically to the HF SRB management, the master controller is in charge of the optimal selection of the cells to be put in series and the others to be bypassed, as well as the transmission of the corresponding commands to the local controllers involved. The selection of the cells is performed according to a priority order provided by a balancing algorithm. This algorithm sorts the cell from multiple criteria as voltage, temperature, state of charge, of health, of power, of energy or even impedance. The master controller’s computing power requirement is similar to central units of classical BMS thanks to the real time architecture described in section 4.1. A 32-bit microcontroller of around 200 DMIPS, running at frequencies up to 200 MHz such as the STM32F7 is sufficient. This kind of microcontroller is available for a target price as low as $2 for 10k units. 3.4 Slave switches choice and thermal considerations Switching losses are theoretically very low due to the low voltage and low switching frequency of each switch. With regard to conduction losses, the internal impedance of switches is generally proportional to their nominal voltage. The sum of the impedances of the switches in series should correspond to that of a conventional inverter. Thus, the conduction losses should be equivalent. Moreover, unlike conventional inverters, the heat distribution over the entire battery pack does not require a specific cooling system. In addition, the choice of the number of switches used in parallel makes it possible to adjust the conduction losses relative to the losses induced by the internal impedance of the cells through which the same current flows. However, there is no point in reducing the conduction losses of the switches too much, as the impedance of the cells generates large joule losses. Thus, an impedance ratio of 1/10 has been targeted in order to make the conduction losses of the switches negligible compared to those presented by the cells themselves. From a thermal point of view, this means that the additional heating near the cells will be of the order of 10%. In other words, 90% of the heating observed in the HF SRB will be due to the self-heating of the cells. In addition to the inverter's cooling specific system saving, the prospect of carrying out cell temperature balancing offers the possibility of relieving the constraints applied on the thermal management system of the battery pack. 4.2 Nearest level control loop There are two different control systems in the HF SRB’s software to manage AC voltage generation during discharge (Fig. 6) and the charge of the batteries with AC voltage (Fig. 7). There are two different control systems in the HF SRB’s software to manage AC voltage generation during discharge (Fig. 6) and the charge of the batteries with AC voltage (Fig. 7). discharge (Fig. 6) and the charge of the batteries with AC voltage (Fig. 7). Figure 6: Synoptic bloc of the Discharge Control System The output of each of them has a System Command Generator (SCG) bloc (Fig. 6) to convert a voltage command to the number of series cells ∆𝑛 to be added or removed from the HF SRB output. 𝑘ெ஺௑ is the maximum number of series cells that the system can be applied at the same time (cells added if positive and cells removed if negative) and 𝑉௖௘௟௟ெ௔௫ is the maximum voltage of one cell (4.2V for NMC Lithium battery). The Discharge Control System (DCS) includes two sub-control loops: a Current Limit Control (CLC) to ensure that 𝐼௢௨௧ amplitude never exceeds 𝐼௅ூெூ், and a Voltage Control (VC) to ensure that 𝑉௢௨௧ follows 𝑉௥௘௙. The target AC voltage data points, that was obtained by sampling a 230VAC-50Hz sinusoidal signal to 20kHz, are stored in a Look-Up-Table (LUT). The latter is then multiplied by a factor 𝛼 to produce 𝑉௥௘௙ with 0 ≤𝛼≤1. 𝛼 is decreased by CLC to lower 𝑉௢௨௧ when the outputting current 𝐼௢௨௧ is too high. The derivative part of the Proportional-Integral-Derivative (PID) controller used in CLC has a primordial function to anticipate the variation of 𝐼௢௨௧ when it lags behind 𝑉௢௨௧ in the case of inductive load. As the derivative function is sensitive to the measurement noises, a Low-Pass Filter (LPF) is added to become a Filtered PID. In VC the Phase Advance Filter (PAF) compensates the delay 𝜑introduced by the HF SRB system response to Figure 6: Synoptic bloc of the Discharge Control System Figure 6: Synoptic bloc of the Discharge Control System The output of each of them has a System Command Generator (SCG) bloc (Fig. 6) to convert a voltage command to the number of series cells ∆𝑛 to be added or removed from the HF SRB output. 4.1 Real time architecture Figure 5: Schedule of a period of the HF SRB control algorithm performed in the master controller Measures Cells fault handler Cells selection Coulometry Control loop Cells CMD Tx Background processes Background processes 1st period part 2nd period part Control process period ( 50 µs ) Figure 5: Schedule of a period of the HF SRB control algorithm performed in the master controller Fig. 5 details the scheduling of those tasks gathered inside a process called “control process” and performed at each control period. One can see that most of the tasks are performed in the order of their dependencies while the balancing task is performed in background over many control periods. The control process is split into two parts. The first period part determines the command frames to send to the slaves. Its duration could have jitter due to the variability of some algorithms as cells selection and coulometry. The second part corresponds to the sending of the command frames. Both parts of the process are triggered by timer events in order to avoid jitter on the frequency of the command frame sending (see Fig. 8.c for real chronograms). 4.1 Real time architecture The master controller performs asynchronous control of the remote modules to make an iterative adjustment of the overall number of active serial levels. This number is determined by a nearest level control method from a reference given by a regulation loop. The reference signal is calculated in real time in order to be able to follow sudden changes in the electrical grid voltage. Each cell is activated following an order of priority established by a balancing algorithm. This one uses cell voltage and temperature measurements as well as cell state indicators provided by the estimation algorithms. A block diagram shows interactions between those tasks in Fig. 4 Figure 4: Block diagram of the HF SRB control algorithm performed in the master controller Cells fault Handler Cells selection and control U, I, P Near level Control loop Coulometry Cells Balancing Cells Ah Serial nb active levels Cells Ranking Cell States HF SRB U/I Cells U/T°C HF SRB U, I, P RMS target Cells CMD Measures Figure 4: Block diagram of the HF SRB control algorithm performed in the master controller Cells fault Handler Cells selection and control U, I, P Near level Control loop Serial nb active levels Cells Ranking Cell States HF SRB U, I, P RMS target Cells CMD Figure 4: Block diagram of the HF SRB control algorithm performed in the master controller EVS33 6 It is then necessary to organise the computation of all of those tasks in order to respect the minimum control time step mentioned before. Real-time computation of the required number of active levels requires measurements of the output voltage and current, at least at the same frequency as that used for serial level control. Moreover, for cost-effective considerations, the overall HF SRB integrates only one current sensor. In our system, we choose to base the states of charge estimator on coulometry. It is then required to perform an individual coulometry estimation of each cell at the control frequency, in order to take into account the serial/bypass state of each cell. Using the same frequency to perform all those tasks simplifies the scheduling of their sequential computation. 4.2 Nearest level control loop The PC bloc determines and controls the delay ∅௔ௗ௩ of the feedback current 𝐼௢௨௧ ∗ with respect to 𝑖௥௘௙ ∗ (simultaneously 𝑉ே) toward to make the charge current and voltage in phase. The LPF is a first order filter where 𝜏= 100µ𝑠. Figure 7: Synoptic Bloc of the Charge Control System Figure 7: Synoptic Bloc of the Charge Control System The Charge Control System (CCS) aims to charge the HF SRB from any electrical AC voltage sources by applying an output voltage 𝑉௢௨௧ with the same pace and in phase with the charge voltage 𝑉ே, while ensuring an instantaneous charge current in phase with the both voltages 𝑉௢௨௧ and 𝑉ே. As a result, the potential reactive power consumed by any inductive components in the system, such as 𝐿ଵ and 𝐿ଶ, will be removed. Hence, the CCS includes three main blocs: a Current Reference Generator (CRG) bloc, a Phase Control (PC) bloc and a Voltage Drop Control (VDC) bloc. The CRG bloc generates two current references signal obtained from 𝑉ே and then, in phase with it: 𝑖௥௘௙ is the instantaneous current that 𝐼௢௨௧ should look like (same pace and effective value 𝐼ாிி) and 𝑖௥௘௙ ∗ is the normalized value of 𝑖௥௘௙. The PC bloc determines and controls the delay ∅௔ௗ௩ of the feedback current 𝐼௢௨௧ ∗ with respect to 𝑖௥௘௙ ∗ (simultaneously 𝑉ே) toward to make the charge current and voltage in phase. The LPF is a first order filter where 𝜏= 100µ𝑠. Finally, the VDC bloc controls the differential voltage ∆𝑉 between 𝑉ே and 𝑉௢௨௧ to keep 𝐼௢௨௧ ∗ close to 𝑖௥௘௙. To obtain ∆𝑉, the control loop estimates the equivalent resistor 𝑅௘௤ between batteries and the AC charger that includes batteries ESR, inductors internal resistance, wire resistance and so on. In practice, 𝑉ே is not directly connected to 𝑉௢௨௧ at the start-up of the control system to avoid short-circuiting and damaging the charger and the HF SRB system. The solution is to synchronize 𝑉ே and 𝑉௢௨௧ when 𝐼௢௨௧= 0𝐴 (𝑆𝑊ଵ off) and to make 𝑉ே’s amplitude slightly higher than 𝑉௢௨௧’s one to allow current flows from AC charger to HF SRB after switching on 𝑆𝑊ଵ. To do this, 𝐼௢௨௧ ∗ is first equal to the modelled current 𝐼௢௨௧ெ௢ௗ௘௟ until \|∅௔ௗ௩\| < 0.01, the necessary condition to switch on the power relay 𝑆𝑊ଵ, and then use 𝐼௢௨௧ as a feedback input current. 4.2 Nearest level control loop 𝑘ெ஺௑ is the maximum number of series cells that the system can be applied at the same time (cells added if positive and cells removed if negative) and 𝑉௖௘௟௟ெ௔௫ is the maximum voltage of one cell (4.2V for NMC Lithium battery). The Discharge Control System (DCS) includes two sub-control loops: a Current Limit Control (CLC) to ensure that 𝐼௢௨௧ amplitude never exceeds 𝐼௅ூெூ், and a Voltage Control (VC) to ensure that 𝑉௢௨௧ follows 𝑉௥௘௙. The target AC voltage data points, that was obtained by sampling a 230VAC-50Hz sinusoidal signal to 20kHz, are stored in a Look-Up-Table (LUT). The latter is then multiplied by a factor 𝛼 to produce 𝑉௥௘௙ with 0 ≤𝛼≤1. 𝛼 is decreased by CLC to lower 𝑉௢௨௧ when the outputting current 𝐼௢௨௧ is too high. The derivative part of the Proportional-Integral-Derivative (PID) controller used in CLC has a primordial function to anticipate the variation of 𝐼௢௨௧ when it lags behind 𝑉௢௨௧ in the case of inductive load. As the derivative function is sensitive to the measurement noises, a Low-Pass Filter (LPF) is added to become a Filtered PID. In VC, the Phase Advance Filter (PAF) compensates the delay 𝜑 introduced by the HF SRB system response to its command ∆𝑛. Therefore, this controller makes the control loop response faster with more stability. In VC, the Phase Advance Filter (PAF) compensates the delay 𝜑 introduced by the HF SRB system response to its command ∆𝑛. Therefore, this controller makes the control loop response faster with more stability. EVS33 7 Figure 7: Synoptic Bloc of the Charge Control System The Charge Control System (CCS) aims to charge the HF SRB from any electrical AC voltage sources by applying an output voltage 𝑉௢௨௧ with the same pace and in phase with the charge voltage 𝑉ே, while ensuring an instantaneous charge current in phase with the both voltages 𝑉௢௨௧ and 𝑉ே. As a result, the potential reactive power consumed by any inductive components in the system, such as 𝐿ଵ and 𝐿ଶ, will be removed. Hence, the CCS includes three main blocs: a Current Reference Generator (CRG) bloc, a Phase Control (PC) bloc and a Voltage Drop Control (VDC) bloc. The CRG bloc generates two current references signal obtained from 𝑉ே and then, in phase with it: 𝑖௥௘௙ is the instantaneous current that 𝐼௢௨௧ should look like (same pace and effective value 𝐼ாிி) and 𝑖௥௘௙ ∗ is the normalized value of 𝑖௥௘௙. 5.2 Waveform generation Fig. 9 shows the experimental waveforms when a battery of 128 VTC6 cells was discharging (a) and when it was charged up at the rated power of 280 W (b). THD measurements has been performed from a HIOKI PW6001-16 used with a current probe HIOKI 20A CT 6841-05. Those are reported as superposed screen captures in Fig. 9. The difference of current displayed on the HIOKI screen capture is due to the use of 5 wire turns around the current probe to improve its precision. One can see that the voltage THD of the HF SRB output is less than 0.3% when it discharges with a serial inductance of 100 µH. For security reason, a rectifier diode stage has been added at the charging input of the HF SRB to prevent outputting current on its connector. Indeed, a male plug is used in order to demonstrate the simplicity of connecting the prototype to the electrical grid. The control loop has been modified to follow the incoming rectified voltage while absorbing a near sinusoidal charging current waveform and in phase with this voltage. The current THD was about 15.6% with a serial inductance of 1.1 mH and a charge current of only 1.2 ARMS. A better current THD should be achieved for higher charge current if more than one parallel cell is used. Figure 9: HF SRB 230Vac 50Hz waveforms 5.3 Efficiency The efficiency is measured in discharge mode and at different current levels on one slave of 4 NMC cells and is extrapolated to estimate the efficiency of the full HF SRB. The slave is driven to generate a sinusoidal voltage of +/- 4 serial levels at 50 Hz. The applied discharge powers vary from 3 WRMS to 125 WRMS using an adjustable power resistor from 100 to 0 Ω. The equipment used to perform the efficiency measurement is a HIOKI PW6001- 16. Each of the 4 cells is instrumented with a current probe HIOKI 20A CT 6841-05 +/- 0.3% and their voltage is measured directly by the measuring device at +/- 0.02%. The output current of the slave is instrumented with a HIOKI 50A probe +/- 0.05% and its voltage is measured directly by the measuring instrument. Figure 9: HF SRB 230Vac 50Hz waveforms 0V 50H f Figure 9: HF SRB 230Vac 50Hz waveforms 5 Experimental results pe e ta esu ts Figure 8: Cells balancing of HF SRB on direct charge with electrical grid and control process period Figure 8: Cells balancing of HF SRB on direct charge with electrical grid and control process period Figure 8: Cells balancing of HF SRB on direct charge with electrical grid and control process period EVS33 8 8 Following experimentations are performed on a real HF SRB of 1 master and 32 slaves integrating 128 NMC cells for a rated power output of 2 kW. An overall capacity of 1 kWh is achieved in a volume of 14.5 L. The Master and one Slave unit are shown in Fig. 8.b and a chronogram of the control process period is shown in (c). 5.1 Cells balancing A complete charge performed at C/2 on the electrical grid from an initial SoC imbalance of 60% shows that the 128 cells are perfectly balanced before the end of charge. The SoC and cell voltage signals transmitted from CAN bus are reported on Fig. 8.a. 5.3 Efficiency The efficiency is measured in discharge mode and at different current levels on one slave of 4 NMC cells and is extrapolated to estimate the efficiency of the full HF SRB. The slave is driven to generate a sinusoidal voltage of +/- 4 serial levels at 50 Hz. The applied discharge powers vary from 3 WRMS to 125 WRMS using an adjustable power resistor from 100 to 0 Ω. The equipment used to perform the efficiency measurement is a HIOKI PW6001- 16. Each of the 4 cells is instrumented with a current probe HIOKI 20A CT 6841-05 +/- 0.3% and their voltage is measured directly by the measuring device at +/- 0.02%. The output current of the slave is instrumented with a HIOKI 50A probe +/- 0.05% and its voltage is measured directly by the measuring instrument. EVS33 EVS33 9 The power losses are depicted on Fig. 10. One can see that the losses curve has a typical quadratic growth pattern. The substantially constant power losses for the lowest powers indicates the static consumption of the slave. This consumption is about 580 mW per slave and includes the power supply of the local control circuits, the cells voltages and temperatures measurement circuits, the cells serial and bypass switches gate drivers as well as the H-bridge switches gate drivers. The power losses are depicted on Fig. 10. One can see that the losses curve has a typical quadratic growth pattern. The substantially constant power losses for the lowest powers indicates the static consumption of the slave. This consumption is about 580 mW per slave and includes the power supply of the local control circuits, the cells voltages and temperatures measurement circuits, the cells serial and bypass switches gate drivers as well as the H-bridge switches gate drivers. Figure 10: Slave HF SRB discharge efficiency over 50Hz staircase shape sine waveform Figure 10: Slave HF SRB discharge efficiency over 50Hz staircase shape sine waveform As shown on Fig. 10, for an output power of 92.069 WRMS, the switching current flowing through the switches is 9.2567 ARMS and power losses are 2.272 WRMS. Knowing the losses offset of 580 mW and that switching losses can be neglected; the conductive losses should be around 1.692 WRMS. At each time, the number of serial switches through which the serial current flows is composed as follows: one for each cell and two for the full H-bridge. 5.3 Efficiency Each switch uses the same reference which have a Ron of 1 mΩ (PSMN0R9-25YLC). Thus, the conducting power losses from those MOSFET should be around 514 mWRMS which is around a third of the amount of measured losses. This tends to show that the total impedance of the conducting tracks is of the same order than the “on” state switch impedances. Therefore, the power losses are well distributed between the switches and power tracks, which allows a good distribution of the heat release over the entire PCB of the local controller. Moreover, those power losses may represent less than 10% of the overall battery loss as the impedance of the cells is more than ten times higher. All this shows that there is no need of a specific cooling for the HF SRB. Efficiency curve of one slave is also depicted on Fig. 10. To extrapolate the efficiency of the HF SRB charging over the electrical network, we multiply the powers on x-axis by the number of slaves required to generate a sine waveform of 230 VRMS (hence a factor of 24 for cells at rated voltage of 3.6 V). The efficiency of the HF SRB is also depicted with subtracting the consumption of the control electronics (24 times 580 mW) as it is usual in efficiency measurements carried out on power converters. It can be seen that the efficiency of the HF SRB is above 98% up to 2300 WRMS of power delivery, which corresponds to a discharge rate of 4.5 C. These results are among the best of the state of the art [21]. References [1] L. Lu, X. Han, J. Li, J. Hua, and M. Ouyang., A review on the key issues for lithium-ion battery management in electric vehicles, Journal of Power Sources, ISSN 0378-7753, 226(2013), 272-288 [2] W.F. Bentley, Cell balancing considerations for lithium-ion battery systems, IEEE (Proceedings of the Annual Battery Conference on Applications and Advances), ISSN 1089-8182, 1997, 223-226 [3] T. Baumhöfer, M. Brühl, S. Rothgang, and D. U. Sauer, Production caused variation in capacity aging trend and correlation to initial cell performance, Journal of power sources, ISSN 0378-7753, 247(2014), 332-338 [4] D. Andrea, Battery Management Systems for large Lithium-Ion battery packs, ISBN 1608071049, Artech House, 2010 [5] S. Ci, N. Lin, and D. Wu, Reconfigurable Battery Techniques and Systems: A Survey, IEEE Access, ISSN 21693536, 4(2016), 1175-1189 [6] L. He, L. Gu, L. Kong, Y. Gu, C. Liu, and T. He, Exploring Adaptive Reconfiguration to Optimize Energy Efficiency in Large-Scale Battery Systems, ISSN 10528725, IEEE Real-Time systems, 2013, 118-127 [7] A. Davis, Z. M. Salameh, and S. S. Eaves, Evaluation of lithium-ion synergetic battery pack as battery charger, IEEE Transactions on Energy Conversion, ISSN 08858969, 14(1999), 830-835 [8] A. Davis, Z. M. Salameh, and S. S. Eaves, Comparison of a synergetic battery pack drive system to a pulse width modulated AC induction motor drive for an electric vehicle, IEEE Trans. Energy Conv., 14(1999), 245-250 [9] A. Genovese, F. Ortenzi, C. Villante, On the energy efficiency of quick DC vehicle battery charging, presented at EVS28 and published in World Electric Vehicle Journal, ISSN 20326653, 7(2015), 540-576 [10] Z. Zheng, K. Wand, L. Xu, and Y. Li, A hybrid cascaded multilevel converter for battery energy management applied in electric vehicles, IEEE Transactions on Power Electronics, ISSN 08858993, 29(2014), 3537-3546 [11] S. D’Arco, M. Quraan, P. Tricoli, and L. Piegari, Low frequency operation of Modular Multilevel Converters with embedded battery cells for traction drives, SPEEDAM, ISBN 978-1-5090-2067-6, 2016, 1375-1382 [12] D. D. Simone and L. Piegari, Integration of stationary batteries for fast charge EV charging stations, Energies, ISSN 19961073, 12(2019) [13] H. Akagi, and L. Maharjan, A battery energy storage system based on a multilevel cascade PWM converter, IEEE, ISSN 2165-0454, 2009, 9-18 [14] M. Quraan, T. Yeo, and P. Tricoli, Design and Control of Modular Multilevel Converters for Battery Electric Vehicles, IEEE Transactions on Power Electronics, ISSN 0885-8993, 31(2016), 507-517 [15] F. Gao, X. Gu, Z. Ma, C. 6 Conclusion The feasibility of a microcontroller based Self-Reconfigurable Battery able to charge itself directly on the electrical grid has been demonstrated. This has been possible thanks to the use of a Near Level Control with iterative commands. The system distribution in Master and Slaves modules has allowed to reduce the complexity of the switch management by adding an abstraction layer. This leads to implement a total number of 128 serial levels, although a greater number of levels is possible. The real-time management of cell switches allows a faulty cell to be replace at any time without disturbing the generated output voltage. The measurements made on the EVS33 10 demonstrator show very good results in term of efficiency (up to 98% including control consumption) and output voltage THD. demonstrator show very good results in term of efficiency (up to 98% including control consumption) and output voltage THD. Authors Authors Remy Thomas received a master's degree in electrical and electronics engineering from the French School Phelma of Grenoble Institute of Technology, France, in 2008. He joined the CEA-LITEN Laboratory in 2010 as research engineer to work on Battery Management Systems as well as Fuel Cell Management Systems, and more specially on real-time embedded systems for which he has developed a good expertise in both software and hardware. Ghislain Despesse graduated (MS degree) in electrical engineering from Ecole Normale Supérieure de Cachan (ENS) in 2002 and he received his Ph.D. Degree in microelectronics from the National Polytechnic Institute of Grenoble (INPG), France, in 2005. He joined the CEA-LETI Laboratory in 2005. He was involved in the development of vibrational energy harvesters using electrostatic or piezoelectric conversion principle. He is implicated for nine years in the development of Battery Monitoring Systems (BMS) including inverter functions for the electrical vehicle and others applications. He holds 70 patents in the fields of energy scavenging, energy conversion and power management. He received his ability to drive research in 2017 and became research director at CEA. Sylvain Bacquet was graduated in 2001 from the National Polytechnic Institute of Grenoble as an engineer in electronics. He is currently working at CEA as a project manager on the topics of energy management innovation and especially on reconfigurable battery packs. Eric Fernandez was graduated in 1991 from the National Polytechnic Institute of Grenoble as an engineer in electronics. He worked in research and development for industry before joining the CEA in 2008. He was involved in the field of battery pack since 2008 and his main work concerns battery management system and self-reconfigurable battery system. Yan Lopez received the B.S. degree in electrical and electronics engineering from Grenoble Institute of Technology, France, in 2006, and the M.S. degree in electronics and information technology from Polytech’ Grenoble, France, in 2009. He is currently an electronic research engineer of CEA Grenoble, France. His current research interest includes battery management and self-reconfigurable battery systems in electric vehicle systems and stationary electric grid storage systems. Prince Ramahefa-Andry received M.S. degree in electrical engineering from the Department of Electrical Engineering, Institut National des Sciences Appliquées de Lyon (INSA Lyon), Lyon, France, in 2013. He is currently a Research Engineer at Commissariat à l’Energie Atomique et aux Energies Alternatives, Grenoble, France. References Zhang, Redistributed Pulsewidth Modulation of MMC Battery Energy Storage System under Submodule Fault Condition, IEEE Trans. on Power Electron., ISSN 0885-8993, (35)2020, 2284-2294 [16] D. D. Simone, L. Piegari, and S. D’Arco, Comparative Analysis of Modulation Techniques for Modular Multilevel Converters in Traction Drives, SPEEDAM, ISBN 978-153864941-1, 2018, 593-600 [17] D. Kraus, E. Specht, T. Merz, M. Hiller, Optimized Real-Time Control for Modular Multilevel Converters using Adaptive Neural Networks, EPE’19 ECCE, ISBN 978-9-0758-1531-3, 2019 [18] H. Kim and K. G. Shin, On Dynamic Reconfiguration of A Large-Scale Battery System, IEEE Real-Time and Embedded Technology and Applications Symposium, ISSN 1545-3421, 2009, 87-96 [19] H. Kim and K. G. Shin, DESA: Dependable, efficient, scalable architecture for management of large-scale batteries, IEEE Transactions on Industrial Informatics, ISSN 1551-3203, 8(2012), 406-417 [20] T. Morstyn et. Al., Distributed Control for State-of-Charge Balancing Between the Modules of a Reconfigurable Battery Energy Storage System, IEEE Transactions on Power Electronics, ISSN 0885-8993, 31(2016), 7986-7995 EVS33 EVS33 11 Authors His current research interests include embedded low energy systems, battery management systems and multilevel converter drives. Leandro Cassarino received the B.S. degree in electronic engineering and the M.S. degree in mechatronic engineering from Polytechnic University of Turin, Italy, in 2010 and 2013, respectively. He was a Visiting Scholar in the department of Electrical Engineering, National Institute of Applied Sciences (INSA), Lyon, France, in 2012. He joined the CEA in 2013, as a research engineer to work on motor control systems and Battery Management Systems. His current research interests include self-reconfigurable battery systems and battery tests for stationary and mobility applications. EVS33 EVS33 12
https://openalex.org/W4392880545	https://neophilology.elpub.ru/jour/article/download/290/275	Russian	null	Automotive culture of China and Russia	Neofilologiâ	2,024	cc-by	5,466	рия и история культуры / Theory and History of Culture рия и история культуры / Theory and History of Culture Неофилология / Neofilologiya = Neophilology  2024;10(1):251-260 ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru НАУЧНАЯ СТАТЬЯ / ORIGINAL ARTICLE УДК / UDC 316.7 https://doi.org/10.20310/2587-6953-2024-10-1-251-260 Шифр научной специальности 5.10.1 Автомобильная культура Китая и России Чуньян ДУ Гуансийский университет науки и технологии 545000, Китайская Народная Республика, Гуанси-Чжуанский автономный район, г. Лючжоу,ул. Вэньчан, 2 duchunyang92@bk.ru Аннотация ц ВВЕДЕНИЕ. Исследование посвящено рассмотрению проблемы специфики китайской и российской автомобильных культур и ориентировано на экспликацию языковых фактов. Цель исследования – проанализировать «лингвокультурологическое пространство» автомо- бильной культуры, способной быть определённым маркёром наименования марок автомо- билей как специфической категории имён собственных в китайской и русской лингвокуль- турах. МАТЕРИАЛЫ И МЕТОДЫ. Эмпирическим материалом для исследования послужи- ли наименования известных марок автомобилей в Китае и России, изъятые из научных и научно-популярных изданий по истории автомобильной культуры. Для описания фактов языка использован метод сплошной выборки, описательный метод и метод интерпретации, дефиниционный анализ. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ. Установлено, что автомобиль- ная культура, являясь составляющей национальной культуры, отражает специфический способ человеческой деятельности. Автомобильная культура укрепляется местными брен- дами автомобилей и играет важную роль в экономике любой страны. В наши дни китайская автомобильная культура играет важную роль на мировом рынке, предоставляя людям всё более конкурентоспособные автомобили и услуги, активнее занимая место в жизни совре- менного общества. Будущее китайской автомобильной промышленности будет двигаться по пути самостоятельного и высококачественного развития, чтобы создавать благоприятную жизненную обстановку для людей. ЗАКЛЮЧЕНИЕ. Развитие автомобильной культуры во многом определяется степенью существенных смысловых показателей, к числу которых от- носятся «престижность», «надёжность», «комфортность», «цена», «мода». Эти показатели для носителя как китайского, так и русского языка закрепляются за наименованиями марок автомобилей и дают представление об автомобильной культуре, отражающейся в виде так называемого «образа мира», который эксплицирует лингвистический компонент с учётом специфики национальной культуры и носителя языка одновременно, отражая важные све- дения о разных лингвокультурах в контексте синхронии. Ключевые слова: лингвокультура, Китай, Россия, автомобильная культура, лингвистиче- ская автомобильность, наименования марок автомобилей Благодарности: Работа выполнена в рамках проекта фонда канцелярии образования Гуанси Китая «Исследование продвижения и развития автомобильной культуры Гуанси в России в контексте инициативы «Один пояс, один путь» (№ 2023KY0337). 本文系 2023 年广西高校中青年教师(科研)基础能力提升项目 “一带一路背景下广西汽车文化在俄罗斯推广与发展研究” 项目资助 (立项编号: 2023KY0337). “一带一路背景下广西汽车文化在俄罗斯推广与发展研究” 项目资助 (立项编号: 2023KY0337). Для цитирования: Ду Чуньян. Автомобильная культура Китая и России // Неофилология. 2024. Т. 10. № 1. С. 251-260. https://doi.org/10.20310/2587-6953-2024-10-1-251-260 Для цитирования: Ду Чуньян. Автомобильная культура Китая и России // Неофилология. 2024. Т. 10. № 1. С. 251-260. https://doi.org/10.20310/2587-6953-2024-10-1-251-260 Контент доступен под лицензией CreativeCommonsAttribution 4.0 License 251 © Ду Чуньян, 2024 Теория и история культуры / Theory and History of Culture 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru Automotive culture of China and Russia Chunyang DU Guangxi University of Science and Technology 2 Wenchang Ave., Guangxi, Liuzhou, 545000, People’s Republic of China duchunyang92@bk.ru Abstract INTRODUCTION. The study is devoted to considering the problem of the specifics of Chinese and Russian automotive cultures and is focused on the explication of linguistic facts. The purpose of the study is to analyze the “linguocultural space” of automotive culture, which can be a certain marker of the names of car brands as a specific category of proper names in Chinese and Russian linguistic cultures. MATERIALS AND METHODS. The empirical material for the study was the names of famous car brands in China and Russia, taken from scientific and popular science publi- cations on the history of automotive culture. To describe the facts of the language, a continuous sampling method, a descriptive method and an interpretation method, and definitional analysis were used. RESULTS AND DISCUSSION. It has been established that automotive culture, being a component of national culture, reflects a specific way of human activity. Automotive culture is strengthened by local car brands and plays an important role in the economy of any country. No- wadays, Chinese automobile culture plays an important role in the global market, providing people with increasingly competitive cars and services, and taking an active place in the life of modern society. The future of the Chinese automotive industry will pursue a path of self-reliant and high- quality development to create a favorable living environment for people. CONCLUSION. The de- velopment of automotive culture is largely determined by the degree of significant semantic indi- cators, which include “prestige”, “reliability”, “comfort”, “price”, “fashion”. These indicators for native speakers of both Chinese and Russian languages are assigned to the names of car brands and give an idea of automotive culture, reflected in the form of the so-called “image of the world,” which explicates the linguistic component, taking into account the specifics of the national culture and the native speaker at the same time, reflecting important information about different linguistic cultures in the context of synchrony. Keywords: linguistic culture, China, Russia, automotive vocabulary culture, linguistic automotivi- ty, names of car brands Acknowledgements: This work is funded by the 2023 Guangxi University Young and Middle- aged Teachers (Scientific Research) Basic Capacity Improvement Project “Research on the Pro- motion and Development of Guangxi Automobile Culture in Russia under the Context of the Belt and Road Initiative” (Project Number: 2023KY0337) 本文系 2023 年广西高校中青年教师 (科研)基础能力提升项目 “一带一路背景下广西汽车文化在俄罗斯推广与发展研究” 项目资助目助（立项编号：2023KY0337）. 助（立项编号：2023KY0337）. For citation: Du Chunyang. Automotive culture of China and Russia. ВВЕДЕНИЕ учёных).Речь идёт о том, что язык, будучи се- миотической системой, объективирует культу- ру и её традиции, то есть национальный язык и культура – это «однопорядковые явления» (О.Р. Очиров, К.К. Васильева). В настоящее время, «когда наметился целенаправленный поворот к углублению и расширению контактов с дружественными странами Азиатско-Тихоокеанского региона» [1, с. 7], исследование автомобильной куль- туры представляется особенно актуальным, поскольку «вещно-предметная репрезента- тивность деятельности обеспечивает эффек- тивность человеческого взаимопонимания в рамках формирующейся языковой реально- сти» [2, с. 148]. Принимая во внимание тот факт, что лингвистическая автомобильность широко заявляет о себе в современной русской пись- менной речи, когда возникают проблемы с правописанием наименований автомобиль- ных средств типа LADA, «Лада», Лада, при характеристике автомобильной культуры следует обратить внимание на её этнодиффе- ренцирующую функцию, которая определяет «оригинальность», «самобытность» и «иден- тичность» этнического сообщества с помо- щью экспликации языковых фактов [6]. Возникает необходимость более углуб- ленного изучения феномена автомобильной культуры, который должен рассматриваться в качестве важнейшего звена для процесса межкультурной коммуникации в различных её проявлениях и укрепления диалога куль- тур, представляющих особую значимость в условиях современной глобализации, щью экспликации языковых фактов [6]. Для современных лингвокультурологи- ческих исследований крайне важен тот факт, что автомобильная культура как компонент этноса по-своему выражает этническую куль- турную специфику в отражении номерных знаков для автомобильных средств, которые имеют свои характерные особенности в Ки- тае и России. Номерные знаки как некие со- общения зашифровываются в языковой код, который нужно распознать. В результате это- го распознавания устанавливается понимание, позволяющее прочитывать информацию с цифровыми и буквенными символами. Куль- турная специфика номерного знака транс- портных средств как номинативной единицы усматривается в его прагматическом компо- ненте – отношении к обозначаемому объек- ту, из чего следует, что автомобильная куль- тура должна рассматриваться как в качестве важнейшего звена для процесса межкультур- ной коммуникации, так и в качестве особого языкового знака, отражающего устойчивое смысловое значение в локальной субкульту- ре, где принято поддерживать национальные традиции. Л.И. Богданова подчёркивает: «При формировании культурной идентично- сти… опора на родной язык является обяза- тельной» [7, с. 12]. В широком понимании термин «автомо- бильная культура», являясь составляющей национальной культуры, включает в себя как уровень изобретения автомобиля, его назва- ние, так и уровень его распространения, ис- пользования, понимания и т. д., можно ска- зать, что автомобильная культура пронизы- вает все аспекты социальной и культурной жизни [3; 4]. Иными словами, автомобильная культура – это совокупность материальных и нематериальных богатств в процессе изобре- тения и развития автомобиля. Автомобильная культура, тесно связан- ная с понятиями пространства и места [5, с. Abstract Neofilologiya = Neophilol- ogy, 2024;10(1):251-260 (In Russ., abstract in Eng.) https://doi.org/10.20310/2587-6953-2024-10- 1-251-260 252 Du Chunyang. Automotive culture of China and Russia Неофилология / Neofilologiya = Neophilology  2024;10(1):251-260 ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru МАТЕРИАЛЫ И МЕТОДЫ Материалом исследования послужили названия известных марок автомобилей в Китае и России, отобранные из научных и научно-популярных публикаций (Цзян Хун- мей, Ли Шизенг, Лю Юй, Ван Сяовэнь, Фё- дор Буцко, В.М. Пахомов), каталогов автомо- билей, размещённых в сети Интернет, Нацио- нального корпуса русского языка (НКРЯ), в связи с чем был использован метод сплош- ной выборки, метод интервьюирования с вла- дельцами автомашин, описательный метод и метод интерпретации. Использовался лингво- культурологический метод, позволяющий ус- тановить факты национального языка и куль- туры как однопорядковые явления. Автомобиль меняет нашу жизнь, он при- носит нам огромное удобство и также во- площает определённую концепцию, отноше- ние и культуру. Автомобиль – это зеркало промышленной цивилизации, он отображает эволюцию и процветание человеческого об- щества и оказывает влияние на экономиче- скую структуру и скорость развития всего общества, а также формирует уникальное культурное явление. С точки зрения общей автомобильной культуры, её истоки восходят к Германии, развиваются в США, где появляется боль- шинство новаций. Как современный промышленный про- дукт, автомобиль отражает ценностные взгля- ды человечества, образ жизни, эмоциональ- ные потребности и т. д., отражает эстетиче- ские предпочтения различных временных периодов и разных слоев населения, образуя уникальную культуру. чувственных и поведенческих компонентов в идентификационных процессах» [8, с. 44]. чувственных и поведенческих компонентов в идентификационных процессах» [8, с. 44]. феномен, обладающей сложной внутренней структурой, свидетельствующей о неразрыв- ном единстве рациональных, бессознательных, чувственных и поведенческих компонентов в идентификационных процессах» [8, с. 44]. ВВЕДЕНИЕ 166-172], структурой нанесения информа- ции на определённый тип номерных знаков транспортных средств в той или иной стране, цветом номерных знаков, индивидуализи- рующих определённый вид транспорта или страну регистрации автомобиля, особенно ярко проявляет себя в буквенных и цифро- вых обозначениях (регистрационных номе- рах автомобилей), отражая тем самым лин- гвокультурологическую маркированность, в основе которой лежит понимание интеграции лингвистических и культурологических мар- кированнных фактов (идеи Н.Д. Арутюновой, В.В. Воробьёва, В.И. Карасика, В.А. Масло- вой, Ю.С. Степанова В.Н. Телия и других Понятие «культурная идентичность» в данном исследовании понимается как «фено- мен, обладающий сложной внутренней струк- турой, свидетельствующий о неразрывном единстве рациональных, бессознательных, 253 Ду Чуньян. Автомобильная культура Китая и России 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ Язык передаёт человеку знания об окру- жающей действительности, знание норм мышления и находит объективацию в языке с помощью имён собственных, номинирую- щих наименования марок автомашин. Осо- бую трудность в современной русской пись- менной речи вызывают наименования авто- мобильных средств, написанные латиницей, «при этом они, как правило, состоят из не- скольких слов и сопровождаются цифровыми обозначениями Audi ТТ Coupe, Toyota Corolla, Audi A8, Lexus IS 250, Renault Scenic, Ford Focus купе, Nissan Almera Classic, среди наименований, написанных кириллицей, также преобладают двойные и тройные на- звания (марка + модель автомобиля, марка + модель + модификация), например «Тойота- Камри», «Рено-Меган», «Ниссан-Аль-мера», «Ниссан-Альмера-Классик» [6, с. 11]. Дан- ный факт весьма существен для письменной практики межкультурной коммуникации, по- скольку за письменным словом стоит «карти- на мира», напрямую зависящая от особенно- стей жизнедеятельности китайского и русско- го сообществ. Развитие культуры автомобилей в Китае можно проследить с середины 1950-х гг. В это время появилась первая китайская автомо- бильная компания – First Automobile Works (FAW), и Китай стал производить и прода- вать собственные автомобили. С экономиче- ским подъёмом Китая, в последние десятиле- тия, китайский автомобильный рынок стал неуклонно расширяться и стал самым круп- ным автомобильным рынком в мире, а также местом расположения штаб-квартир не- скольких крупнейших автомобильных про- изводителей в мире. С расширением китайского рынка авто- мобилей увеличилось число людей, которые владеют автомобилями и интересуются ими. В Китае появляются различные автомобиль- ные реалии, такие, как автосалоны, автомо- бильные гонки, автомобильные клубы и фо- румы. Китайская культура имеет влияние на автомобильный дизайн (например, присутст- вие национальных узоров на сиденьях и ру- левых колёсах). Понятие «культурная идентичность оп- ределяется как социально-психологический 254 Du Chunyang. Automotive culture of China and Russia Неофилология / Neofilologiya = Neophilology  2024;10(1):251-260 ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru Развитие автомобильной промышленно- сти Китая можно условно разделить на три этапа. паний. Во-вторых, изучаются автомобильные выставки, фестивали, музеи и т. д. Кроме того, одни учёные обращают внимание на техноло- гический аспект развития автомобилей и уде- ляют больше внимания применению новых технологий в автомобильной промышленно- сти, другие учёные исследуют автомобиль- ную культуру с точки зрения экологичного развития автомобильной промышленности и подчёркивают необходимость создания ус- тойчивого и гармоничного общества [9]. Первый этап – рождение автомобильной промышленности Китая. Второй этап – с 1978 г. до конца ХХ ве- ка. В этот период китайская автомобильная промышленность получила существенный рост и сформировала систему производства автомобилей. Отгрузочные автомобили пре- вратились в легковые автомобили и начали активно развиваться. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ Это было время перехо- да от плановой экономической системы к ры- ночной экономической системе. Особенностью этого периода является быстрый рост развития коммерческого транспорта, постепенное раз- витие серийного производства автомобилей, повышение производственной мощности, на- личие квалификационных ресурсов при раз- работке самостоятельных продуктов автомо- бильной промышленности и основание фун- дамента производства легковых автомоби- лей. Производственная система автомобиль- ной промышленности в Китае становится более совершенной. Коммерческие органи- зации автомобильной промышленности по- степенно освободились от ограничений адми- нистративного управления; создавались со- вместные предприятия, автомобильная про- мышленность в Китае существенно повысила качество производства автомобилей. Автомобили марки “解放牌” (Jiěfàng pái – Цзефан пай) – это первый автомобиль, про- изведённый в Китае после основания Нового Китая в 1949 г. Марка “解放牌” был первым изготовленным серийно автомобилем в Ки- тае и был запущен в производство в 1956 г. Название “解放牌” означает «знак освобож- дения». В это название была заложена мысль о начале развития автомобильной промыш- ленности в новом Китае, конец истории, ко- гда Китай не мог массово производить авто- мобили. Первые автомобили марки “解放” были изготовлены на заводе «Первый автомобиль- ный завод» в Чанчуне и представлены на всеобщее обозрение на площади Тяньань- мэнь в Пекине. Эти автомобили стали одним из символов китайского автопрома, они ши- роко использовались в различных областях, в том числе для строительства китайской же- лезной дороги. Третий этап – с начала XXI века. После вступления Китая в ВTO автомобильная промышленность вошла в период быстрого расширения рынка и производства, а затем полностью влилась в мировую автомобиль- ную промышленность. Поскольку Китай по- ка не обладает автономным проектированием и разработкой автомобилей, трудно говорить о том, что в Китае развита полноценная ав- томобильная промышленность. Проблемы с качеством автомобилей и доверием к брендам являются главными препятствиями для разви- тия китайской автомобильной индустрии, по- этому она до сих пор не занимает прочной позиции на мировом рынке автомобилей. Среди марок китайских автомобилей об- ращает на себя внимание автомобиль под наименованием “Hongqi” («Хунци»). Этот автомобиль, созданный в 1958 г., стал свиде- тельством создания нового Китая и его роста. Марка “Hongqi” всегда придерживалась кон- цепции «независимости, инноваций и по- бед», производители автомашин стремилась к высокому качеству и представляли один за другим роскошные седаны для общественно- сти. История развития “Hongqi” была широ- ко известна в мире и отражала высокие дос- тижения автомобильной промышленности Китая. Китайские учёные обычно исследуют ав- томобильную культуру с двух точек зрения. Во-первых, изучается история развития авто- мобилей, культура бренда и концепции ком- Марка “Wuling” (Улин) появилась в 1985 г. 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru вание автомобиля марки “Wuling” воплотило в себе дух предпринимательства «упорного труда и стремление к сильному выражению». На данный момент эта марка стала одним из наиболее известных брендов автомобильной индустрии Китая. Компания “Geely” начина- ла свою деятельность с производства мото- циклов и с тех пор стала одним из лидеров автомобильных производителей Китая. Более того, учитывая спрос рынка и активную инно- вационную деятельность, автомобили произ- водства компании “Geely” активно покупа- ются, имея доверие потребителей, поскольку обладают такими показателями, как «пре- стижность», «надёжность», «комфортность», «умеренная цена», «мода». вание автомобиля марки “Wuling” воплотило в себе дух предпринимательства «упорного труда и стремление к сильному выражению». На данный момент эта марка стала одним из наиболее известных брендов автомобильной индустрии Китая. Компания “Geely” начина- ла свою деятельность с производства мото- циклов и с тех пор стала одним из лидеров автомобильных производителей Китая. Более того, учитывая спрос рынка и активную инно- вационную деятельность, автомобили произ- водства компании “Geely” активно покупа- ются, имея доверие потребителей, поскольку обладают такими показателями, как «пре- стижность», «надёжность», «комфортность», «умеренная цена», «мода». В то же время производители китайских автомобилей активно включаются в тренд цифровизации и интеллектуализации, посто- янно содействуя развитию автоматической езды, альтернативных источников энергии, интеллектуального транспорта и других сфер, становятся организаторами продвиже- ния всемирных интеллектуальных автомо- бильных технологий. В целом, китайские марки автомобилей, пройдя испытания и преодоление трудно- стей, стабильно продвигаются вперед. В бу- дущем они будут продвигаться более стре- мительно и с широкой перспективой, про- должая содействовать инновациям и разви- тию автомобильной промышленности Китая. Можно предположить, что будущее китай- ской автомобильной промышленности будет продолжать двигаться по пути самостоятель- ного развития и высококачественного разви- тия, особенно в туриндустрии. Кроме того, китайское правительство принимает ряд мер, чтобы поддержать развитие и укрепление ме- стных брендов автомобилей. Думается, что в будущем китайская автомобильная промыш- ленность продолжит играть заметную роль на мировом рынке, укрепляя маркетинговые стратегии и предоставляя людям более конку- рентоспособные автомобили и услуги. В автомобильной промышленности Китая появилось множество известных брендов, та- ких как “Geely”, “Great Wall Motors”, “Chery”, “BYD” и многие другие. Эти бренды имеют свою привлекательность на внутреннем и меж- дународных автомобильных рынках, способст- вуют дальнейшему развитию и укреплению автомобильной промышленности в Китае. В последние годы китайская автомо- бильная промышленность достигла значи- тельного прогресса и прорыва в области са- мостоятельного развития, интеллектуализа- ции, экологической устойчивости и т. д. 1 Буцко Фёдор. Откуда берутся названия автомо- билей? // «Мiръ имёнъ и названiй»: 45 выпусков науч- но-просветительской газеты по ономастике. 2006– РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ и стала одним из брендов автомо- бильной промышленности Китая. Наимено- 255 Ду Чуньян. Автомобильная культура Китая и России 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru Неофилология / Neofilologiya = Neophilology  2024;10(1):251-260 ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru Неофилология / Neofilologiya = Neophilology  2024;10(1):251-260 ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru ряд, в который вошли как русские, так и заим- ствованные слова: апгрейд, доработка, мод- динг, настройка, улучшение. Автомобильная культура – это уникаль- ный культурный феномен. С приходом моды на путешествия появляются автомобильные модели с географическими названиями (мо- дели «Капри», «Гранада»), с названиями вет- ров (модели «Сирокко», «Пассат»). «Авто- мобильные номинаторы» изначально давали простые имена типа «Гольф» (автомобиль немецкой компании “Volkswagen”, «Кадет» (немецкой компанией “Opel”), но со време- нем появились малопонятные наименования, например, «Тигуан» (образовано из слов «тигр» и «игуан» (крупная ящерица), «Каш- кай» (восходит к имени древнего иранского кочевого племени). За словом ТЮНИНГ закрепилось обще- употребительное значение «техническое усо- вершенствование, доработка чего-либо с це- лью изменения внешнего или внутреннего оборудования автомобиля» под желания и возможности конкретного водителя, который старается «обжить машину», сделать её для себя максимально комфортной или чтобы чем-то выделиться (изменить внешнюю от- делку, увеличить мощность двигателя, уста- новить пластмассовые накладки на кузове, тонировать стекла, внести детали интерьера и т. д.). В этом случае можно утверждать, что автомобиль служит объектом самовыраже- ния. А.З. Исаханян подчёркивает: «Нужно отметить, что современные водители стара- ются создать в салоне автомобиля особую атмосферу уюта. Зачастую можно наблю- дать, как салон автомобиля украшают раз- личными аксессуарами, будь то брелки, иг- рушки подушки и т. д., хотя все эти предме- ты не обладают функциональностью и нико- им образом не влияют на технические харак- теристики автомобиля» [5, с. 171]. Российские фанаты и любители автомо- билей организуют различные выставки авто- мобилей, ралли, съезды автоводителей и со- ревнования с тюнингом машин, такие, как «Международный автомобильный и води- тельский праздник России» – ежегодное ме- роприятие, объединяющее участников из России и других стран, демонстрирующих передовые автомобили. В целом, автомо- бильная культура России отражает уникаль- ное понимание и любовь русских к автомо- билям, а также демонстрирует сторону рос- сийского духа и инновационного мышления. Как известно, для обозначения русских номерных знаков транспортных средств ис- пользуются 12 букв русского алфавита, сходных по написанию с латинскими (А, Б, Е, К, М, Н, О, Р, С, Т, У, Х). Для обозначения китайских номерных знаков транспортных средств используются иероглифы, указы- вающие провинцию, буквы алфавита и пять цифр или букв (не используется буква О, ко- торую можно перепутать с цифрой 0). Ср.: китайские и русские регистрационные но- мерные знаки транспортных средств: 京C A1235; 浙B AB987) и МХТ *** 68. С развитием автомобильной культуры в русском языке стали появляться новые заим- ствованные слова, ср. 2 Крысин Л.П. Иллюстративный толковый сло- варь иностранных слов. М.: Эксмо, 2008. С. 732. 2010 гг. / авт.-сост. М.В. Горбаневский, В.О. Максимов / под общ. ред. А.С. Щербак. М.: МАСКА, 2023. С. 207. 2 Крысин Л.П. Иллюстративный толковый сло- варь иностранных слов. М.: Эксмо, 2008. С. 732. 2010 гг. / авт.-сост. М.В. Горбаневский, В.О. Максимов / под общ. ред. А.С. Щербак. М.: МАСКА, 2023. С. 207. 2 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru Ки- тайские автомобильные предприятия, будучи более открытыми, толерантными и иннова- ционными, активно вступают в глобальную конкуренцию и постепенно создают новые модели с учётом своих собственных брендов, внедряя международные передовые техноло- гии и используя стратегическое сотрудниче- ство как дополнительные элементы. р у у Автомобильная культура России восхо- дит к началу ХХ века. Когда экономика и инфраструктура транспорта начали отставать от других промышленных стран, русские на- чали самостоятельно производить автомоби- ли, заниматься ремонтом и модификацией автомобилей, привезенных из США и Евро- пы. Этот дух самостоятельности и любви к автомобилям продолжает пронизывать авто- мобильную культуру России до сегодняшне- го дня. Заметим, что в начале XXI века «мно- гие автоконцерны начали использовать в на- званиях модельного ряда типичные сочета- ния букв и слогов. К примеру, у Citroеn это буква икс в названиях Saxo, Xantia или Xsara. Toyota часто использует окончание <is: Auris, Yaris, Avensis»1. После периода накопления и качествен- ного прорыва на первом этапе, становления и быстрого развития на втором этапе китай- ские марки автомобилей уже находятся в бо- лее зрелой и стабильной стадии развития. Несмотря на жёсткую конкуренцию на миро- вом автомобильном рынке со стороны миро- вых брендов, китайское автомобильное про- изводство постоянно повышает уровень ка- чества, учитывает новые технологии, посто- янно открывает новые рынки и группы поль- зователей, учитывая спрос на услуги авто- сервисов. 256 Du Chunyang. Automotive culture of China and Russia 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru отражение разных моделей национально- культурной специфики языкового сознания, формирующих автомобильную культуру в широком смысле. ной культуры, поскольку является её состав- ляющей, и отражает специфический способ человеческой деятельности. Автомобильная культура укрепляется местными брендами автомобилей и играет важную роль в эконо- мике любой страны. р Обращает на себя внимание тот факт, что правописание названий автомобилей на русском языке для носителей китайской культуры вызывает особые трудности: с про- писной или со строчной буквы, в кавычках или без кавычек (ср.: «Запорожец», «запоро- жец»). Заметим, что Э.В. Шаламова приво- дит перечень номинаций автомобилей без кавычек: «Лада приора, Соболь, Таврия, УАЗ патриот, ЛУАЗ; из иностранных – Додж, Исузу, Ламборгини, Ниссан, Рено Дастер, Форд Фиеста, Ховер [11, с. 13]. В Нацио- нальном корпусе русского языка (НКРЯ) приводятся примеры написания названия ма- рок машин в кавычках, но и с прописной, и со строчной буквы, например: «Чуть по- одаль виднелся чёрный «Запорожец» Дегтя- рёва, из окон которого выглядывали…» (Да- рья Донцова «Доллары царя Гороха»); «Представляете, «запорожец» тогда стоил тысячу шестьсот, «жигули» – пять» (Юрий Башмет «Вокзал мечты»). В наши дни китайская автомобильная культура играет важную роль на мировом рынке, предоставляя людям всё более конку- рентоспособные автомобили и услуги, ак- тивнее занимая место в жизни современного общества. Будущее китайской автомобиль- ной промышленности будет двигаться по пути самостоятельного и высококачествен- ного развития, чтобы создавать благоприят- ную жизненную обстановку для людей. Обращение к основным характеристикам номерных знаков транспортных средств, имеющих хождение в КНР и России, показа- ло основные тенденции языковых фактов в автомобильной культуре. В обозначении ки- тайских и русских номерных знаков транс- портных средств как речевых продуктов от- ражается специфика национальной культу- ры: в китайской лингвокультуре желательны цифры 6, 8 и нежелательна цифра 4, эти циф- ры в русской лингвокультуре для обозначе- ния номерных знаков транспортных средств не имеют такой чёткой дифференциации. В.М. Пахомов отмечает: «Ни один из справочников и учебников (в том числе Ро- зенталь) не даёт однозначного ответа. Форд или форд? «Форд» или «форд»? А как, к примеру, писать Форд экс-плорер 517 JP?»3 Для наименования марок автомобилей в русском языке нет устоявшихся правил, что представляет особую трудность для носите- лей китайского языка при осмыслении орто- логии русского языка. Неофилология / Neofilologiya = Neophilology  2024;10(1):251-260 ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru слово ТЮНИНГ (от английского tuning – «настраивать, «регули- ровать») в значении «доводка, доработка ав- томобильного оборудования, осуществляе- мая обычно перед продажей автомобиля»2, ЯГТАЙМЕР (автомобиль, как правило, не моложе 20, но не старше 30 лет), ОЛДТАЙ- МЕР (автомобиль старше 30 лет), СТАЙ- ЛИНГ (усовершенствование только внешне- го вида автомобиля). Слово «тюнинг» легло в основу произ- водного АВТОТЮНИНГ, способствовало об- разованию прилагательных (словосочетания ТЮНИНГОВАННЫЙ АВТОМОБИЛЬ, ТЮ- НИНГОВАННЫЙ САЛОН), глагола (ТЮ- НИНГОВАТЬ), расширило синонимический Почеркнём, что в китайской лингвокуль- туре для номера автомобиля очень нежела- тельно присутствие цифры 4, которая ассо- циируется с образно-символическим смыс- лом слова 死 «смерть». Восточный нацио- нальный менталитет, складывающийся века- ми, до сих пор сохраняет числовые предрас- судки и стереотипы [10]. Следовательно, обозначения русских и китайских номерных знаков транспортных средств являют собой 257 Ду Чуньян. Автомобильная культура Китая и России 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru 3 Пахомов В.М. Языковой тюнинг. Как писать на- звания автомобилей? // «Мiръ имёнъ и названiй»: 45 выпусков научно-просветительской газеты по оно- мастике. 2006–2010 гг. / авт.-сост. М.В. Горбаневский, В.О. Максимов / под общ. ред. А.С. Щербак. М.: МАСКА, 2023. С. 358. 1. Богуславская В.В., Будник Е.А., Голубь А.Р. и др. Коммуникации с учётом национальной культуры адресата / под ред. А.С. Мамонтова, А.Г. Ратниковой. М.: РИОР, 2023. 230 с. 2. Очиров О.Р., Васильева К.К. Забайкальская школа китайского языка и культуры: история и совре- менность // Язык и культура. 2022. № 57. С. 145-157. https://doi.org/10.17223/19996195/57/7, https://elibrary.ru/retdol ЗАКЛЮЧЕНИЕ Автомобильная культура способствует углубленному пониманию кода националь- Автомобильная культура Китая активно проникает в Россию: спрос на китайские ма- шины заметно вырос. К самым продаваемым автомашинам на российском рынке относятся кроссовер “Chery” («Чери»), городской крос- совер “Haval Jolion” («Хавал Джолион»), кроссовер “Geely Coolray” («Джили Кулрей»). адреса а / од ред. .С. а о о а, . . а о о . .: О , 0 3. 30 с. 2. Очиров О.Р., Васильева К.К. Забайкальская школа китайского языка и культуры: история и совре- менность // Язык и культура. 2022. № 57. С. 145-157. https://doi.org/10.17223/19996195/57/7, https://elibrary.ru/retdol Список источников 258 Du Chunyang. Automotive culture of China and Russia ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru 3. Цзян Хунмей, Ли Шизенг. Разговор о построении автомобильной культуры с китайской спецификой на основе текущей ситуации потребления автомобилей // Технологии производства оборудования. 2021. № 05. С. 282-284 (蒋红梅, 黎仕增. 从汽车消费现状谈中国特色汽车文化建设 [J]. 装备制造技术 2021(05 C. 282-284). ) 4. Лю Юй, Ван Сяовэнь. Анализ текущей ситуации автомобильной культуры моей страны // Исследова- ние автомобильной промышленности. 2010. № 06. С. 44-48. (刘宇, 万晓文. 我国汽车文化现状分析[J]. 汽车工业研究 , 2010 (06). C . 44 -48). ) 5. Исаханян А.З. Автомобильная культура в контексте современной концепции пространства // Вестник Томского государственного университета. Философия. Социология. Политология. 2016. № 4 (36). С. 166-173. https://doi.org/10.17223/1998863X/36/17, https://elibrary.ru/xqrsob ) 5. Исаханян А.З. 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Mezhduna- rodnyi zhurnal issledovanii kul’tury = International Journal of Cultural Research, 2023, no. 2 (51), pp. 44-55. 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru ISSN 2587-6953 (Print), ISSN 2782-5868 (Online)  https://neophilology.elpub.ru 10. Gurevich T.M., Izotova N.N. Linguosemiotic analyses of numbers in Japanese phraseology. Voprosy kogni- tivnoi lingvistki = Issues of Cognitive Linguistics, 2023, no. 3, pp. 125-133. (In Russ.) https://doi.org/ 10.20916/1812-3228-2023-3-125-133, https://elibrary.ru/eitnwd 10. Gurevich T.M., Izotova N.N. Linguosemiotic analyses of numbers in Japanese phraseology. Voprosy kogni- tivnoi lingvistki = Issues of Cognitive Linguistics, 2023, no. 3, pp. 125-133. (In Russ.) https://doi.org/ 10.20916/1812-3228-2023-3-125-133, https://elibrary.ru/eitnwd p y 11. Shalamova Eh.V. Avtomobil’nayaleksikavsovremennomrusskomyazyke (sostav, funktsionirovanie, tendentsiirazvitiya): avtoref. dis. ... kand. filol. nauk [Automotive vocabulary in modern Russian (Composition, Functioning, Development Trends): PhD (Philology) diss. abstr.]. Moscow, 2018, 22 p. (In Russ.) 11. Shalamova Eh.V. Avtomobil’nayaleksikavsovremennomrusskomyazyke (sostav, funktsionirovanie, tendentsiirazvitiya): avtoref. dis. ... kand. filol. nauk [Automotive vocabulary in modern Russian (Composition, Functioning, Development Trends): PhD (Philology) diss. abstr.]. Moscow, 2018, 22 p. (In Russ.) Received Аugust 24, 2023 Revised January 11, 2024 Accepted January 18, 2024 References (In Russ.) https://doi.org/10.52173/2079-1100_2023_2_44, https://elibrary.ru/pgfrts 9 Shuai Jinshi Car Culture Beijing Tsinghua University Press 2007 (In Chinese) ( ) p g _ _ _ , p y pg 9. Shuai Jinshi. Car Culture. Beijing, Tsinghua University Press, 2007. (In Chinese) 259 Ду Чуньян. Автомобильная культура Китая и России 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology 2024;10(1):251-260  Неофилология / Neofilologiya = Neophilology Information about the author Du Chunyang, PhD (Philology), Lecturer, Guangxi University of Science and Technology, Guangxi, Liuzhou, People’s Republic of China, https://orcid.org/0000-0001- 8511-475X, duchunyang92@bk.ru Ду Чуньян, кандидат филологических наук, пре- подаватель, Гуансийский университет науки и техноло- гии, Гуанси-Чжанский автономный район, г. Лючжоу, Китайская Народная Республика, https://orcid.org/0000- 0001-8511-475X, duchunyang92@bk.ru Contribution: existing research in the field of Chi- nese and Russian automotive culture review, factual ma- terial analysis, research methodology development, manu- script text drafting, text editing. Вклад в статью:обзор имеющихся исследований в области китайской и русской автомобильной культу- ры, анализ фактического материала, разработка мето- дологии исследования, написание текста статьи, редак- тирование текста. Received Аugust 24, 2023 Revised January 11, 2024 Accepted January 18, 2024 Поступила в редакцию 24.08.2023 Поступила в редакцию 24.08.2023 Поступила в редакцию 24.08.2023 Поступила после рецензирования 11.01.2024 Принята к публикации 18 01 2024 у р Поступила после рецензирования 11.01.2024 П б 18 01 2024 Поступила после рецензирования 11.01.2024 у р р Принята к публикации 18.01.2024 Принята к публикации 18.01.2024 260
https://openalex.org/W2741099554	https://scielo.conicyt.cl/pdf/ssa/v17n1/0719-0948-ssa-17-01-00113.pdf, https://www.redalyc.org/pdf/3379/337952287005.pdf	es		La relación bilateral entre Chile y el Perú: La propuesta del fallo de la Corte Internacional de Justicia como punto de inflexión histórico y su posterior fracaso	Si somos americanos	2,017	cc-by	9,130	La relación bilateral entre Chile y el Perú: La propuesta del fallo de la Corte Internacional de Justicia como punto de inflexión histórico y su posterior fracaso The bilateral relationship between Chile and Peru: the proposal of the International Court of Justice judgment as a historical turning point and its posterior failure Rocío Arenas Riveros* Consejo Académico Consultivo de Estudios e Investigación Militar Pablo Rivas Pardo** University of Groningen, Reino de los Países Bajos Recibido: 17 de julio de 2015. Aprobado: 20 de mayo de 2017 Resumen Entre los meses de enero de 2008 y enero de 2014 se vivió entre el Perú y Chile un contencioso en la Corte Internacional de Justicia. Si bien el fallo no dio todo lo solicitado al Perú, este país sí logró la incorporación de un nuevo espacio marítimo en desmedro de Chile. En este lapso de seis años surgió la propuesta de que el fallo de la Corte fuera un punto de inflexión que cambiaría una relación que históricamente ha sido de confrontación e inconstancia integrista a una, más bien, de base estructural cooperativa. La hipótesis no se aprobó, puesto que ocurrido el fallo inmediatamente se levantó un * ** Cientista Política por la Universidad Diego Portales; Diplomado en Relaciones Internacionales, Universidad de Chile; Diplomado en Seguridad Multidimensional, Academia Nacional de Estudios Políticos y Estratégicos. Actualmente se desempeña como asesora en el Consejo Académico Consultivo de Estudios e Investigación Militar del Ejército de Chile. Correo electrónico: rocio.arenas@gmail.com Cientista Político y Licenciado en Ciencia Política de la Universidad Diego Portales. Diplomado en Operaciones de Paz y Seguridad Internacional, Diplomado Seguridad y Defensa, ambos en la ANEPE; Diplomado Corresponsal de Defensa, Academia de Guerra del Ejército; y Diplomado La relación chileno-peruana: puntos de encuentro, Universidad de Chile. Actualmente es alumno del Master of Art International Relations and International Organization, University of Groningen, Reino de los Países Bajos. Correo electrónico: p.rivas.pardo@ gmail.com Si Somos Americanos. Revista de Estudios Transfronterizos 113 nuevo conflicto, el denominado “triángulo terrestre”, que eliminó la propuesta del punto de inflexión. En virtud de lo anterior, este artículo explica por qué no se logró cumplir con el punto de inflexión, en razón del denominado triángulo terrestre. El análisis del rechazo del punto de inflexión incluye los principales eventos de esta relación bilateral, explicando así, por qué un triángulo de 3,7 hectáreas tuvo un valor cualitativo mayor que cambiar una relación bilateral caracterizada por su inconstancia. Palabras clave: Relación Chile-Perú, punto de inflexión, triángulo terrestre. Abstract During January 2008 and January 2014, Peru and Chile lived a dispute in the International Court of Justice. Although the ruling did not give everything Peru request, this country achieved incorporated a new maritime space at the expense of Chile. In this six years, arose the hypothesis that the Court’s decision would be a turning point that would change a confrontational and inconstancy relationship to new bilateral relationship with cooperative structure. The hypothesis was not approved, because immediately a new conflict arose, the called “terrestrial triangle” which eliminated the thesis of the inflection point. In view of the above, this article explains why it was not possible to comply with the turning point, because of the terrestrial triangle. The analysis of the turning point includes the main events of this bilateral relationship, it explaining in this way, why a triangle of 3,7 hectares has a qualitative value bigger than to change a bilateral relationship characterized by its inconstancy. Keywords: Chile-Peru relationship, turning point, land triangle. Introducción El Perú interpuso ante la Corte Internacional de Justicia (CIJ) en enero de 2008, una demanda contra el Estado de Chile. Tal demanda, en síntesis, solicitó que la CIJ definiera el límite marítimo entre ambos países; especificara el origen de tal demarcación; que realizara la proyección de una línea equidistante; y que tal proyección abarcara aguas internacionales como parte de sus aguas nacionales (CIJ, 2008). En medio de este contexto 114 Si Somos Americanos. Revista de Estudios Transfronterizos y previo al fallo de la CIJ, se desarrolló y difundió, durante el gobierno del Presidente de Chile, Sebastián Piñera (2010-2014) y del Perú, Alan García (2006-2011) y luego Ollanta Humala (2011-2016), la hipótesis de aprovechar la coyuntura de la demanda como un momento oportuno para generar un cambio en la relación bilateral respecto a la conformación territorial, es decir “un punto de inflexión”. Lo anterior fue una iniciativa que se adoptó a nivel presidencial, entre cancillerías y el plano académico. Finalmente, la Corte dio a conocer su fallo el 27 de enero de 2014 (ver anexo I). Si bien el origen del límite no fue el propuesto por el Perú, ya que fue un punto ubicado al norte del solicitado, y la línea equidistante comienza 80 millas marítimas después de la línea paralela que va desde el origen, el Estado peruano incorporó espacio marítimo a su soberanía territorial, económica y jurisdiccional, que el Estado de Chile consideraba como propio (CIJ, 2014). Sin embargo, tras el fallo, y a semanas de ser conocido el resultado de la demanda, surgió una nueva situación de tensión, el “denominado triángulo terrestre” (ver anexo II), que ambos países consideraron como propio, no siendo efectiva la propuesta de que el fallo sería el punto de inflexión en las relaciones bilaterales. Luego de dicho desencuentro, la posibilidad de aprovechar la coyuntura de la demanda como un punto de inflexión que concluyera con el ciclo de disputas a lo largo de la historia de la relación bilateral no funcionó, en el inmediato ni en el corto plazo. Cabe distinguir que se usa la expresión “denominado triángulo terrestre” dado que el espacio territorial no corresponde geométricamente a un triángulo y que además, ambos países lo consideran como propio, por lo cual, la existencia o no de un triángulo terrestre pendiente corresponde a que ambos Estados así lo digan. El artículo utiliza una metodología cualitativa de investigación, y el método de estudio de caso, para examinar una relación específica en un período de tiempo determinado entre dos actores: la relación bilateral entre Chile y el Perú. El objetivo general de este artículo es explicar por qué el fallo de la CIJ no fue un punto de inflexión en la relación bilateral que se esperaba, y consecuentemente, identificar el motivo del fracaso del “triángulo terrestre”. Los objetivos específicos son 1) Especificar el concepto punto de inflexión, 2) Analizar la propuesta del triángulo terrestre como punto de inflexión, 3) Examinar los ciclos de integración y diputas de la relación bilateral con el objeto de comprender la incidencia del pasado en la situación actual. Estos objetivos buscan responder a la Si Somos Americanos. Revista de Estudios Transfronterizos 115 siguiente pregunta: ¿Qué explica que el triángulo terrestre no permitiera la posibilidad de punto de inflexión histórico en la relación bilateral? Punto de inflexión: definición, propuestas políticas y académicas El concepto punto de inflexión posee diferentes significados según el contexto en que se utiliza, por ejemplo, en geometría inflexión significa “el punto de una curva en que cambia de sentido su curvatura” (RAE, 2014). En el lenguaje anglosajón, punto de inflexión se denomina turning point y se define como “el momento en que una situación comienza a cambiar en un sentido importante” (Cambridge Dictionary, 2014) en otras palabras “punto en el que se produce un cambio significativo” (Merriam Dictionary, 2014) o “verdaderos cambios que han transformado la manera en que son percibidas las sociedades humanas” (Guillén y Ontveros, 2012: 9), es decir, en el plano de las ciencias sociales, esto se considera como un cambio importante en la dirección de un fenómeno social. Desde la historia, Roger Launius señala que los puntos de inflexión “son un evento o varios de ellos que son capaces de cambiar la historia; es un hecho particular y no necesariamente debe tener la cualidad de dramático” (Launius, 2007: 19). En este artículo se utiliza el concepto como un evento que demarca un cambio significativo que circunscribe un antes y después en la historia de la relación bilateral entre Chile y el Perú. En el caso citado, durante el gobierno del presidente Piñera por Chile y los presidentes García (2006-2011) y Humala (2011-2016) por el Perú, se propuso transformar el problema de la demanda en una oportunidad o “punto de inflexión” para lograr un cambio en la relación que a lo largo del tiempo se ha caracterizado por ser intermitente, teniendo un carácter tenso y de confrontación. Marcos Robledo, entonces académico de la Universidad Diego Portales y actual Subsecretario de Defensa de Chile, fue explícito al escribir sobre esta idea, señalando que: “La sentencia de la Corte Internacional de Justicia podría, e idealmente debería, convertirse en un punto de inflexión histórico que marque una nueva etapa, en que la conformación territorial de los Estados peruano y chileno es aceptada por ambas partes” (Robledo, 2012: 30). 116 Si Somos Americanos. Revista de Estudios Transfronterizos Si bien la cita apunta a que se puede lograr un cambio, se infiere que no existiría o de plano no existe entre ambos países, una opinión compartida sobre la conformación territorial. Lo anterior fue comprobado tras el fallo de la CIJ, ya que el denominado “triángulo terrestre” no dio espacio al punto de inflexión esperado. Relacionado con lo anterior, en noviembre de 2012, durante la VI Cumbre de UNASUR -a tres días antes de la fase oral del juicio- ambos Presidentes hicieron declaraciones con la intención de mejorar la relación bilateral, por su parte el Presidente Humala indicó: “Hoy día estamos trabajando por fortalecer la integración, la relación bilateral entre Chile y el Perú, fortalecer el dinamismo de nuestras economías y en general, hacer más fructífera esta relación bilateral (…) Señalamos la importancia de trabajar una agenda después de La Haya” (El Mercurio: 2012). El Presidente Piñera respondió: “La agenda del pasado a veces nos ha dividido, pero la agenda del futuro definitivamente nos une, hemos conversado con el Presidente Humala cómo seguir fortaleciendo los lazos de unidad, amistad y colaboración entre Perú y Chile” (El Mercurio: 2012). A nivel de Cancilleres, en enero de 2013, estos también manifestaron su voluntad de conciliar un futuro auspicio en las relaciones bilaterales: “Los Ministros reafirmaron, en ese sentido, la voluntad de sus gobiernos de trabajar coordinadamente en la generación de las condiciones más propicias para consolidar y proyectar en el mediano y largo plazo una relación bilateral de estabilidad, confianza y cooperación” (Perú, Ministerio de Relaciones Exteriores: 2013). Durante los años 2010 y 2013, se realizaron, en ambos países, considerables charlas, mesas de trabajo y seminarios sobre la relación de Chile y Perú, orientado a las posibles sentencias de la Corte y las repercusiones a futuro para las dos naciones. De esto se destacan los encuentros binacionales de distintas personalidades de Chile y el Perú, tanto en Lima como en Santiago y la invitación de Académicos peruanos y ex Ministros. En esto, se reconoce en particular el aporte a la materia que realizó el Instituto de Ciencias Sociales de la Universidad Diego Portales (ICSO UDP), el Instituto de Estudios Internacionales de la Universidad de Chile (IEI), el Instituto de Estudios Internacionales Si Somos Americanos. Revista de Estudios Transfronterizos 117 de la Pontificia Universidad Católica del Perú (IDEI PUCP), la Fundación Chile 21 y la Fundación Konrad Adenauer (KAS). Dentro de las anteriores se encuentra la charla realizada en el ICSO UDP, donde expusieron los ex Cancilleres Rafael Roncagliolo del Perú (2011-2013) y Gabriel Valdés por Chile (1999-2000). Ambos coincidieron en ver el fallo de la CIJ como oportunidad. Aun así se plantearon incertidumbres, la del Perú consistía en un eventual armamentismo chileno, y por parte de Chile, el que esta no fuese la última disputa territorial por parte del Perú (ICSO UDP, 2013). Reforzando aquello, el historiador peruano Daniel Chasqui aseveró frente a la idea de un cambio en la relación: “¡Claro que se puede lograr (el punto de inflexión) y ya se está logrando! Si ambos países acatamos el fallo y lo ejecutamos con prontitud y humildad, las buenas historias nos buscarán a nosotros. En cambio, si comenzamos a ponerle “peros” al fallo y comenzamos a dilatar la ejecución, la desconfianza se va a acrecentar” (Publimetro Perú, 2014). Hay claridad en que, desde el plano político como académico, hubo una real intención de que el punto de inflexión histórico, que invitaba a cambiar la relación bilateral en el contexto de la demanda del Perú contra Chile, fuese concebido, sin embargo, en razón del denominado “triángulo terrestre” no fue posible en el inmediato o corto plazo. El triángulo terrestre como impedimento para cambiar la relación bilateral Inmediatamente dado el resultado de la CIJ, en enero de 2014, surgió una nueva controversia: el denominado triángulo terrestre (ver anexo II). Fue así que toda hipótesis de concluir los temas pendientes en materia de conformación territorial quedó desestimada. En el contexto de la VIII Cumbre de la Alianza del Pacífico, realizada en febrero de 2014 en Cartagena de Indias, los Presidentes Humala y Piñera hablaron sobre el triángulo. Tras el encuentro bilateral Humala señaló «hemos conversado, cada uno tiene su posición, pero llevando la voz del Estado peruano le hemos ratificado con firmeza al presidente Piñera la soberanía de Perú sobre el denominado triángulo terrestre» (El 118 Si Somos Americanos. Revista de Estudios Transfronterizos Mercurio, 2014), a lo que el gobierno de Chile por medio de su Presidente contestó con nota de protesta y de manera pública: “Le hemos expresado a (Ollanta) Humala que la posición peruana no está de acuerdo al derecho, ni siquiera a lo que ha sido la jurisprudencia durante más de 60 años respecto de ese triángulo terrestre (...) Estoy convencido de que los argumentos chilenos en esta materia son muy sólidos” (La Tercera, 2014). Meses después, el 19 de agosto de 2014, el Presidente Humala, en acto público, en el Salón Cáceres, mostró una nueva cartografía de los límites peruanos. En esta, incluyó el denominado triángulo terrestre como parte de la soberanía peruana e indicó: “Esta carta recoge la sentencia de La Haya del 27 de enero de este año y los trabajos que ha venido realizando una Comisión Mixta peruano-chilena para determinar las coordenadas del límite marítimo (…) esta Carta señala claramente que el inicio de la frontera entre Perú y Chile es el Punto Concordia y no hay otro” (Perú, Presidencia, 2014). Lo anterior deja explícito la falta de intención de promover una buena relación o dejar lugar a que se produjera el punto de inflexión. La respuesta del Estado de Chile, otorgada por el Canciller Heraldo Muñoz, señaló que “el triángulo terrestre es incuestionablemente chileno” (La Tercera, 28 de agosto de 2014), a propósito de una marcha convocada por un grupo nacionalista peruano para ir desde Tacna al triángulo terrestre, iniciativa que finalmente no llegó al destino propuesto. En este contexto, vemos que la propuesta de un punto de inflexión, como instancia para concluir la conformación territorial de ambos Estados, no se logró, en primer lugar, en razón de un triángulo terrestre -que mide 3,7 hectáreas- y en segundo lugar, a que ninguno de los gobiernos está dispuesto a priorizar unas buenas relaciones bilaterales por sobre la oportunidad de reclamar un espacio territorial como propio, puesto que es parte de su idiosincrasia. Respecto a la pregunta de investigación, este artículo tiene la hipótesis de que la propuesta del triángulo terrestre como punto de inflexión, que concluiría el ciclo de disputas a lo largo de la historia de la relación bilateral respecto a la conformación territorial, no fue viable de acuerdo a la incidencia del pasado en la situación actual. Si Somos Americanos. Revista de Estudios Transfronterizos 119 Ciclos de integración y diputas de la relación bilateral entre Chile y el Perú El artículo considera como un factor fundamental estudiar la incidencia del pasado en la situación actual, puesto que permite comprender que no es ajeno a la historia entre Chile y el Perú, el que no se terminen los asuntos pendientes en el ámbito territorial. Por tanto, que surja un nuevo tema en la agenda y que no se haya concretado la propuesta del punto de inflexión, no es una novedad, aún más, considerando que hubo académicos que advirtieron que dicho triángulo sería un nuevo punto en cuestión. Se considera importante repasar dos contextos históricos. El primero es el origen de esta relación, desde la época colonial hasta el siglo XIX, la cual muestra que desde sus cimientos la relación bilateral ha estado marcada por desentendimientos. El segundo, consiste en los principales hitos del siglo XX, que denota la dificultad de la aplicación e implementación de los tratados y de los procesos de integración. El origen de la relación: colonia, independencia y las guerras El inicio de la relación política entre ambos países data del Siglo XVI, cuando los españoles instalaron, en el nuevo continente, las colonias. En esta etapa el Perú se constituyó en un Virreinato, principal unidad administrativa en las nuevas tierras, destacándose por ser un gran proveedor de oro para España. De manera distinta, Chile fue una gobernación que funcionó bajo la lógica militar, a causa de la lucha contra los mapuches, constituyéndose así en un reducto que demandaba recursos al Rey. En aquella época, el Virreinato no sólo tenía una situación de desarrollo superior sobre la Gobernación de Chile, este último, además, se encontraba dependiente a las decisiones políticas y económicas que se le dictaran. Como señaló Alberto Wagner de Reyna: “La Capitanía General o reino de Chile dependió de Lima hasta 1798, la élite de sus hombres se educó en Lima, y separado de la Argentina por la cordillera, su vecino más efectivo era el Perú” (Winter, 2013: 7). A juicio de varios autores, tal situación administrativa (de dependencia) generó la instauración de una visión negativa entre ambas sociedades, la que habría sido heredada al conformarse y confrontarse las repúblicas. Esto se ejemplifica en la reseña que realizó Paz Milet al citar una entrevista del ex Canciller peruano, García Belaúnde: 120 Si Somos Americanos. Revista de Estudios Transfronterizos “Podría sostenerse que el establecimiento de las primeras instituciones civiles en el Perú tuvo como antecedente una guerra civil entre los pizarristas de Lima y Cuzco y “los de Chile”, como eran llamados los almagristas, que derivó en el asesinato de Pizarro” (Milet, 2005: 7). Con esta cita, se demuestra que junto con existir un origen competitivo y de subordinación entre el Virreinato del Perú y la Gobernación de Chile, existió tempranamente un enfrentamiento armado entre ambas comunidades, asunto que culturalmente es heredado una vez finalizada la época colonial. El Embajador peruano, Juan Miguel Bákula, refuerza esta idea en su libro “Perú en el Reino Ajeno” como se puede apreciar en la siguiente cita: “La “herencia colonial”, como consecuencias de la concepción política reinante, de las costumbres administrativas y de un ejercicio de la autoridad destinado a sostener una relación radial (…) Fue característico que la vinculación entre las dependencias coloniales estuviera marcada por el signo de la subordinación y, en otras circunstancias, por la prohibición” (Bákula, 2006: 97). El caso de los almagristas y del monopolio económico que duró casi dos siglos y medios, caló en la categorización y rivalidad de las sociedades, desde un plano social y económico. Respecto a lo primero, existía una mirada despectiva hacia los “mapochinos” es decir, los residentes de Santiago, por parte de la sociedad limeña, como sinónimo de incivilizados; y en lo económico, el control que ejercía el Virreinato sobre la gobernación era total, inclusive abarcaba hasta el Puerto de Buenos Aires. Entonces, el cambio de Gobernación a Capitanía, para la élite peruana se interpretó como una manifestación de rivalidad, puesto que para los entonces “chilenos” este paso implicó tener una mejor y mayor autonomía, junto con una relación directa con la Corona y sus instituciones, que pasaron a ser representantes para el continente y ya no con el Virrey. Cerrada la colonia, se observa claramente que esta no legó una relación cooperativa para ambos países al iniciarse la emancipación americana, al contrario, las elites tenían una mirada de rivalidad. Posteriormente, la independencia de América del Sur, dada entre los años 1809 y 1826, tampoco heredó una estructura de buena vecindad a pesar de la ayuda prestada por Chile a la independencia del Perú, tal y como lo señala el decreto “Tratado de Auxilio al Perú” de 1823 por el cual se ayuda con medios militares a la guerra. De ese documento se destaca la inspiración política del momento: Si Somos Americanos. Revista de Estudios Transfronterizos 121 “Queriendo manifestar la persuasión invariable en que se halla este gobierno y todo chileno, de que la Guerra del Perú es la Guerra de Chile, no solo por la consideración de su seguridad, sino porque la gloria que ha resultado a Chile de haber sido el primero que la emprendió, se mancharía si floja o caprichosamente abandonemos el campo” (Chile. Biblioteca del Congreso Nacional, 1823: 1). Es común suponer que la participación de Chile para lograr la expulsión de los españoles en el último reducto castellano llevó a una valorización entre las partes, al mirarse como gobiernos amigos, pero aquello es totalmente erróneo. Si se observa bien, el proceso de independencia del Perú es exportado desde el sur y del norte, no es un proceso innato a sus líderes políticos. Vale observar que son San Martín, bajo el financiamiento de O´Higgins, y Simón Bolívar con las tropas grancolombianas, los que se encuentran en Guayaquil para decidir el futuro del Virreinato. Por lo cual, el paso de Virreinato a República no deja ser una sensación errada de común ideario de vecindad. Al decenio siguiente, las nacientes repúblicas se encuentran en la “Guerra contra la Confederación Perú-Boliviana”, dada entre 1836 y 1839. Tal episodio es visto, por la historiografía chilena más tradicional, como un punto positivo. En palabras simples, se expresa que Chile se alió con los peruanos restauradores y logró reintegrar al Perú republicano en contra del enemigo común: la Bolivia del Mariscal Santa Cruz, liberándolos por segunda vez, ya que antes había sido de España. Una segunda mirada que se presenta en parte de la historiografía peruana, es mirar la Confederación como un proyecto de integración, al cual Chile, y en especial, Diego Portales, de manera preventiva atacó con peruanos que se oponían a otros peruanos. En cita de Percy Cayo Córdova: “Cierto es también que aun en la historiografía peruana y boliviana hay voces que proclaman su desacuerdo o antagonismo, su cuestionamiento o inconformidad con el gran proyecto confederacional (…) Más creemos que ya es tiempo de apuntar a una reflexión más certera y comprensiva del Gran Mariscal Andrés de Santa Cruz. Loas, alabanzas y encarecimientos tantas veces presentes en estas ocasiones no deben postergar la reflexión serena sobre lo que quiso realizar” (Cayo, 2010: 194). La interpretación de este autor apunta a ver a Santa Cruz como un hombre que buscó la integración, como consecuencia natural de un origen común entre ambos países. Entrando en las fases de esta guerra, las campañas contra la Confederación fueron dos. La primera, liderada por Blanco Encalada que fracasó, debiendo capitular frente a Andrés de Santa Cruz, con la firma del “Tratado de Parcaupata”. Si bien el tratado obligaba a que 122 Si Somos Americanos. Revista de Estudios Transfronterizos las tropas chilenas se retirasen, poniendo así fin al conflicto, el gobierno chileno hizo una nueva campaña liderada por Manuel Bulnes, que llegó al triunfo en la Batalla de Yungay. Entonces, al no haber respetado la firma de Parcaupata, se levantó la tesis de algunos historiógrafos peruanos de que “los chilenos no respetan los tratados cuando estos no les convienen” (Zapata, 2012) idea reforzada –años más tarde- al no realizarse el arbitraje por Tacna y Arica al cumplirse los 10 años del Tratado de Ancón, detallado más adelante. En años posteriores, el Perú vivió serios problemas con sus vecinos del norte, teniendo entre medio un conflicto con Ecuador en 1851, el que venía arrastrándose desde los años 20 contra la Gran Colombia, por lo que la preocupación principal del Perú era tener seguridad y buenas relaciones con su frontera noreste, Chile no era un problema. Ya en la década de los 60 tanto Bolivia, Chile y Ecuador fueron a la ayuda del Perú durante la Guerra contra España entre 1865 y 1866. Sin embargo tal momento solidario no llevó a institucionalizar una buena vecindad, ya que por esa misma fecha comenzó la competencia económica entre los países por el salitre, teniendo como antesala la disputa del guano. El ejemplo de la competencia fue la contención entre los puertos del Callao y de Valparaíso con miras para consolidarse como el principal puerto del Pacífico Sur y de principal plataforma exportadora de salitre. En esa misma década, se firmó entre Chile y Bolivia el Tratado de 1866, el cual fijó entre las partes como límite internacional el paralelo 24° de Latitud Sur implicando para Chile y el Perú no ser países vecinos (Bolivia. Dirección Estrategia de Reivindicación Marítima-DIREMAR, 2014). A finales de la década siguiente, en 1879, comenzó la Guerra del Pacífico con la ocupación de Antofagasta, por lo cual las hostilidades fueron primeramente entre Chile y Bolivia. Pero a causa del Tratado Secreto para una asistencia militar (Pardo por el Perú y Frías por Bolivia) entró el Perú, extendiéndose la guerra hasta la Sierra peruana. El fin de las hostilidades llegó con el Tratado de Ancón de 1883, pero no así el conflicto en un plano diplomático, al contrario, este se extendió hasta 1929. Tal extensión y dilatación de una solución definitiva implicó que el problema no sea únicamente la manera en que se gana la guerra y los tributos de ella, sino también, la manera como se construyó la paz y la cultura que ella generó. En detalle, el 20 de octubre de 1883 se firma el Tratado de Paz de Ancón, el que fijó una tregua entre las partes, la provincia de Tarapacá pasó a Chile, y se dejó pendiente la situación de Tacna y Arica: “Dicho tratado en su artículo 3 establecía que las provincias de Tacna y Arica continuarían en posesión de Chile y sujetas a la legislación y autoridades de ese país por el término de 10 años y, una vez expirado el plazo, un plebiscito (votación popular), celebrado en estas dos provincias, decidiría si las mismas quedaban definitivamente Si Somos Americanos. Revista de Estudios Transfronterizos 123 bajo el dominio y la soberanía de Chile, o si continuaban siendo parte del territorio peruano. Dicho plebiscito nunca llegó a realizarse y las provincias de Tacna y Arica continuaron bajo soberanía chilena, pese a que la ocupación debió terminar en 1894» (Sandoval, 2011, web). La Cancillería peruana en su página de Internet señala sobre la Guerra del Pacífico: “Las relaciones diplomáticas entre el Perú y Chile se establecieron inmediatamente después de la independencia. La Guerra del Pacífico motivó la interrupción de las mismas hasta la firma del Tratado de Ancón de 1883. Una nueva interrupción se produjo entre los años 1910 y 1928 como resultado de las diferencias sobre la ejecución del artículo 3 de dicho Tratado, referido a la realización de un plebiscito para definir la soberanía de las provincias cautivas de Tacna y Arica” (Perú. Ministerio de Relaciones Exteriores, 2010). Bákula hace una reflexión muy interesante sobre los momentos en que el Perú tuvo fuertes cambios en su vida como unidad territorial y en dos de ellos Chile es parte integrante: “Nunca el “Perú” volvió a ser el mismo en el tiempo siguiente, cuanto por los cruentos sucesos ocurridos. Y, al igual, porque en esos tres momentos las causas determinantes fueron exógenas, o sea que vinieron de fuera. Así fue en la conquista y subsiguiente colonización; en la revolución de la independencia, al imponerse un nuevo régimen; y en la guerra del Pacífico, al invadirse y cercenarse su territorio” (Bákula, 2006: 156). El legado negativo de la guerra se expresa en la cultura, siendo este el mayor escollo a destrabar. El Perú consagra una identidad común y nacional por medio de sus héroes, implicando una construcción de identidad en oposición de Chile y de los chilenos. El territorio cercenado, como señala Bákula, es un cambio del cual Chile es protagonista y el antagonista de un país, el cual mantiene unas provincias prisioneras, como lo señala el Escudo del Ejército del Perú, que tiene dos hojas de su rama de laurel dobladas en señal de “dos lugares cautivos” (Perú. Ejército, 2014) interpretándose como Arica y Tarapacá. La relevancia de la Guerra del Pacífico o Guerra contra Chile, y que no entrega la Independencia ni otras guerras para el Perú, es la construcción de héroes propios que pasan a ser los primeros próceres de su vida como República, encarnados en el Almirante Miguel Grau y el General Francisco Bolognesi, ambos Héroes de la defensa del Perú. 124 Si Somos Americanos. Revista de Estudios Transfronterizos Sin embargo, y como se señaló antes, el tema no quedaba allí, el cómo se construyó la paz también es un problema, y quizás, aún mayor. Ernesto Yepes (1999), destacado historiador peruano, en su libro “Un plebiscito Imposible. Tacna y Arica 1925-1926” describe lo vivido por los árbitros estadounidenses, los cuales tenían como misión solucionar la situación vivida entre las partes. Ellos en sus memorias transmiten como traba para efectuar el plebiscito en los años 20 la actitud hostil de Chile para realizar un plebiscito legítimo y sin vicios. A modo de síntesis, entre 1879 y 1929 transcurrieron 50 años, en que hubo una Guerra de cinco años, no se hizo el plebiscito, se rompieron las relaciones y se solucionó la soberanía de Tacna y Arica de manera transaccional. La académica Paz Milet, a su vez, señala que la relación bilateral ha sido fluctuante debido a que ha estado: “Afectada permanentemente por la herencia del pasado y sus consecuencias más visibles -pérdida territorial, invasión chilena y consecuente obtención de trofeos de guerra- que aún son un factor determinante en el avance y desarrollo de los vínculos entre Chile y Perú” (Milet, 2004: 228). Complementando la idea anterior, esta guerra es considerada como una de las más relevantes en la historia de la región junto con la de Triple Alianza y la del Chaco en los siglos XIX y XX respectivamente. Fernando Cavieres, historiógrafo chileno, subraya que la vida económica, post Guerra del Pacífico, se restableció rápidamente, entre Chile, Perú y Bolivia, pero no fue así en la política y quedó plasmada en la historia: “Una política de desconfianza que se fue traspasando hacia todos los sectores de la sociedad, especialmente cuando, además, los procesos históricos de los países se fueron desarrollando con ritmos y profundidades diferentes” (Cavieres, 2006: 22). Rodríguez Elizondo indica que el orgullo vencedor chileno no ayudó a mejorar la relación bilateral, e incluso: “En contrapunto con el rencor peruano, amarró el desarrollo futuro de ambos países a una íntima enemistad, que se expresaría, para unos, en la obligación de conservar lo ganado y, para otros, en la necesidad de recuperar lo perdido. Ese amarre impediría Si Somos Americanos. Revista de Estudios Transfronterizos 125 asomarse a las posibilidades de una cooperación que los potenciara a ambos conjuntamente” (en Milet, 2004: 229). Cerrando esta sección y explicando por qué se puede afirmar la existencia de un “vicio de origen” en la relación Chile-Perú, vale recordar la frase “el Estado hace la guerra y la guerra hace al Estado” acuñada a Charles Tilly. La primera parte de ella indica lo lógico, que son los Estados los que declaran, hacen y deciden una guerra, la segunda parte, ahonda en el problema antes mencionado, que la guerra haga al Estado implica que esta diseña una identidad común. Bajo la lógica recién explicitada, para Chile fue Yungay la que creó al roto chileno y la identidad chilena, para el Perú fue la capitulación de Ancón de 1883 y el Tratado de 1929 lo que creó una cruzada e identidad nacional, siendo esto el vicio de origen. Vemos que ambos países no tuvieron una integración bilateral institucionalizada antes de la guerra, por lo cual, la conflagración copó un espacio vacío entre ambos países, sumada la herencia del periodo colonial. Principales hitos del Siglo XX desde el Tratado de 1929 La relación bilateral entre Chile y el Perú, en este lapso, se puede distinguir en tres períodos. Se considera al primero, desde 1929 a la década del 1950 con la firma del Tratado de Lima. Esta fase se destacó por significantes hitos en el ámbito territorial y marítimo que permiten describir este período como uno de importantes acuerdos y de buenas relaciones vecinales, que mirado en retrospectiva pudo haber sido una oportunidad para haber desarrollado un punto inflexión, tras la solución de la soberanía de Tacna y Arica. El segundo es a partir de la década de 1960, con el surgimiento de los regímenes militares, primero en Perú en 1968 y luego en Chile en 1973, donde las relaciones fueron de alta tensión y crisis político-militar, hasta el retorno a la democracia en 1990. El tercero se desarrolla durante la década de 1990, período en que se pone en aplicación el Tratado de 1929, y que pudo haber sido otro momento oportuno para lograr el punto de inflexión en la relación, pero tampoco fue el caso. El primer período se caracterizó por importantes tratados y declaraciones en el ámbito territorial, marítimo y político. Comenzando por el ámbito territorial, el Tratado de Lima 126 Si Somos Americanos. Revista de Estudios Transfronterizos y su Protocolo Complementario, firmado el 3 de junio de 1929, ratificaron los límites entre ambas naciones dejando Arica en Chile y Tacna para el Perú. El Tratado de 1929 puso fin, momentáneamente, a los asuntos pendientes que tenían ambos países desde el fin a la Guerra del Pacífico (1879-1883). El artículo primero indica: “Queda definitivamente resuelta la controversia originada por el Artículo Tercero del Tratado de Paz y Amistad del veinte de octubre de mil ochocientos ochenta y tres, que era la única dificultad pendiente entre los Gobiernos signatarios” (Dirección de Frontera y Límites, 2014). El Tratado de 1929 tiene la peculiaridad de haber sido firmado por Augusto Leguía, quien gobernó en el Perú entre los años 1919 y 1930, y que durante su gestión dio solución a los complejos problemas limítrofes que tenía con sus vecinos. Vale destacar que Leguía siempre fue un duro en las negociaciones con Chile, por lo que, la solución adoptada en su gobierno fue más bien pragmática, antes que representativa de la idea política que se manejaba en relación a lo justo (Bákula, 2006). Otro evento significativo en la relación bilateral, y una primera demostración de buena vecindad, fue la participación de Chile junto a Brasil, Estados Unidos y Argentina como garantes del protocolo de Río de Janeiro de 1942, que ponía fin a la guerra acontecida un año antes entre el Perú y Ecuador. Entre las décadas de 1940 y 1950, el ámbito marítimo fue el tema principal de la agenda bilateral. En ellas se levantó una inédita propuesta entre Chile y Perú para evaluar y extender la proyección marítima vigente, con el fin de crear las condiciones necesarias de subsistencia, fortalecer el desarrollo económico y cuidar la conservación y protección de los recursos naturales. Dicha iniciativa conjunta generó cambios en el Derecho Internacional del Mar. En el año 1947 se realizaron declaraciones jurisdiccionales unilaterales, entre los dos Estados, con el fin de cambiar las 3 millas marítimas territoriales a 200 millas. El 18 de agosto del año 1952 ambos países, junto a Ecuador, celebran y ratifican la “Declaración sobre Zona Marítima” o “Declaración de Santiago” donde: “proclaman como norma de su política internacional marítima, la soberanía y jurisdicción exclusivas que a cada uno de ellos corresponde sobre el mar que baña las costas de sus respectivos países, hasta una distancia mínima de 200 millas marinas desde las referidas costas” (Chile. Dirección de Fronteras y Límites b, 2014). Si Somos Americanos. Revista de Estudios Transfronterizos 127 Este nuevo hito en soberanía quedaría incluido en el Convenio de Derecho del Mar del año 1982. Manuel Rodríguez Cuadros, relata el hecho en su libro “Delimitación marítima con equidad. El caso de Perú y Chile” de la siguiente forma: “El verdadero impulso que inició el proceso de cambio del Derecho Internacional del mar provino de las iniciativas de Chile y del Perú en 1947. Ambos países aprobaron sendos decretos que, por primera vez en la historia, reivindicaron la soberanía y jurisdicción exclusivas sobre las columnas de aguas oceánicas hasta una distancia de 200 millas, incluidos el suelo, el subsuelo, con la finalidad de preservar, proteger y explotar sus recursos marinos renovables y no renovables” (Rodríguez Cuadros, 2007: 167-168). Como continuación del “Convenio sobre Zona Especial Fronteriza Marítima” de 1954 se realizó el “Acta de los Representantes de Chile y del Perú” de 1968 y el “Acta de la Comisión Mixta Chileno-Peruana” de 1969 (Chile. Dirección de Fronteras y Límites c, 2014). Esta fue una década en que la continuidad de integración se encaminó tras la creación de la “Corporación Andina de Fomento” actualmente la “Comunidad Andina de Naciones”. El ex Canciller de Chile, Valdés Subercaseaux, señala ese episodio en sus memorias, describiendo su experiencia como Canciller tras una reunión de la ALALC, en Uruguay, en la que vio que el camino no era promisorio y de vuelta del viaje junto con sus pares del Perú, Ecuador, Colombia y Venezuela, se les ocurrió crear la CAF: “El pacto fue firmado en Lima y se agregó el gobierno de Bolivia, cuyo ministro de Relaciones -un militar- nos retrasó la firma varias horas, porque él no quería que antes nos prometiéramos a darle a su país una salida soberana al mar. Después de una larga discusión, yo mantuve mi negativa y la frase que el delegado boliviano deseaba incluir fue puesta al final, después de nuestras firmas, como una condición que sólo obligaba a Bolivia” (Valdés, 2009: 157). Desde que se firmó el Tratado de 1929, Chile con el Perú lograron construir una dinámica relación que partió con la aclaración de los asuntos territoriales y luego marítimos, que la visión realista de las relaciones internacionales considera vitales para controlar el conflicto. Como complemento, se creó una institución de cooperación económica, cualidad importante para lograr una integración bajo la óptica liberal. Tomando lo expuesto al inicio del artículo, este primer período pudo ser considerado como otro punto de inflexión en la relación bilateral, puesto que se estaban respetando los tratados, se consolidaban nuevos y se actuaba de forma conjunta por un bien mayor 128 Si Somos Americanos. Revista de Estudios Transfronterizos como fue el Protocolo de Río de 1942 y la Declaración de Santiago por la delimitación marítima. Pero, como se advertirá a continuación, con la instauración de los regímenes militares se rompen las relaciones bilaterales y se retrocede a un estado de crisis. En el segundo período, que comienza en la década del 60, se produce la convergencia en Presidentes ideológicamente símiles, Eduardo Frei Montalva (1967-1970) y Fernando Belaúnde (1963-1968), y luego entre el “Gobierno Revolucionario de las Fuerzas Armadas” de Velasco Alvarado (1968-1975) y Salvador Allende (1970-1973), pero que se rompe tras el inicio del Régimen Militar del General Augusto Pinochet (1973-1990), que le tocó estar con Velasco Alvarado; Francisco Morales Bermúdez (1975-1980), Belaunde Terry (1980-1985) y Alan García (1985-1990). Especialmente, entre los años 1973 y 1978, surgió un fuerte ambiente de desconfianza y alejamiento, debido en gran parte a que se acercaba el centenario de la Guerra del Pacífico. En 1974 hubo una tensión evidente, puesto que ambas fuerzas armadas estaban movilizadas y desplegadas en la frontera (Robles, 2006). El cuadro militar estratégico estaba totalmente hecho para una acción bélica. La revista ¿Qué Pasa? en su edición del 3 de julio de 1993, en un reportaje afirma: “Con préstamos soviéticos a largo plazo y de baja tasa de interés, Perú inició sus órdenes de compra en Moscú adquiriendo 200 tanques T-54 y T-55, aviones de combate sofisticados, artillería pesada, etc. se irían acumulando año a año en los regimientos peruanos” (Revista ¿Qué Pasa? 1993: 4). Tal idea también es sostenida por Rodríguez Elizondo (2004) en su libro “El Siglo que vivimos en peligro”, en donde señala que el armamentismo peruano no llegó a la guerra por la situación interna del país, pero que en 1975 pudo haber habido guerra, o inclusive, años después al celebrarse el centenario de la Guerra del Pacífico en 1979. En el invierno de 1975, va a disminuir la tensión de la relación bilateral, luego que el general Francisco Morales Bermúdez tomara el poder del gobierno peruano, ante un General Velasco muy deteriorado por motivos de una enfermedad. Dichos tiempos de serenidad se quebraron en 1978 cuando en diciembre: “Dos oficiales navales chilenos del petrolero Beagle, ubicado en Talara, fueron detenidos bajo la acusación de haber sido sorprendidos fotografiando instalaciones militares. El 20 de diciembre, la Cancillería peruana informó sobre la expulsión de seis chilenos, incluyendo cuatro funcionarios de rango diplomático. El 19 de enero de Si Somos Americanos. Revista de Estudios Transfronterizos 129 1979, se declaró persona non grata al embajador de Chile en Lima y se solicitó su retiro inmediato (…) el 20 de enero de 1979 es fusilado un suboficial de la Fuerza Aérea del Perú acusado de haber entregado información militar a los chilenos” (Gallardo, 2005: 216-217). La tensión político militar no decreció hasta el retorno de la democracia con los presidentes Aylwin en Chile (1990-1994) y Fujimori en Perú (1990-2000). El tercer período entre los Estados comienza con el retorno a la democracia, donde se retoman las relaciones y vuelve a la agenda bilateral el Tratado de 1929, puesto que se quiere concluir con los asuntos que aún no se ejecutaban en su totalidad. Luego de negociaciones bilaterales entre las Cancillerías, en noviembre del año 1999, se firma el “Acta de Ejecución” y se declaran por terminados los temas pendientes entre ambos Estados. El Ministro peruano de Relaciones Exteriores de la época, Fernando de Trazeginies, indicó en su discurso posterior a la firma que: “Hemos presenciado hace unos minutos, el fin a las últimas secuelas de un conflicto entre Perú y Chile que tuvo lugar hace 120 años y que, felizmente, se encuentra hoy totalmente superado. Desde esa época hasta nuestros días, ha tenido lugar un proceso de curación de las heridas ocasionadas por la guerra y de restablecimiento de las relaciones normales entre nuestros países” (Sandoval, 2011). De este modo se concluyó de manera definitiva con los asuntos pendientes del mencionado Tratado y su Protocolo Complementario, preservando el Perú sus derechos de usufructo y servidumbre de un muelle, de un malecón de atraque, establecimientos y zonas de operación dentro del Complejo Portuario de la ciudad fronteriza de Arica; así como el libre tránsito de personas, mercaderías y armas entre Tacna y el Muelle de Arica y viceversa (Sandoval, 2011). Luego de que se firmó el Acta de Ejecución y su respectivo Reglamento, el 7 de marzo 2000, se inauguró el Cristo de la Concordia sobre el Morro de Arica, hito que simbolizaba el fin de los asuntos pendientes y daba paso a un nuevo escenario de las relaciones entre ambos Estados. A su vez, el entonces Ministro de Relaciones Exteriores de Chile, Juan Gabriel Valdés, declaró: “Mediante este acto, completamos la ejecución de algunas cláusulas del Tratado de 1929 y de su Protocolo Complementario, respectivamente los Artículos 5º y 2º. Después de 70 años, hemos encontrado la forma de terminar una tarea en esta materia, y ofrecer a nuestras comunidades nacionales, una perspectiva que permita mirar hacia el futuro con renovada confianza y en un marco de sólida cooperación” (Sandoval, 2011). 130 Si Somos Americanos. Revista de Estudios Transfronterizos Concluye el siglo XX con un promisorio horizonte para la relación bilateral. Se acordó la creación del Comité de Seguridad y Defensa (COSEDE), la instauración de mecanismos de intercambio y coordinación entre Estados Mayores de la Defensa Nacional y de las Fuerzas Armadas. Estas instancias, implementadas el año 2001, contaron con versiones posteriores, pero de manera discontinua hasta el día de hoy. Este tercer período fue idóneo para lograr un punto de inflexión, considerando la ejecución completa del acta complementaria del Tratado de 1929 y los avances en las relaciones bilaterales al punto de crear un proyecto conjunto en materias de seguridad y defensa, pero el debate por límite marítimo volvería a desechar la posibilidad de poner fin a la conformación territorial de los Estados. Conclusiones El fallo de la Corte Internacional de Justicia no cambió la relación bilateral entre Chile y el Perú. La propuesta de punto de inflexión se desmanteló, quedó en el plano especulativo, a causa del denominado “triángulo terrestre”. Vale destacar que la propuesta de punto de inflexión fue afirmada públicamente por los Presidentes de ambas Repúblicas, no fue únicamente una idea del mundo público o académico, demostrando que no valieron las buenas intenciones declarativas de los entonces Presidentes Piñera y Humala principalmente. Retomando la pregunta de investigación ¿Qué explica que el triángulo terrestre no permitiera la posibilidad de punto de inflexión histórico en la relación bilateral? Se responde positivamente con la hipótesis presentada: La propuesta del triángulo terrestre como punto de inflexión que concluiría el ciclo de disputas a lo largo de la historia de la relación bilateral respecto a la conformación territorial, no fue viable de acuerdo a la incidencia del pasado en la situación actual. Esta afirmación se basa en dos elementos expuestos en el artículo y que se resumen a continuación. Primero, frente al contexto de la búsqueda de un punto inflexión, el artículo expone dos momentos en que pudo haber existido y no se produjo: tras la firma del Tratado de 1929 y tras el Acta de Ejecución a fines de los años 90. Ambos momentos tienen como elemento común, que se desprenden de la Guerra del Pacífico y de los tratados que los secundaron. En consecuencia, que tras el fallo de la CIJ no se haya producido el punto de inflexión Si Somos Americanos. Revista de Estudios Transfronterizos 131 histórico, no es una novedad en la historia bilateral, al contrario es un intento fallido adicional en la búsqueda de finiquitar los asuntos pendientes de aspecto territorial, lo que deja manifiesto la inconstancia en la integración bilateral. La hipótesis de punto de inflexión fue rechazada dado que la conformación territorial de los Estados volvió a ser incompatible y que en el corto y mediano plazo no se le ve solución. Esto se explica por la carga histórica que tienen ambos países, dada por el origen de la relación, muy marcada por la herencia negativa de la Colonia, la Guerra del Pacífico con sus consecuentes tratados y la falta de integración continua durante el Siglo XX. Cabe mencionar que respecto a la Guerra del Pacifico, ésta trajo beneficios territoriales a Chile en desmedro del Perú; marcó una parte de la identidad nacional del Perú en oposición a Chile; y forjó una identidad política bilateral que no ha sido modificada hasta el día de hoy y que tiene como cualidad la crisis y la confrontación diplomática por la conformación territorial. Segundo, el denominado triángulo terrestre, que es de 3,7 hectáreas, vale más que un cambio en la relación bilateral para ambos Estados, lo cual tiene una relación directa con la conformación de ambos Estados y sus territorios. Como señala Robledo en la cita tomada en este artículo, el punto de inflexión dependía de la aceptación de la conformación territorial entre ambas partes del fallo de la Corte, asunto que no ocurrió. El denominado “triángulo terrestre” no tiene un valor cuantitativo al día de hoy, pero su cualidad histórica y simbólica es muy relevante. En razón de ello ninguno de los Estados está dispuesto a priorizar unas buenas relaciones bilaterales por sobre la oportunidad de reclamar un espacio territorial como propio. Según la discusión conceptual realizada en la primera sección de este artículo, un punto de inflexión es aquel evento que demarca un cambio significativo que circunscribe un antes y después. En este caso, se traduce como un antes y un después en la historia de la relación bilateral entre Chile y el Perú que ha estado demarcada por situaciones de crisis y conflicto más que de cooperación. Contrariamente, entre ambos Estados existen ciclos de integración y conflicto diplomático, no una relación lineal que se consolide en el tiempo. 132 Si Somos Americanos. Revista de Estudios Transfronterizos Bibliografía Bákula, J. (2006). El Perú en el Reino Ajeno. Historia interna de la acción externa. Lima: Universidad de Lima y Fondo Editorial. Bolivia. Dirección Estrategia de Reivindicación Marítima-DIREMAR. (2014). “Tratado de 1866” (en línea). (Fecha de consulta 10 de diciembre de 2014). Disponible en: http:// www.diremar.gob.bo/node/81 Cambridge Dictionary. (2014). “Turning points” (en línea). (Fecha de consulta 10 de diciembre de 2014) Disponible en: http://dictionary.cambridge.org/dictionary/english/ turning-point / Cavieres, F. (2006). Chile-Perú, La Historia y La Escuela. Conflictos Nacionales, Percepciones Sociales. Valparaíso: Ediciones Universitarias de Valparaíso. Cayo, P. (2010). “Perú y Bolivia: una historia común” Lima: Editorial de la Universidad del Pacifico. Chile. Biblioteca del Congreso Nacional. (1823). “Acuerdo de auxilio al Perú 1823” (en línea). (Fecha de consulta 22 de septiembre de 2014) Disponible en: https://www.leychile. cl/Consulta/listaresultadosimple?cadena=Acuerdo+de+auxilio+al+Perú+1823. Chile. Dirección de Fronteras y Límites. (2014). “Tratado de 1929” (en línea). (Fecha de consulta 22 de septiembre de 2014) Disponible en: http://www.difrol.gob.cl/instrumentosjuridicos/peru.html. Chile. Dirección de Fronteras y Límites. (2014b). “Declaración sobre Zona Marítima, 18 de agosto de 1952” (en línea). (Fecha de consulta 30 de septiembre de 2014) Disponible en: http://www.difrol.gob.cl/instrumentos-juridicos/peru.html. Chile. Dirección de Fronteras y Límites. (2014c). “Acta de los Representantes de Chile y del Perú, 26 de abril de 1968” (en línea). (Fecha de consulta 30 de septiembre de 2014) Disponible en: http://www.difrol.gob.cl/instrumentos-juridicos/peru.html. Corte Internacional de Justicia. (2008). “Peru institutes proceedings against Chile with regard to a dispute concerning maritime delimitation between the two States”, en Si Somos Americanos. Revista de Estudios Transfronterizos 133 International Court of Justice. Cases. List of all cases (en línea). (Fecha de consulta 10 de octubre de 2014). Disponible en: http://www.icj-cij.org/docket/index.php?p1=3&p2=3& code=pch&case=137&k=88&p3=0. Corte Internacional de Justicia. (2014). “The Court determines the course of the single maritime boundary between Peru and Chile”, en International Court of Justice. Press Relase, 2014 (en línea). (Fecha de consulta 10 de octubre de 2014). Disponible en: http:// www.icj-cij.org/docket/index.php?p1=3&p2=3&k=88&case=137&code=pch&p3=5. El Mercurio. (2012) “Presidentes de Chile y Perú se comprometen a respetar fallo de La Haya” en Diario EMOL, 30 de noviembre de 2012, (en línea). (Fecha de consulta 10 de octubre de 2014). Disponible en: www.emol.com. El Mercurio. (2014). “Ollanta Humala asegura que el “triángulo terrestre” pertenece a Perú”, en Diario EMOL, 10 de febrero de 2014, (en línea). (Fecha de consulta 10 de octubre de 2014) Disponible en: www.emol.com. ICSO-UDP. (2013). “Charla en la Universidad Diego Portales, de los ex Cancilleres de Chile, Gabriel Valdés y del Perú, Rafael Roncagliolo”. Santiago de Chile, 25 de Septiembre de 2013. Gallardo, A. (2005). Crisis Internacionales en Sudamérica: Teoría y Análisis. Santiago de Chile: Ediciones de la Academia Nacional de Estudios Políticos y Estratégico– ANEPE. Launius, R. (2007). What Are Turning Points in History, and What Were They for the Space Age. En: Launius, R. y Dick, J. (editores) Societal Impact of Spaceflight (pp. 19-40). Washington D.C.: NASA. Launius, R. (2007). What Are Turning Points in History, and What Were They for the Space Age. En: Societal Impact of Spaceflight, Washington D.C. La Tercera. (2014). “Canciller responde a Ministro del Interior de Perú: triangulo terrestre es incuestionablemente chileno” en Diario La Tercera, 28 de agosto de 2014, (en línea). (Fecha de consulta 10 de octubre de 2014). Disponible en: www.latercera.cl. Merriam Dictionary. (2014). “Turning Point” (en línea). (Fecha de consulta 10 de octubre de 2014) Disponible en: http://www.m-w.com/dictionary/turning%20point. 134 Si Somos Americanos. Revista de Estudios Transfronterizos Milet, P. (2004). Chile-Perú: Las dos caras de un espejo. Revista de Ciencia Política 24(2), 228-235. Milet, P. (2005). Chile-Perú: las raíces de una difícil relación. Revista de Estudios Internacionales 38(150), 59-73. Perú. Ejército. (2014). “Significado del Escudo del Ejército del Perú” (en línea). (Fecha de consulta 22 de septiembre de 2014). Disponible en: www.ejercito.mil.pe/index.php/ nuestra-heraldica. Perú. Ministerio de Relaciones Exteriores. (2010). “Relaciones bilaterales con Chile” (en línea). (Fecha de consulta 22 de septiembre de 2014). Disponible en: http://www.rree.gob. pe/SitePages/america_sur.aspx#chile. Perú, Ministerio de Relaciones Exteriores del Perú. (2013). “Declaración de los Ministro de Relaciones Exteriores de Chile y el Perú 24 de enero de 2013” en Declaraciones conjuntas (en línea). (Fecha de consulta 10 de octubre de 2014). Disponible en: http://www.rree. gob.pe/SitePages/comunicados.aspx#conjuntos. Perú, Presidencia. (2014). “Presidente Humala presenta Carta del Límite del a frontera sur” (en línea). (Fecha de consulta 10 de octubre de 2014) Disponible en: http://www. presidencia.gob.pe/lista-notas-prensa. Publimetro Perú. (2014). “Daniel Parodi: tenemos mucho en común. En Publimetro Perú” 27 de enero de 2014 (en línea). (Fecha de consulta 10 de octubre de 2014). Disponible en: http://publimetro.pe/actualidad/noticia-daniel-parodi-tenemos-mucho-comun-19926. Real Academia Española. (2014). “Inflexión” (en línea). (Fecha de consulta 8 de octubre de 2014) Disponible en: http://dle.rae.es/srv/search?m=30&w=inflexión. Revista ¿Qué Pasa? (1993). “Los años que remecieron Chile” Edición del 3 de julio de 1993, 1-14. Robles, M. J. (2006). De la disuación a la cooperación: dos siglos en la relación PerúChile. Revista Fuerzas Armadas y Sociedad 20(1), 31-50. Robledo, M. (2012). Chile-Perú ¿punto de inflexión? Revista Mensaje 608, 29-31. Si Somos Americanos. Revista de Estudios Transfronterizos 135 Rodríguez Cuadros, M. (2007). Delimitación marítima con equidad, el caso de Perú y Chile. Lima: Peisa. Rodríguez Elizondo, J. (2004). Chile-Perú, el siglo que vivimos en peligro. Santiago de Chile: La Tercera Mondadori. Rodríguez Elizondo, J. (2009). De Charaña a La Haya. Chile entre la aspiración marítima de Bolivia y la demanda marítima de Perú. Santiago de Chile: La Tercera Mondadori. Sandoval, A. (2011). “Perú-Chile. Instrumentos jurídicos internacionales para el establecimiento de su frontera (1883-1999)”, (en línea). (Fecha de consulta 11 de noviembre de 2011). Disponible en: http://www4.congreso.gob.pe/comisiones/1999/exteriores/ chile/INDICE.html. Valdés, G. (2009). Gabriel Valdés, sueños y memorias. Santiago de Chile: Editorial Taurus. Yepes, E. (1999). Un plebiscito imposible. Tacna y Arica 1925-1926. Lima: Ediciones Análisis. Winter, L. (2013). La defensa de Chile en La Haya. Santiago de Chile: Ediciones LYD. Zapata, A. (2012). “Clases Diplomado “Chile-Perú: puntos de encuentro” realizado por la Fundación Konrad Adenauer y el Instituto de Estudios Internacionales de la Universidad de Chile. 136 Si Somos Americanos. Revista de Estudios Transfronterizos Anexo I: Mapas del Fallo de la Corte Internacional de Justicia, en el caso del Perú contra Chile, en relación de la zona en controversia y de la resolución de la Corte Si Somos Americanos. Revista de Estudios Transfronterizos 137 Corte International de Justicia. (2014) Perú against Chile, Press release. (Fecha de consulta 11 de noviembre de 2011). (en línea) Disponible en: http://www.icj-cij.org/ docket/files/137/17928.pdf 138 Si Somos Americanos. Revista de Estudios Transfronterizos Anexo II: Mapa alegórico del denominado triángulo terrestre LatinoAmericaHoy. (2014). La frontera marítima Perú-Chile y el triángulo terrestre. (Fecha de consulta 11 de noviembre de 2011). (en línea) Disponible de: https:// latinamericahoy.es/2014/09/04/la-controversia-en-la-frontera-maritima-peru-chile-yel-triangulo-terrestre/ Si Somos Americanos. Revista de Estudios Transfronterizos 139
https://openalex.org/W3214084150	https://www.frontiersin.org/articles/10.3389/fendo.2021.752944/pdf	English	null	Autonomic Imbalance Increases the Risk for Non-alcoholic Fatty Liver Disease	Frontiers in endocrinology	2,021	cc-by	7,491	ORIGINAL RESEARCH published: 08 November 2021 doi: 10.3389/fendo.2021.752944 Autonomic Imbalance Increases the Risk for Non-alcoholic Fatty Liver Disease Inha Jung 1†, Da Young Lee 2†, Mi Yeon Lee 3, Hyemi Kwon 1, Eun-Jung Rhee 1, Cheol-Young Park 1, Ki-Won Oh 1, Won-Young Lee 1, Sung-Woo Park 1 and Se Eun Park 1* Inha Jung 1†, Da Young Lee 2†, Mi Yeon Lee 3, Hyemi Kwon 1, Eun-Jung Rhee 1, Cheol-Young Park 1, Ki-Won Oh 1, Won-Young Lee 1, Sung-Woo Park 1 and Se Eun Park 1 Inha Jung 1†, Da Young Lee 2†, Mi Yeon Lee 3, Hyemi Kwon 1, Eun-Jung Rhee 1, Cheol-Young Park 1, Ki-Won Oh 1, Won-Young Lee 1, Sung-Woo Park 1 and Se Eun Park 1* 1 Division of Endocrinology and Metabolism, Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea, 2 Division of Endocrinology and Metabolism, Department of Internal Medicine, Korea University College of Medicine, Seoul, South Korea, 3 Division of Biostatistics, Department of R&D Management, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea Keywords: heart rate variability, autonomic nervous system, sympathetic nervous system, parasympathetic nervous system, fatty liver disease (FLD) Edited by: Giovanna Muscogiuri, University of Naples Federico II, Italy Edited by: Giovanna Muscogiuri, University of Naples Federico II, Italy Background: Although autonomic imbalance is associated with an increased risk for metabolic disease, its effects on nonalcoholic fatty liver disease (NAFLD) remains unclear. We aimed to evaluate whether autonomic dysfunction predicts the risk for nonalcoholic fatty liver disease (NAFLD). Reviewed by: Goh Eun Chung, Seoul National University Hospital, South Korea Evelyn Frias-Toral, Catholic University of Santiago de Guayaquil, Ecuador Reviewed by: Goh Eun Chung, Seoul National University Hospital, South Korea Evelyn Frias-Toral, Catholic University of Santiago de Guayaquil, Ecuador Methods: A total of 33,899 participants without NAFLD who underwent health screening programs between 2011 and 2018 were enrolled. NAFLD was identiﬁed by ultrasonography. Autonomic activity was estimated using heart rate variability (HRV). Time domain [standard deviation of the normal-to-normal interval (SDNN) and root mean square difference (RMSSD)]; frequency domain [total power (TP), low frequency (LF), and high frequency (HF), and LF/HF ratio were analyzed. *Correspondence: Se Eun Park seeun0630@gmail.com †These authors have contributed equally to this work Findings: A total 6,466 participants developed NAFLD within a median of 5.7 years. Subjects with incident NAFLD showed decreased overall autonomic modulation and vagal activity with lowered SDNN, RMSSD, HF, normalized HF, compared to those without NAFLD. As the SDNN, RMSSD, TP, LF, and HF tertiles increased, the risk of NAFLD decreased with tertile 1 being the reference group [the hazard ratios (95% conﬁdence intervals) of tertile 3 were 0.90 (0.85–0.96), 0.83 (0.78–0.88), 0.91 (0.86-0.97), 0.93 (0.87- 0.99) and 0.89 (0.83-0.94), respectively] after adjusting for potential confounders. The risk for NAFLD was signiﬁcantly higher in subjects in whom sustained elevated heart rate, normalized LF, and LF/HF ratio values than in those with sustained decrease in these parameters during follow-up. Specialty section: This article was submitted to Obesity, a section of the journal Frontiers in Endocrinology Received: 03 August 2021 Accepted: 21 October 2021 Published: 08 November 2021 ORIGINAL RESEARCH published: 08 November 2021 doi: 10.3389/fendo.2021.752944 Abbreviations: ANS, autonomic nervous system; BMI, body mass index; HOMA– IR, homeostasis model assessment of insulin resistance; HRV, heart rate variability; IR, insulin resistance; LDL, low-density lipoprotein; NAFLD, nonalcoholic fatty liver disease; SNS, sympathetic nervous system; T2DM, type 2 diabetes mellitus; TG, triglyceride. Citation: Jung I, Lee DY, Lee MY, Kwon H, Rhee E-J, Park C-Y, Oh K-W, Lee W-Y, Park S-W and Park SE (2021) Autonomic Imbalance Increases the Risk for Non-alcoholic Fatty Liver Disease. Front. Endocrinol. 12:752944. Conclusions: Overall autonomic imbalance, decreased parasympathetic activity, and recently increased sympathetic activity might increase the risk of NAFLD. November 2021 \| Volume 12 \| Article 752944 1 Frontiers in Endocrinology \| www.frontiersin.org Autonomic Imbalance and NAFLD Jung et al. Anthropometric and Laboratory Measurements The aim of the present study was to explore the longitudinal association between heart rate variability (HRV), which is a noninvasive measure of autonomic imbalance, and the risk for NAFLD in Asian adults. We also investigated the relationship between HRV and the possibility of advanced liver ﬁbrosis. Data on medical history, medication use, and health-related behaviors were collected through a self-administered questionnaire. Physical measurements and serum biochemical parameters were obtained by trained staff after 12 hours of fasting. The questionnaire was based on the fourth Korea National Health and Nutritional Examination Surveys (12) and the Korean version of International Physical Activity Questionnaire short form (13). Body mass index (BMI) was deﬁned as body weight in kilograms divided by height in meters squared. A BMI cutoff of 25 kg/m2 was used to deﬁne obesity in this Korean study population. Systolic and diastolic blood pressures (BPs) were measured with a standardized sphygmomanometer. Study Population and Design y p g This longitudinal cohort study comprised participants from a Kangbuk Samsung Health Study who underwent comprehensive health examinations annually or biennially at the Kangbuk Samsung Hospital Total Healthcare Centers in Seoul and Suwon, South Korea. In Korea, it is mandatory to participate in regular health check-up programs for every employee and companies to enhance the early detection of disease (11). For this study, we selected subjects who underwent health checkup, aged ≥18 years, including abdominal ultrasonography and HRV measurements, at least twice between January 1, 2011 and December 31, 2018 (Supplementary Figure 1). The initial study population comprised 80,784 subjects. We excluded subjects with fatty liver conﬁrmed by abdominal ultrasonography upon the initial examination (n = 13,281) and positive hepatitis viral serology at the baseline (n = 681); male and female participants with alcohol consumption of >30 g/day and 20 g/day, respectively (n = 7,957); and subjects who self- reported a history of hepatitis and/or liver disease (n = 9,027). To minimize the inﬂuence of chronic diseases and medications on HRV, participants who had any of the following conditions at baseline were excluded: age of >65 years (n = 48); anemia (n = 81); estimated glomerular ﬁltration rate (eGFR) <60 mL/min/ 1.73 m2 (n = 84); serum C-reactive protein (CRP) level >1.0 mg/dL Glycated hemoglobin (HbA1c), fasting plasma glucose (FPG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglyceride, high-density lipoprotein cholesterol(HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured after 12 hours of fasting. Dyslipidemia was deﬁned as total cholesterol level of ≥240 mg/ dL (≥6.21 mmol/L) or intake of antihyperlipidemic medications. Diabetes was deﬁned as FPG level ≥126 mg/dL, HbA1c level ≥6.5%, and/or current use of antihyperglycemic medications (14). eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation (15). Serum hs-CRP level was analyzed by a BNII nephelometer (Dade Behring, Deerﬁeld, IL, USA). Insulin resistance was assessed with the homeostatic model assessment–insulin resistance (HOMA-IR) equation (16). INTRODUCTION (n = 3); abnormal thyroid function and history of thyroid disease with the related medications (n = 6,012); and history of chronic obstructive pulmonary disease and/or bronchial asthma (n = 1,101), heart disease (n = 351), history of hypertension and/or intake of any antihypertensive agent or individuals with abnormal blood pressure (systolic/diastolic blood pressure of 140/90 mmHg or higher) (n = 4,796), and malignancy (n = 1,361). Heart disease was deﬁned as angina, myocardial infarction, or arrhythmia that needed treatment. Subjects with any missing data were excluded (n = 3,223). Total 33,899 participants were included in ﬁnal analyses. Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in modernized countries and has a rapidly increasing global prevalence (1–3). Therefore, many studies have focused on the pathogenesis and ﬁnding the predictive factors of NAFLD. Obesity, dyslipidemia, and insulin resistance have been regarded as well-known risk factors for fatty liver (4). Recently, we provided evidence that decreased vagal activity and deviations in sympathetic activity were associated with the development of diabetes (5). Imbalance in the autonomic nervous system (ANS) as an early signal of insulin resistance and metabolic diseases has been reported to have clinical implications (6). Previous studies have shown a relationship between hepatic steatosis and autonomic dysfunction (7–10). However, these were small-sized, single-center, and cross- sectional studies. All participants provided written informed consent for the use of their data in this study. This study was approved by the institutional review board of Kangbuk Samsung Hospital (IRB No. KBS12089). Statistical Analysis y Continuous variables were presented as mean ± standard deviation. Categorical variables were expressed as numbers (percentages). Right-skewed variables (i.e., HOMA–IR, hs- CRP, AST, ALT, alcohol intake, TSH, free T4, and free T3) underwent logarithmic transformation for analysis. Student’s t- test for continuous variables and chi-square test for categorical variables were used to compare the baseline characteristics between subjects who developed NAFLD and those who did not. To estimate hazard ratios (HRs) and 95% conﬁdence intervals (95% CI) for the development of NAFLD, Cox proportional hazards models were used according to tertiles of HRV variables at baseline, with tertile 1 being the reference group. Model 1 was adjusted for age, sex, body mass index, current smoking, alcohol intake, regular exercise, aspartate transaminase, low-density lipoprotein cholesterol levels, and systolic blood pressure. Model 2 was adjusted for the same parameters as model 1 plus high-sensitivity C-reactive protein, homeostasis model assessment for insulin resistance score, and presence of diabetes. Continuous variables were presented as mean ± standard deviation. Categorical variables were expressed as numbers (percentages). Right-skewed variables (i.e., HOMA–IR, hs- CRP, AST, ALT, alcohol intake, TSH, free T4, and free T3) underwent logarithmic transformation for analysis. Student’s t- test for continuous variables and chi-square test for categorical variables were used to compare the baseline characteristics between subjects who developed NAFLD and those who did not. To estimate hazard ratios (HRs) and 95% conﬁdence intervals (95% CI) for the development of NAFLD, Cox proportional hazards models were used according to tertiles of HRV variables at baseline, with tertile 1 being the reference group. Model 1 was adjusted for age, sex, body mass index, current smoking, alcohol intake, regular exercise, aspartate transaminase, low-density lipoprotein cholesterol levels, and systolic blood pressure. Model 2 was adjusted for the same parameters as model 1 plus high-sensitivity C-reactive protein, homeostasis model assessment for insulin resistance score, and presence of diabetes. Diagnosis of Nonalcoholic Fatty Liver Disease and Advanced Liver Fibrosis Group 0: V1 exam (baseline) < median, V2 exam (follow-up) < median: reference group; sease a d d a ced e b os s (RMSSD; ms). SDNN is a marker of overall autonomic modulation, whereas RMSSD reﬂects cardiac parasympathetic drive (18). For the spectral (frequency) domain, total power (TP) (0–0.4 Hz, ms2), low frequency (LF) (0.04–0.15 Hz, ms2), and high frequency (HF) (0.15–0.4 Hz, ms2) were analyzed. LF is generally considered an index of both sympathetic and parasympathetic activities, whereas HF represents parasympathetic activity in the sinus node. Normalized LF norm and HF norm were calculated from the LF/(LF + HF) and HF/(LF + HF) percentile units, with emphasis on changes in sympathetic and parasympathetic (vagal) regulation, respectively. Moreover, the LF/HF ratio, which represents sympathovagal balance was calculated (19). Group 1: V1 exam < median, V2 exam ≥median; Group 2: V1 exam ≥median, V2 exam < median; and Group 3: V1 exam ≥median, V2 exam ≥median. Future risk for NAFLD was explored using the above- mentioned cox analysis. In addition, subgroup analysis was conducted according to sex and the presence of diabetes or dyslipidemia. All reported p values were two-tailed, and p values <0.05 were considered statistically signiﬁcant. Bonferroni correction was performed for multiple comparisons. Statistical analyses were performed using STATA version 16.0 (StataCorp, College Station, TX, USA). The study endpoint was development of the ﬁrst NAFLD diagnosis by abdominal ultrasonography (Logic Q700 MR; GE, Milwaukee, WI, USA) or December 31, 2018. NAFLD was identiﬁed based on known standard criteria, including hepatorenal echo contrast, liver brightness, and vascular blurring using a 3.5-MHz probe. Eleven experienced radiologists who were unaware of this study and were blinded to the clinical status of the subjects performed the ultrasound examinations. The inter- and intraobserver reliabilities for fatty liver diagnosis were high, with kappa statistics of 0.74 and 0.94, respectively (20). The follow-up period was designated as the time from the baseline examination to the ﬁrst diagnosis of NAFLD or December 31, 2018. As a surrogate marker for advanced liver ﬁbrosis, NAFLD Fibrosis Score (NFS) and Fibrosis 4 (FIB 4) index were used (21, 22). NFS of less than −1.455 or FIB 4 index of less than 1.3 excluded advanced liver ﬁbrosis with high accuracy. Baseline Characteristics and HRV Indices of the Participants During 168,413 person-years of follow-up [median (interquartile range) 5.7 (4.3–6.3) years], a total of 6,466 subjects developed NAFLD. As shown in Table 1, compared with the subjects in the control group, the subjects who developed NAFLD were more likely to be obese, had higher levels of liver enzymes, and had metabolically worse lipid levels. The proportion of subjects with comorbid diabetes and obesity was higher in the NAFLD group than in the control group. g The HRV indices SDNN, RMSSD, HF, and HF norm were lower, whereas heart rate, LF, LF norm, and LF/HF ratio were higher in the NAFLD group than in the control group (Table 2). Among the participants who were grouped according to the NFS and FIB 4 index (21, 22), those who had advanced liver ﬁbrosis according to NFS showed signiﬁcantly lower SDNN, RMSSD, TP, LF, and HF, compared with the values in the participants who did not have advanced liver ﬁbrosis. The heart rate was higher in participants with advanced liver ﬁbrosis deﬁned by NFS than in the control group, not reaching statistical signiﬁcance. Those who had advanced liver ﬁbrosis according to FIB 4 index, showed lower heart rate, SDNN, TP, LF, and HF compared with the values in the control group. Measurement of Heart Rate Variability Measurement of Heart Rate Variability As a component of the health screening exam, HRV was measured by three-minute recordings using a 3000P analyzer (Medicore Co., Ltd., Seoul, Korea), while the subject was seated in a quiet room. Participants were asked to stay still with their eyes open, remain silent, and breathe normally during the procedure. According to the methodological standards recommended by the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology for Heart Rate Variability, HRV was analyzed in both the time and frequency domains (17). November 2021 \| Volume 12 \| Article 752944 Frontiers in Endocrinology \| www.frontiersin.org 2 Jung et al. Autonomic Imbalance and NAFLD A sensitivity analysis was performed on 9,673 individuals in whom HRV examination was repeated within two years. These subjects were stratiﬁed into four groups, based on the median value of each HRV variable, as follows: The time domain parameters included standard deviation of the normal-to-normal interval (SDNN; ms) and the square root of the mean squared difference of successive RR intervals. Frontiers in Endocrinology \| www.frontiersin.org Relationship Between HRV Indices and the Risk for NAFLD We investigated the risk for NAFLD according to the tertile of HRV variables. As shown in Figures 1, 2, the fully adjusted HRs (Model 2) of the highest tertiles of SDNN, RMSSD, TP, LF, and HF were signiﬁcantly decreased, compared with those of the lowest tertiles. For heart rate, a positive relationship between each tertile and the risk for incident NAFLD was observed. November 2021 \| Volume 12 \| Article 752944 Frontiers in Endocrinology \| www.frontiersin.org 3 Autonomic Imbalance and NAFLD Jung et al. TABLE 1 \| Baseline characteristics of participants according to development of non-alcoholic fatty liver disease. Relationship Between HRV Indices and the Risk for NAFLD Variable Total (N = 33, 899) Control (N = 27,433) NAFLD (N = 6, 466) P valuea Age (years) 35.6 ± 5.7 35.4 ± 5.7 36.3 ± 5.6 <0.001 Sex, Men (%) 14632 (43.2) 9953 (36.3) 4679 (72.4) <0.001 BMI (kg/m2) 21.9 ± 2.7 21.5 ± 2.5 23.8 ± 2.5 <0.001 Waist circumference (cm) 77.8 ± 8 76.5 ± 7.6 83.9 ± 7.0 <0.001 FBG (mg/dL) 92.6 ± 9.5 92 ± 9 95.3 ± 10.8 <0.001 Hemoglobin A1c (%) 5.6 ± 0.3 5.5 ± 0.3 5.6 ± 0.4 <0.001 HOMA-IR 1.20 ± 0.70 1.13 ± 0.62 1.46 ± 0.91 <0.001 SBP (mmHg) 105.2 ± 11 104.1 ± 10.8 109.9 ± 10.7 <0.001 AST (IU/L) 19.2 ± 8.4 18.9 ± 8.5 20.5 ± 7.6 <0.001 ALT (IU/L) 17.1 ± 11.1 16 ± 10.3 21.8 ± 13.1 <0.001 Total cholesterol (mg/dL) 187.5 ± 31.2 185.5 ± 30.5 196.3 ± 32.4 <0.001 Triglyceride (mg/dL) 112.3 ± 28.8 109.4 ± 28 124.3 ± 29.3 <0.001 HDL-C (mg/dL) 90 ± 49 83.3 ± 41.9 118.4 ± 64.3 <0.001 LDL-C (mg/dL) 61.2 ± 14.2 62.9 ± 14.1 53.9 ± 12.2 <0.001 hs-CRP (mg/dL) 0.09 ± 0.28 0.08 ± 0.28 0.11 ± 0.30 <0.001 Hemoglobin (g/dL) 14.2 ± 1.5 14 ± 1.5 15 ± 1.5 <0.001 TSH (mIU/mL) 2.09 ± 1.003 2.10 ± 1.01 2.04 ± 0.98 <0.001 Free T4 (ng/dL) 1.3 ± 0.2 1.3 ± 0.2 1.3 ± 0.2 <0.001 Free T3 (pg/mL) 3.2 ± 0.4 3.1 ± 0.4 3.3 ± 0.4 <0.001 Alcohol intake (g/day) 6.1 ± 6.6 5.7 ± 6.3 8.1 ± 7.5 <0.001 Current smoker (%) 5042 (14.9) 3265 (11.9) 1777 (27.5) <0.001 Regular exercise (%)b 4330 (12.8) 3473 (12.7) 857 (13.3) 0.073 Obesity (%)c 4204 (12.4) 2330 (8.5) 1874 (29) <0.001 DM (%) 234 (0.7) 146 (0.5) 88 (1.4) <0.001 Dyslipidemia (%) 3146 (9.3) 2184 (8) 962 (14.9) <0.001 TABLE 1 \| Baseline characteristics of participants according to development of non-alcoholic fatty liver disease. Data are presented as mean ± standard deviation or number (%). TABLE 2 \| Comparison of heart rate variability indices in participants according to development of non-alcoholic fatty liver disease and possibility of advanced liver ﬁbrosis. Relationship Between HRV Indices and the Risk for NAFLD NAFLD NFSb FIB-4c Contro l (N = 27433) NAFLD (N = 6466) P valuea <-1.455 (N = 6, 372) ≥-1.455 (N = 89)d P valuea <1.3 (N = 6, 293) ≥1.3 (N = 170)e P valuea Heart rate 64.5 ± 8.4 65.1 ± 8.5 <0.001 65.7 ± 8.7 66.7 ± 7.6 0.206 65.7 ± 8.7 64 ± 8.5 0.009 SDNN (ms) 45.6 ± 16.6 44.8 ± 16.1 <0.001 44.5 ± 16.5 36.9 ± 13.4 <0.001 44.5 ± 16.4 41.6 ± 17.8 0.018 RMSSD (ms) 42.8 ± 19 39.6 ± 17.9 <0.001 38.9 ± 17.8 30.5 ± 12.2 <0.001 38.8 ± 17.7 36.4 ± 18.5 0.080 TP (ms2) 1618.2 ± 1387.4 1596.1 ± 1407.3 0.250 1596.4 ± 1450.1 1021.7 ± 866.8 <0.001 1594.1 ± 1444.1 1331.9 ± 1440.7 0.005 LF (ms2) 442.1 ± 579 476.1 ± 632.6 <0.001 475.8 ± 635.1 248.2 ± 245.3 <0.001 473.7 ± 624.6 413.8 ± 854.4 0.008 HF (ms2) 540 ± 520.3 467.5 ± 456.3 <0.001 455 ± 451.7 272.9 ± 229.4 <0.001 455 ± 451 343.9 ± 377.4 0.006 LF norm 42.3 ± 20.6 47.2 ± 20.5 <0.001 47.9 ± 20.7 46.5 ± 19.6 0.951 47.9 ± 20.6 47.2 ± 20.8 0.951 HF norm 56.9 ± 20.9 52.1 ± 20.6 <0.001 51.4 ± 20.7 51.8 ± 19.8 0.529 51.4 ± 20.7 51.8 ± 20.9 0.843 LF/HF ratio 1.2 ± 2 1.4 ± 2.1 <0.001 1.5 ± 2 1.3 ± 1.4 0.727 1.5 ± 2 1.5 ± 1.8 0.892 HF, high-frequency; HF norm, normalized high-frequency; LF, low-frequency; LF norm, normalized low-frequency; NAFLD, non-alcoholic fatty liver disease; NFS, NAFLD ﬁbrosis score; RMSSD, root mean square difference; SDNN, standard deviation of the normal-to-normal interval; TP, total power. aStudent’s t-tests were used to compare heart rate variability indices. bIn patients with NFS < -1.455, advanced liver ﬁbrosis can be excluded with high accuracy. cIn patients with FIB-4 < 1.3, advanced liver ﬁbrosis can be excluded. dFive patients with missing data during follow-up was excluded when calculating NFS. eThree patients with missing data during follow-up was excluded when calculating FIB-4 index. articipants according to development of non-alcoholic fatty liver disease and possibility of advanced liver ﬁbrosis. who had sustained decrease in heart rate, LF norm, and LF/HF ratio (reference group, group 0); individuals who had continuously increased HF norm (group 0) exhibited lower risk (Figure 3). Data are presented as mean ± standard deviation or number (%). AST, aspartate transaminase; ALT, alanine aminotransferase; BMI, body mass index; DM, diabetes mellitus; FBG, fasting blood glucose; HDL-C, high density lipoprotein cholesterol; HOMA-IR, homeostatic model assessment-Insulin resistance; hs-CRP, high-sensitivity c-reactive protein; HTN, hypertension; LDL-C, low-density lipoprotein cholesterol; NAFLD, non- alcoholic fatty liver disease; SBP, systolic blood pressure; TSH, thyroid-stimulating hormone. aStudent’s t-tests for continuous variables and Chi-square tests for categorical variables were used to compare characteristics of the study subjects at baseline. Right-skewed variables (HOMA-IR, AST, ALT, hs-CRP, TSH, Free T4, Free T3, and Alcohol intake) were log-transformed for Student’s t-tests. bRegular exercise was deﬁned as performing > 20 minutes of vigorous physical activity at least three times per week. cBMI cutoff of 25kg/m2 was used to deﬁne obesity for Korean population in this study. Relationship Between HRV Indices and the Risk for NAFLD The subjects who had follow-up heart rate, LF In the sensitivity analysis for subjects who had follow-up HRV measurements within two years, the risk for NAFLD was signiﬁcantly higher in those who had sustained elevations in heart rate, LF norm, and LF/HF ratio (group 3) than in those November 2021 \| Volume 12 \| Article 752944 Frontiers in Endocrinology \| www.frontiersin.org Autonomic Imbalance and NAFLD Jung et al. FIGURE 3 \| Risk of incident diabetes according to change of HRV measurement in the participants who underwent follow-up HRV exam within two years. Data shown in this ﬁgure is included in supplemental materials (Supplementary Table 5). FIGURE 1 \| Risk of incident non-alcoholic fatty liver disease according to tertiles of heart rate variability indices. FIGURE 3 \| Risk of incident diabetes according to change of HRV measurement in the participants who underwent follow-up HRV exam within two years. Data shown in this ﬁgure is included in supplemental materials (Supplementary Table 5). FIGURE 1 \| Risk of incident non-alcoholic fatty liver disease according to tertiles of heart rate variability indices. group was signiﬁcantly lower in those with sustained elevation of HF norm (group 3) than in group 0 (Figure 3). Original data shown in the Figures 2, 3 is included in supplemental materials (Supplementary Tables 4, 5, respectively). norm, and LF/HF ratio that were newly elevated, compared with the median values (group 1), showed higher risk for NAFLD, compared with the risk of group 0. The HRs of group 1 were higher, compared with those of group 3. The risk for NAFLD A B D C FIGURE 2 \| Kaplan–Meier curves for the risk for incident NAFLD according to HRV measurement. Each curves represent the risk for incident NAFLD according to tertiles of (A) heart rate, (B) root mean square difference (RMSSD), (C) low frequency (LF), and (D) high frequency (HF). Original data shown in the is included in supplemental materials (Supplementary Table 4). A B B A D C D C FIGURE 2 \| Kaplan–Meier curves for the risk for incident NAFLD according to HRV measurement. Each curves represent the risk for incident NAFLD according to tertiles of (A) heart rate, (B) root mean square difference (RMSSD), (C) low frequency (LF), and (D) high frequency (HF). Original data shown in the is included in supplemental materials (Supplementary Table 4). DISCUSSION This longitudinal cohort study revealed that decreased overall autonomic function and parasympathetic activity were associated with the development of NAFLD and liver ﬁbrosis in Asian participants, even after adjusting for potential confounders. This signiﬁcance was especially observed in men and in subjects without diabetes or dyslipidemia. Also, we observed recent autonomic imbalance, especially increased sympathetic activity, might be related to the development of NAFLD. Previous studies have suggested that autonomic imbalance might be a component in the pathogenesis of NAFLD (23, 24). In 497 Taiwanese who had health checkups, SDNN showed a negative correlation with the presence NAFLD (25). Several cross-sectional studies have suggested that the grade of NAFLD and diabetic status contributed to the decrease in cardiovascular autonomic function and overall activity (7). Consistent with these results, lower cardiovagal tone was found to be associated with hepatic steatosis in patients with recent- onset T2DM (8). In addition, a small cohort study based on magnetic resonance spectroscopy and liver biopsy reported that compared with controls, individuals with hepatic steatosis had higher proportion of autonomic dysfunction and that inﬂammation was independently related with autonomic dysfunction (9). However, all these previous studies were conducted with a small number of subjects and had a cross- sectional design. The ﬁndings of this present study were in line with those of previous studies mentioned above. In addition, our study extended the previous ﬁndings by investigating the longitudinal association between autonomic imbalance, based on HRV measurements, and the risk for incident NAFLD. The results of sensitivity analysis is quite interesting. The subjects with recently elevated HR, LF norm, and LF/HF ratio, showed higher HRs for NAFLD, compared with those with sustained elevated HR, LF norm and LF/HF ratio. Recent deviation in sympathovagal imbalance to sympathetic activity and decreased parasympathetic activity might be related to increased risk of developing NAFLD. To our knowledge, no studies have investigated whether recent alterations in HRV variables have different effects on clinical outcomes than sustained alterations in HRV variables. In the subgroup analysis by sex, the more signiﬁcant ﬁndings in men than in women might be explained by the higher incidence rate of NAFLD in men than in women and the suggested susceptibility of men to autonomic dysfunction (36). Prior studies reported that sex difference in HRV indices correlated with insulin resistance and diabetic neuropathy (36, 37). Relationship Between HRV Indices and the Risk for NAFLD FIGURE 2 \| Kaplan–Meier curves for the risk for incident NAFLD according to HRV measurement. Each curves represent the risk for incident NAFLD according to tertiles of (A) heart rate, (B) root mean square difference (RMSSD), (C) low frequency (LF), and (D) high frequency (HF). Original data shown in the is included in supplemental materials (Supplementary Table 4). November 2021 \| Volume 12 \| Article 752944 5 Frontiers in Endocrinology \| www.frontiersin.org Autonomic Imbalance and NAFLD Jung et al. have shown that direct SNS innervation of adipocytes can stimulate lipolysis, thereby, possibly contributing to insulin resistance from the elevated free fatty acid levels. About 60% of liver triglycerides in patients with NAFLD are known to be derived from white adipose tissue lipolysis, which is stimulated by sympathetic activation and insulin resistance (23, 30). In rats, excessive elevations in efferent hepatic sympathetic outﬂow promoted hepatic steatosis, whereas hepatic sympathetic denervation reduced obesity-induced hepatic steatosis (31). On the other hand, several studies have reported anti-inﬂammatory properties of vagus nerve stimulation (32) and suggested that vagus nerve mediated cholinergic activation had anti-infprotective effects against obesity-related inﬂammation and other metabolic complications (33, 34). Nishio et al. (35) have shown that vagus nerve-mediated cholinergic signaling played a key role in regulating inﬂammatory responses in Kupffer cells and eventually contributed to the suppression of nonalcoholic steatohepatitis progression. In addition, the hepatic parasympathetic branch could inhibit the secretion of very low- density lipoprotein and might contribute to hepatic steatosis in the early stages of NAFLD (23, 26). Consistent with laboratory data, our study showed that decreased vagal activity and recently increased sympathetic activity might lead to the development of NAFLD. Furthermore, the degree of the fatty liver condition, which was determined by elevated markers of liver ﬁbrosis, was relatively severe in subjects with overall autonomic imbalance and decreased vagal activity. Subgroup Analyses g p y In the subgroup analyses, there was a signiﬁcant interaction between sex or presence of diabetes and the risk for incident NAFLD for all HRV indices (Supplementary Tables 1, 2). In cases with dyslipidemia, incident NAFLD had a signiﬁcant interaction with heart rate, SDNN, RMSSD, TP, LF, and HF (Supplementary Table 3). The relatively low risk for NAFLD in tertiles 3 of SDNN, RMSSD, TP, LF, and HF and the high risk in heart rate were consistently observed in men and in subjects without diabetes or dyslipidemia. In women and in subjects with diabetes and dyslipidemia, this trend was not observed, except for heart rate, RMSSD, HF (only in patients with dyslipidemia), and LF/HF ratio (only in patients with diabetes). Frontiers in Endocrinology \| www.frontiersin.org CONCLUSIONS we assessed the relationship between noninvasive indices of hepatic ﬁbrosis and autonomic dysfunction. we assessed the relationship between noninvasive indices of hepatic ﬁbrosis and autonomic dysfunction. Autonomic imbalance was associated with an increased risk for incident NAFLD. In addition, decreased parasympathetic activity or recently increased sympathetic activity increased the risk for the development of NAFLD. These ﬁndings suggested that ANS impairment might play a role in the pathogenesis of NAFLD. However, this study had several limitations. First, fatty liver was diagnosed by abdominal ultrasonography not by liver biopsy. Therefore, we could not assess the severity of fatty liver in this study. Although liver biopsy is the gold standard method for the diagnosis of fatty liver, it is invasive and unsuitable as a screening tool, especially in a large-scale cohort study. On the other hand, diagnosis of fatty liver by ultrasonography has been widely used both clinically and in population-based studies because of its noninvasive nature and acceptable degree of diagnostic accuracy for steatosis (38). Second, we conducted three-minute not ﬁve- minute HRV measurements, and we did not perform repeated measurements during the health checkup program because of the standardized short-term HRV measurement protocols (17). Nevertheless, a previous study demonstrated that three minutes was the required minimal recording period that would correlate with ﬁve-minute HRV measurements (39). Regarding information on the physical activity, history of comorbidities and medication was based on a self-reported questionnaire, which is subject to recall response bias. Regarding the issue of implementation of repeated HRV measurements, we showed consistent results for individuals who had prospective follow-up HRV tests within two years. Finally, our results were derived from a sample of relatively healthy young and middle-aged educated Koreans who participated in a health checkup program regularly. Those who regularly receive a health checkup are likely to have healthier lifestyles involving good diets. We included smoking status, alcohol consumption and levels of physical activity in regression models but diet is another important factor related with pathogenesis of NAFLD; therefore, the lack of data regarding calorie intake and diet is another limitation of our study. Therefore, generalization of our results to other ethnicities or age groups might be limited. Additional studies should be performed in multiple institutes and on different ethnicities in the future. ETHICS STATEMENT This study was approved by the Institutional Review Board of Kangbuk Samsung Hospital of Korea (IRB No. KBS12089). Participants who underwent national health check-up examinations provided written informed consent for the use of their data for research purposes. All personal information was deleted and only non-identiﬁable data were included for analysis. DATA AVAILABILITY STATEMENT The data analyzed in this study is subject to the following licenses/ restrictions: The original dataset in this study are available from Kangbuk Samsung Health Study database. The availability of these data, which were used under approval for this current study, and so are not publicly available. Requests to access these datasets should be directed to sh703.yoo@samsung.com. ACKNOWLEDGMENTS The authors acknowledge the efforts of Department of R&D Management at Kangbuk Samsung Hospital, Korea for editing ﬁgures and tables. AUTHOR CONTRIBUTIONS IJ and DYL wrote and edited the original draft and did formal analysis. MYL performed data curation and investigation. HK performed data curation. E-JR supervised the study design. C-YP performed data curation. K-WO revised the manuscript critically for intellectual content. W-YL was in charge of methodology and did review and editing. S-WP performed data curation. SEP conceptualized the study and approved the ﬁnal manuscript. All authors contributed to the article and approved the submitted version. Although the causal relationship between altered HRV and risk for fatty liver development could not be fully addressed in the present study, we observed that autonomic imbalance preceded incident NAFLD. These ﬁndings have clinical implications, possibly extending the utility of HRV measurement for risk assessment of NAFLD. Given the complexity of the pathophysiology of NAFLD, no pharmacological agents has been approved by regulatory authorities and organizations. At present, lifestyle modiﬁcations including weight reduction is recommended for prevention and treatment of NAFLD. There are increasing numbers of researches reporting the risk of various diseases and mortality among patients with NAFLD, even with mild, early stages of the disease (40–42). Therefore, detecting the alterations in HRV in advance or at patients’ earlier stages of NAFLD would be helpful for clinicians identifying patients who need interventions such as lifestyle modiﬁcations. Though additional studies are needed to clarify the roles of autonomic imbalance in the development of NAFLD, our study further suggests a new perspective for therapeutic potential of bioelectrical medicine for NAFLD. DISCUSSION Interestingly, compared with individuals without diabetes or dyslipidemia, those with diabetes or dyslipidemia had less association with incident NAFLD, despite the higher incidence. Either hyperglycemia or dyslipidemia, might have played more critical roles in the development of NAFLD rather than autonomic imbalance (7). A diagram describing the hypothetical mechanisms of this study is shown in Supplementary Figure 2. The mechanisms linking autonomic dysfunction with NAFLD are not yet fully elucidated. However, there had been several laboratory ﬁndings that described the role of ANS in hepatic lipid metabolism (26). The pathophysiology of NAFLD is closely related with insulin resistance (27). Subjects with insulin resistance exhibit a defect in suppression of lipolysis in adipose tissue, resulting in increased free fatty acid efﬂux from adipose tissue to the liver (28). Zeng et al. (29) This present study had several distinguishing features. To the best of our knowledge, this was the ﬁrst longitudinal study that examined the association between autonomic imbalance and incident NAFLD. 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Hernaez R, Lazo M, Bonekamp S, Kamel I, Brancati FL, Guallar E, et al. Diagnostic Accuracy and Reliability of Ultrasonography for the Detection of Fatty Liver: A Meta-Analysis. Hepatology (2011) 54(3):1082–90. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2021. 752944/full#supplementary-material November 2021 \| Volume 12 \| Article 752944 Frontiers in Endocrinology \| www.frontiersin.org Autonomic Imbalance and NAFLD Jung et al. 40. Paik JM, Henry L, De Avila L, Younossi E, Racila A, Younossi ZM. Mortality Related to Nonalcoholic Fatty Liver Disease Is Increasing in the United States. Hepatol Commun (2019) 3(11):1459–71. doi: 10.1002/hep4.1419 41. Xu J, Dai L, Zhang Y, Wang A, Li H, Wang Y, et al. Severity of Nonalcoholic Fatty Liver Disease and Risk of Future Ischemic Stroke Events. 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NAFLD, and Cardiovascular and Cardiac Diseases: Factors Inﬂuencing Risk, Prediction and Treatment. Diabetes & Metabolism (2021) 47:101215. doi: 10.1016/j.diabet.2020.101215 Frontiers in Endocrinology \| www.frontiersin.org November 2021 \| Volume 12 \| Article 752944 REFERENCES doi: 10.1002/hep.24452 18. Liu Y, Dai M, Bi Y, Xu M, Xu Y, Li M, et al. Active Smoking, Passive Smoking, and Risk of Nonalcoholic Fatty Liver Disease (NAFLD): A Population-Based Study in China. J Epidemiol (2013) 23(2):115–21. doi: 10.2188/jea.JE20120067 39. Shaffer F, Shearman S, Meehan ZM. The Promise of Ultra-Short-Term (UST) Heart Rate Variability Measurements. Biofeedback (2016) 44(4):229–33. doi: 10.5298/1081-5937-44.3.09 19. Malik M, Bigger JT, Camm AJ, Kleiger RE, Malliani A, Moss AJ, et al. Heart Rate Variability. Standards of Measurement, Physiological Interpretation, and November 2021 \| Volume 12 \| Article 752944 Frontiers in Endocrinology \| www.frontiersin.org 8 Autonomic Imbalance and NAFLD Jung et al. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2021 Jung, Lee, Lee, Kwon, Rhee, Park, Oh, Lee, Park and Park. 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. 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. November 2021 \| Volume 12 \| Article 752944 Frontiers in Endocrinology \| www.frontiersin.org 9
https://openalex.org/W2150536784	https://dash.harvard.edu/bitstream/1/14368995/1/jbacter00076-0330.pdf	English	null	Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis	Journal of bacteriology	1,992	cc-by	5,704	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:14368995 Published Version http://jb.asm.org/content/174/10/3416.full.pdf+html Citation Citation Eisen, J. A., S. W. Smith, and Colleen M. Cavanaugh. 1992. "Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis." Journal of Bacteriology 174, no. 10: 3416-3421. Phylogenetic Relationships of Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`* Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2 Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Received 4 November 1991/Accepted 9 March 1992 The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria. the 5S rRNA molecule (-120 bp) precludes resolution that can be attained with larger molecules such as 16S rRNA (-1,550 bp) (16). Species of the genus Solemya are, to date, the only bivalves of the subclass Protobranchia in which chemoautotrophic symbiosis has been documented. The protobranchs represent an important component of studies of chemoautotrophic symbioses, since they may be the closest living group to the ancestral bivalve condition, be- cause they dominate the deep sea and are present along a gradient from the deep sea bottom to the shore (1). the 5S rRNA molecule (-120 bp) precludes resolution that can be attained with larger molecules such as 16S rRNA (-1,550 bp) (16). Species of the genus Solemya are, to date, the only bivalves of the subclass Protobranchia in which chemoautotrophic symbiosis has been documented. The protobranchs represent an important component of studies of chemoautotrophic symbioses, since they may be the closest living group to the ancestral bivalve condition, be- cause they dominate the deep sea and are present along a gradient from the deep sea bottom to the shore (1). Procaryote-eucaryote associations in which marine inver- tebrates harbor chemoautotrophic bacteria as endosym- bionts appear to be widespread in marine habitats such as deep-sea hydrothermal vents and coastal sediments (8, 15). In such symbioses, the procaryotes utilize the energy re- leased by the oxidation of reduced inorganic substrates, such as hydrogen sulfide, to fix carbon dioxide via the Calvin- Benson cycle (7, 13). The hosts appear to derive nutrition from their endosymbionts and in turn provide the symbionts simultaneous access to the substrates from anoxic and oxic environments which are necessary for energy generation. Phylogenetic Relationships of Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`* Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2 Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Maintenance of such intracellular symbionts presents a novel metazoan acquisition of procaryotic energy generation and autotrophic carbon fixation. g p PCR amplification. We used the polymerase chain reaction (PCR) (28) to amplify 16S rRNA coding regions from a mixture of procaryotic and eucaryotic DNA extracted from the symbiont-containing gills of S. velum. S. velum were collected from eelgrass beds near Woods Hole, Mass., and placed in filtered (passed through filters with a pore size of 0.2 ,um) seawater to cleanse body surfaces prior to dissec- tion. The gills, which contain -109 bacterial symbionts per g (wet weight), and feet, in which symbionts have not been observed (7), were dissected, frozen in liquid nitrogen, and stored at -85°C. Frozen tissue was homogenized in lysis buffer, and DNA was isolated by using hexadecyltrimethy- lammonium bromide (4). DNA from Escherichia coli JM109, prepared by the miniprep method (4), was used as a positive control. p While the existence of chemoautotroph-invertebrate sym- bioses is now generally accepted, little is actually known about the symbionts observed in the tissues of any of the hosts because none have been cultured. Comparison of rRNA sequences has greatly facilitated the identification of bacteria, including unculturable microorganisms, and the elucidation of their natural relationships (38). Phylogenetic analysis of 16S rRNA sequences enabled Distel et al. (12) to establish that the chemoautotrophic symbionts of the hydro- thermal vent tubeworm and five species of bivalves of the subclass Lamellibranchia are related and cluster in the gamma subdivision of the Proteobacteria (formerly purple photosynthetic bacteria), one of the 11 major groups of eubacteria (30). Amplification of 16S rRNA genes by PCR was carried out essentially by the method of Weisburg et al. (34) using eubacterial universal primers and 200 ng of template DNA. DNA products (Fig. 1) amplified from S. velum gill tissue (lane 1) and from the positive-control E. coli (lane 4) were prominent single bands of approximately 1,500 bp. Amplifi- cation was not detected when DNA template was not added (lane 2), nor when DNA from S. velum foot tissue was used as the template (lane 3). ( ) In this investigation we sought to establish the phyloge- netic relationships and the species specificities of the sym- bionts of the protobranch bivalve Solemya velum Say, an Atlantic coast clam which has been studied as a shallow- water model of invertebrate-chemoautotroph associations (7, 9, 10). The phylogenetic placement of the S. * Corresponding author. t Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Vol. 174, No. 10 JOURNAL OF BACTERIOLOGY, May 1992, p. 3416-3421 0021-9193/92/103416-06$02.00/0 Copyright © 1992, American Society for Microbiology Phylogenetic Relationships of Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`* Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2 Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 These results, coupled with the finding of identical partial sequence in three animals, indicated that the 16S rRNA gene was amplified from the S. velum symbionts and not from a free-living surface contaminant. fragment patterns resulting from this set of enzymes were unique to the sequence of the amplified gene, as determined by a search of all sequences of the 16S rRNA data base (24) and of the other chemoautotrophic symbionts (12). Genomic DNA (5 ,ug) was digested with restriction en- zymes according to the manufacturer's instructions. Gill tissue DNA from nine different animals was cut with three to nine enzymes, with at least three animals used for each enzyme. Southern blots were performed by the method of Maniatis et al. (21). The 16S rRNA gene of E. coli, amplified using PCR (see above), was used as a probe. The probe was radioactively labelled by using the Random Priming Labeling Kit (Bethesda Research Laboratory) and [a-32P]dATP (6,000 Ci/mmol; New England Nuclear) according to the supplier's instructions and purified using a Sephadex G50 column (21). Blots were hybridized at 68°C and washed at 65°C in a solution of 0.1x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate. E. coli genomic DNA, cut with PstI, was used as a positive control producing expected rRNA gene fragment patterns as de- scribed (5) (data not shown). g Southern blot and hybridization analysis. Restriction en- zyme analysis of S. velum genomic DNA was conducted for three reasons: (i) to confirm that the 16S rRNA gene ampli- fied was that of the gill endosymbionts, (ii) to examine restriction site variability between individual S. velum and (iii) to determine the number of rRNA gene copies in the symbiont genome. Enzymes were chosen on the basis of a restriction map predicted from the sequence of the 16S rRNA gene amplified from S. velum gills (Fig. 2). Restriction The use of PCR of such a highly conserved bacterial gene with universal eubacterial primers (34) made it necessary to determine that the gene amplified was that of the endosym- bionts and not other bacteria such as free-living surface contaminants. The fragment sizes produced by probing genomic S. velum DNA with E. coli 16S rDNA exactly matched those predicted by the sequence of the PCR prod- uct (Fig. 2 and 3). This suggested that the gene amplified was dominant in the S. Phylogenetic Relationships of Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`* Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2 Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 velum sym- biont-containing gill genomic DNA with an E. coli 16S rRNA gene probe. DNA from different animals (I to V) was digested with individual or pairs of restriction enzymes and separated by electro- phoresis on 1.3% (A) and 0.8% (B) agarose gels. Lanes: 1, AvaI; 2, Bcl; 3, AvaI plus Bcll; 4, EcoNI; 5, EcoRI; 6, XhoI; 7, PvuII; 8, BglII. Enzymes were selected on the basis of the predicted restric- tion map of symbiont 16S rRNA coding regions (Fig. 2). The high-molecular-weight bands inAvaI and BglII digests are a result of partial digestion (data not shown). Numbers to the sides of the blots indicate molecular size standards (in kilobases). FIG. 3. Southern blot hybridization of restricted S. velum sym- biont-containing gill genomic DNA with an E. coli 16S rRNA gene probe. DNA from different animals (I to V) was digested with individual or pairs of restriction enzymes and separated by electro- phoresis on 1.3% (A) and 0.8% (B) agarose gels. Lanes: 1, AvaI; 2, Bcl; 3, AvaI plus Bcll; 4, EcoNI; 5, EcoRI; 6, XhoI; 7, PvuII; 8, BglII. Enzymes were selected on the basis of the predicted restric- tion map of symbiont 16S rRNA coding regions (Fig. 2). The high-molecular-weight bands inAvaI and BglII digests are a result of partial digestion (data not shown). Numbers to the sides of the blots indicate molecular size standards (in kilobases). a 16S probe to genomic digests of foot DNA (data not shown). a 16S probe to genomic digests of foot DNA (data not shown). 16S rRNA gene sequence. Amplified DNA, purified using low-melting-point agarose (29), was sequenced directly using synthetic oligonucleotide primers (19, 36) and the U.S. Biochemical Sequenase Kit with glycerol (34% final concen- tration) added to the DNA denaturation step (37). Both strands of the amplified 16S rRNA gene were sequenced completely for one animal, a total of 1,460 bp. The 16S genes amplified from two other individual clams were partially sequenced (nucleotides 102 to 306, with numbers corre- sponding to those for E. coli [6]). The sequences of all three animals were identical in this region, which includes both variable and conserved portions of the molecule. Variability in the 16S rRNA sequences of symbionts from an individual, which would show up as either ambiguous sites or sites with high background, was not observed for any of the animals, indicating that only one bacterial rDNA was amplified. Phylogenetic Relationships of Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`* Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2 Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 velum symbionts, to date limited to sequence analysis of the 5S rRNA, indicates that these symbionts also fall in the Proteo- bacteria gamma subdivision (31). However, the small size of p The strong amplification from gill tissue DNA and lack of amplification from foot tissue DNA (Fig. 1) supports the conclusions from studies of enzyme activity, electron mi- croscopy (9), and 5S rRNA sequences (31) that the bacteria are abundant within, and specific to, the gill tissue. This conclusion was further supported by lack of hybridization of 3416 NOTES 3417 I II III IV V 1 2 3 4 1 2 3 4 1 2 3 4 5 6 7 8 5 6 7 8 23.1 9.4 6.6 4.4 2.3 - 2.0 - 1.4 - 1.08- 0.87- -23.1 - 9.4 - 6.6 - 4.4 2.3 2.0 FIG. 3. Southern blot hybridization of restricted S. velum sym- biont-containing gill genomic DNA with an E. coli 16S rRNA gene probe. DNA from different animals (I to V) was digested with individual or pairs of restriction enzymes and separated by electro- phoresis on 1.3% (A) and 0.8% (B) agarose gels. Lanes: 1, AvaI; 2, Bcl; 3, AvaI plus Bcll; 4, EcoNI; 5, EcoRI; 6, XhoI; 7, PvuII; 8, BglII. Enzymes were selected on the basis of the predicted restric- tion map of symbiont 16S rRNA coding regions (Fig. 2). The high-molecular-weight bands inAvaI and BglII digests are a result of partial digestion (data not shown). Numbers to the sides of the blots indicate molecular size standards (in kilobases). VOL. 174, 1992 I II III IV V 1 2 3 4 1 2 3 4 1 2 3 4 5 6 7 8 5 6 7 8 23.1 9.4 6.6 4.4 2.3 - 2.0 - 1.4 - 1.08- 0.87- -23.1 - 9.4 - 6.6 - 4.4 2.3 2.0 1 2 3 4 5 23.1 6.6 - 2.0 - 14 0.9 0.3 1 2 3 4 5 23.1 6.6 - 2.0 - 14 0.9 0.3 FIG. 1. PCR-mediated amplification of the 16S rRNA coding regions of the S. velum symbionts. DNA samples from symbiont- containing S. velum gill tissue (lane 1), S. velum foot tissue (lane 2), and E. Phylogenetic Relationships of Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`* Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2 Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 coli (lane 4) as well as negative controls with no DNA template (lane 3) were submitted to PCR amplification primed by oligonucleotides complementary to conserved regions at opposite ends of the bacterial 16S rRNA gene (see "PCR amplification" section). Products were separated by electrophoresis in 1% agarose and stained with ethidium bromide. The sizes (in kilobases) of some of the DNA molecular weight standards (lane 5) are indicated to the right. FIG. 1. PCR-mediated amplification of the 16S rRNA coding regions of the S. velum symbionts. DNA samples from symbiont- containing S. velum gill tissue (lane 1), S. velum foot tissue (lane 2), and E. coli (lane 4) as well as negative controls with no DNA template (lane 3) were submitted to PCR amplification primed by oligonucleotides complementary to conserved regions at opposite ends of the bacterial 16S rRNA gene (see "PCR amplification" section). Products were separated by electrophoresis in 1% agarose and stained with ethidium bromide. The sizes (in kilobases) of some of the DNA molecular weight standards (lane 5) are indicated to the right. FIG. 1. PCR-mediated amplification of the 16S rRNA coding regions of the S. velum symbionts. DNA samples from symbiont- containing S. velum gill tissue (lane 1), S. velum foot tissue (lane 2), and E. coli (lane 4) as well as negative controls with no DNA template (lane 3) were submitted to PCR amplification primed by oligonucleotides complementary to conserved regions at opposite ends of the bacterial 16S rRNA gene (see "PCR amplification" section). Products were separated by electrophoresis in 1% agarose and stained with ethidium bromide. The sizes (in kilobases) of some of the DNA molecular weight standards (lane 5) are indicated to the right. FIG. 1. PCR-mediated amplification of the 16S rRNA coding regions of the S. velum symbionts. DNA samples from symbiont- containing S. velum gill tissue (lane 1), S. velum foot tissue (lane 2), and E. coli (lane 4) as well as negative controls with no DNA template (lane 3) were submitted to PCR amplification primed by oligonucleotides complementary to conserved regions at opposite ends of the bacterial 16S rRNA gene (see "PCR amplification" section). Products were separated by electrophoresis in 1% agarose and stained with ethidium bromide. The sizes (in kilobases) of some of the DNA molecular weight standards (lane 5) are indicated to the right. FIG. 3. Southern blot hybridization of restricted S. FIG. 2. Restriction map of S. velum symbiont 16S rRNA coding region based on the sequence of 16S rDNA (1,460 bp) amplified from S. velum gill tissue. Additional enzymes used in analyses predicted not to restrict the gene included BglII, PvuII, and XhzoI. Phylogenetic Relationships of Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis JONATHAN A. EISEN,lt STEVEN W. SMITH,2 AND COLLEEN M. CAVANAUGH`* Department of Organismic and Evolutionary Biology, 1 and Harvard Genome Laboratory,2 Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 velum gills. For example, enzymes predicted to cut twice within the gene produced the pre- 5 m U 8 Lu FIG. 2. Restriction map of S. velum symbiont 16S rRNA coding region based on the sequence of 16S rDNA (1,460 bp) amplified from S. velum gill tissue. Additional enzymes used in analyses predicted not to restrict the gene included BglII, PvuII, and XhzoI. J. BACTERIOL. 3418 J. BACTERIOL. NOTES 'awvfawnu,s v VanoZIJJOUO N luoiaisoiasw d ZI9N sunp!xoouiif :1 !xabuvy A d wnsid -v S wnsid -V iatunq J avnuownaud -7 naAt.u J zU-- umils ds widso.iwonltj wAs v3jfUsVw j wAs snl!ydouuaq, g wAs wnlat -S wAs vjildAipvd wAs sLvjlnd?qJo i) wAs vwivnuuv 7 wAs vjvuozrnbav 7 c 00 c) 00 ..- 0 cC 0 C() tn r- aN \O M 00 It r-4 W) 00W 4k &- &~o 6 4 4 W) Wor1i cl 4 - 4 r- t r 0000000000000000000000000000000000ICW)W)00 'IC 0000000000000000000000000000000000 el; 00 0000 00 0000 000000 00000000000000 '- -4 ClCl r- 00 00 00 00 0000 0000000 0000000c~000r- r-4"-C4 0000000000000000000000ON0\" C4 W)00 ''W)r4,-rC ICI 00 00 00 0000 00 0000 00 00 00 N00 C14 ICIC (1 N ON(7 0N Cl C CI ON r4V- -4 - r- rO \1 Nr Cr -O r4C )c O0-ON ONO(1 N ON ON ON00000000ONr-4r4r4 r-4 -4-4- ON.ONON= .CN'-4 NClONON0eC - rON\OO ONONONOON 0 00 000ONr-4r- -4 ,-4--4 -4 ,4 OCNON00ONON00ON000000 r-qr4r- -4 --4 .-4 ' -4 CO r-NCD O 0C)-4 " -ic %C r00NCfN00 ONr 0000000000 O V ~ --4 '-4 rl~4- O OON NN cl 00 r -- 4 r--q .- -4r~ -4r4 - O OON N ,-4-4 r- -4 4 r- 00 NON C "C)C)C00O0r -00 ett l r C00N11 ON , r-4r- -4 '- v-4-4 r-4r --4 r- CC) C4 co~ - ,- ` rj i ZC- E C) ~ C)' ~ 0s "C) U) 0 C) u r. C) r. (D z C) - '0 0j 0) Q 00 ca .0 .0 0 0 U .) 0 ' u, . C) U0o Du v: u U,'0 i= .2 C) C)0 U._ CuX ._ Ut a) ;C U) 0 C u r C) r. (D z C) - '0 0j 0) Q 00 ca .0 .0 0 0 U .) 0 ' u, . C)* U0o Du v: u U,'0 i= .2 C) C)0 U._ CuX ._ Ut a) ;C NOTES 3419 3419 NOTES VOL. 174, 1992 A. pisum P A. piswn S Tx. nivea L awaaa sym LL equizenata syr Cud orbgcdar s,ym rs. gesgosterorn - I /\ -- V I N. gonorrhoeae B. Uhar.opkiuns sym 5% C. magncisca sym Tns. sp. L-12 A. tnefaciens R. ricketsil FIG. 4. Unrooted phylogenetic tree showing the position of the S. velum symbionts in relation to that of other Proteobacteria species on the basis of 16S rRNA gene sequences. The tree was constructed from evolutionary distances in Table 1. Members of the alpha and beta subclasses of the Proteobacteria are bracketed; all others are of the gamma subclass. Chemoautotrophic symbionts (sym) are listed in boldface type. Full species names listed in Table 1. Scale bar represents percent similarity. A. pisum P Tns. sp. L-12 FIG. 4. Unrooted phylogenetic tree showing the position of the S. velum symbionts in relation to that of other Proteobacteria species on the basis of 16S rRNA gene sequences. The tree was constructed from evolutionary distances in Table 1. Members of the alpha and beta subclasses of the Proteobacteria are bracketed; all others are of the gamma subclass. Chemoautotrophic symbionts (sym) are listed in boldface type. Full species names listed in Table 1. Scale bar represents percent similarity. it should be noted that a large duplication of the region containing the rRNA operon with no subsequent changes at any of the nine restriction sites could escape detection by this analysis. i dicted size bands for S. velum genomic DNA (Fig. 3A): AvaI and BclI, 1,080 bp; EcoNI, 1,109 bp; and Nco and Stul, 998 bp (data not shown). We suggest that this technique is generally useful for the confirmation of the presence of PCR-generated sequences in cells with multiple types of DNA. y These results suggest that the symbiont genome contains but a single rRNA operon. Bacterial rRNA operons (rm), which include the 5S, 16S, and 23S rRNA genes, vary considerably in number among bacteria. In contrast to free-living species of Proteobacteria, which have 4 to 7 rm loci (18), only one copy has been detected in other endosym- bionts including both the primary (P) and secondary (S) symbionts of the pea aphid, Acyrthosiphon pisum (33) (in- cluded in Fig. 4). Multiple rRNA operons have generally been thought necessary to support a high rate of rRNA synthesis in rapidly dividing cells (3, 22). REFERENCES 1. Allen, J. 1978. Evolution of the deep sea protobranch bivalves. Philos. Trans. R. Soc. Lond. B 284:387-401. 1. Allen, J. 1978. Evolution of the deep sea protobranch bivalves. Philos. Trans. R. Soc. Lond. B 284:387-401. 2. Allen, J. 1985. Recent bivalvia: their form and evolution, p. 337-403. In E. Trueman and M. Clarke (ed.), The mollusca, vol. 10. Evolution. Academic Press, Inc., Orlando, Fla. g p p g ( ) A phylogenetic tree based on evolutionary distances (Fig. 4) indicates that the symbionts fall within the gamma subdi- vision of the Proteobacteria (30) and is consistent with earlier results based on comparison of 5S rRNA (31). This topology is also supported by parisimony analysis with bootstrapping. The 16S rRNA gene sequence indicates that the S. velum symbionts fall between the two branches of the chemoautotrophic symbiont cluster (Fig. 4). One branch includes the vent tubeworm Riftia pachyptila, and three lucinid clams, Lucinoma annulata, Lucinoma aequizonata, and Codakia orbiculanis, and the other branch includes the symbionts of the vent clam Calyptogena magnifica, the vent mussel Bathymodiolus thermophilus, and the free-living chemoautotroph Thiomicrospira sp. strain L-12. Alterations of the positions analyzed and variations in order of sequence addition always resulted in the same topology of branch points of the chemoautotrophic symbionts. While the Pro- teobactena class includes many species found in association with eucaryotes, including both parasitic and mutualistic symbionts of plants (e.g., agrobacteria and rhizobia) and animals (e.g., enteric organisms and rickettsiae) (30), the positions of S. velum symbionts in the tree supports the conclusion of Distel et al. (12) that the chemoautotrophic symbionts are from an evolutionarily distinct group of bac- teria. i , , , 3. Amikam, D., S. Razin, and G. Glaser. 1982. Ribosomal RNA genes in Mycoplasma. Nucleic Acids Res. 10:4215-4222. g y p 4. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. John Wiley & Sons, New York. gy y , 5. Boros, I., A. Kiss, and P. Venetianer. 1979. Physical map of the seven ribosomal RNA genes of Eschenichia coli. Nucleic Acids Res. 6:1817-1830. 6. Brosius, J., J. L. Palmer, J. P. Kennedy, and H. F. Noller. 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75:4801- 4805. 7. Cavanaugh, C. M. 1983. REFERENCES Symbiotic chemoautotrophic bacteria in marine invertebrates from sulfide-rich habitats. Nature (Lon- don) 302:58-61. ) 8. Cavanaugh, C. M. 1985. Symbiosis of chemoautotrophic bacte- ria and marine invertebrates from hydrothermal vents and reducing sediments. Bull. Biol. Soc. Wash. 6:373-388. g 9. Cavanaugh, C. M., M. S. Abbott, and M. Veenhuis. 1988. Immunochemical localization of ribulose-1,5-bisphosphate car- boxylase in the symbiont containing gills of Solemya velum (Bivalvia: Mollusca). Proc. Natl. Acad. Sci. USA 85:7786-7789. 10. Conway, N., and J. Capuzzo. 1991. Incorporation and utilization of bacterial lipids in the Solemya velum symbiosis. Mar. Biol. 108:277-292. 11. De Soete, G. 1983. A least squares algorithm for fitting additive trees to proximity data. Psychometrika 48:621-626. 12. Distel, D. L., D. J. Lane, G. J. Olsen, S. J. Giovannoni, B. Pace, N. R. Pace, D. A. Stahl, and H. Felbeck. 1988. Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences. J. Bacteriol. 170:2506-2510. While the symbionts as a whole appear to have a common origin, the evolution within the cluster is complex. Distel et al. (12) suggested that the symbiosis has evolved separately in many host lineages, because the symbiont trees do not parallel the host classifications which are, unfortunately, not based on explicit phylogenies. The evolutionary separation between the bivalve subclasses Lamellibranchia and Proto- branchia, which are considered to be closest to the ancestral bivalve condition, is well characterized morphologically and in the fossil record (2, 27). If lamellibranchs truly arose from protobranchs, then the topology of this tree placing the symbionts of a protobranch between two branches of lamel- libranchs (Fig. 4) lends strong support to the idea of multiple origins of the symbioses. Further studies of host invertebrate evolutionary histories are needed so that the phylogenetic relationships of symbionts may be compared with those of their hosts, enabling us to resolve the possibility of polyphyl- etic origin of chemoautotrophic symbioses. In addition, future characterization of the 16S rRNA of symbionts from several species within a given genus such as Solemya, will help determine whether the symbiosis has evolved a single time within a particular lineage, as suggested for the lucinid bivalves (12). 13. Felbeck, H. 1981. Chemoautotrophic potential of the hydrother- mal vent tube worm, Riftia pachyptila Jones (Vestimentifera). Science 213:336-338. 14. Felsenstein, J. 1989. PHYLIP-phylogeny inference package (version 3.2). Cladistics 5:164-166. 15. Fisher, C. 1990. Chemoautotrophic and methanotrophic symbi- oses in marine invertebrates. Rev. Aquat. Sci. 2:399-436. U) 0 C u r C) r. (D z C) - '0 0j 0) Q 00 ca .0 .0 0 0 U .) 0 ' u, . C)* U0o Du v: u U,'0 i= .2 C) C)0 U._ CuX ._ Ut a) ;C Homologous nucleotide positions of 16S rRNA sequences of selected bacteria were aligned with that of the S. velum symbiont using conserved regions of sequence and secondary structure as a guide. Percent similarities and evolutionary distances of 16S rRNA for S. velum and selected free-living and symbiotic bacteria are shown in Table 1. Phylogenetic trees were constructed from evolu- tionary distances by the least-squares method of De Soete (11) and by parsimony analysis with bootstrapping (14), using the Treetool computer program (20). for helpful advice and discussion; and Wally Gilbert for the use of equipment. q p This work was supported by National Science Foundation grant DCB 8718799. q p This work was supported by National Science Foundation grant DCB 8718799. U) 0 C u r C) r. (D z C) - '0 0j 0) Q 00 ca .0 .0 0 0 U .) 0 ' u, . C)* U0o Du v: u U,'0 i= .2 C) C)0 U._ CuX ._ Ut a) ;C Unterman and Baumann (32) suggested that the aphid symbionts therefore grow slowly, with doubling times of 2 days to parallel the growth rate of the aphid host. They further speculated that the single rRNA operon in the aphid symbiont genome is a consequence of the adaptation to a symbiotic existence, which necessitates a slow growth rate. Although the division rate of S. velum symbionts is not known, it is unlikely that they grow slowly, since they must produce all of the biomass for their invertebrate host. Studies of rn copy number and growth rates of endosymbionts and their free-living relatives from a variety of phylogenetic groups may help resolve the significance of rRNA operon redundancy. The restriction patterns of 16S rRNA coding regions for DNA extracted from S. velum gills were identical for all nine clams examined; representative results are shown in Fig. 3. This, along with the lack of variability in the partial sequence of 16S rDNA for three individuals, suggests that there is a single dominant bacterial species within S. velum and that the host-symbiont association is species specific. This result is in agreement with the findings of Distel et al. (12) for lamellibranch bivalve and tubeworm chemoautotrophic sym- bionts. Single bands were evident for all enzymes predicted to cut outside or near the ends of the gene such as AvaI, Bcll, EcoNI, PvuII, XhoI (Fig. 3), and NcoI (band size, 9,600 bp; data not shown). Some of these enzymes generated restric- tion fragments larger than that of a typical bacterial ribo- somal operon (which includes the 5S, 16S, and 23S rRNA genes [-5 kb]), indicating that the single bands observed were not generated by double cuts within multiple operons. Furthermore, only two bands were observed for enzymes predicted to cut near the middle of the 16S rRNA gene such as EcoRI (Fig. 3B) and StuI (bands of 4,400 and 19,500 bp; data not shown). Thus, all enzymes in all animals generated patterns consistent with the presence of only one copy of the 16S rRNA gene in the symbiont genome (Fig. 3). However, g p y Phylogenetic analysis of the S. velum symbionts. Phyloge- netic analysis was conducted using the Genetic Data Envi- ronment program (Steve Smith, Harvard Genome Laborato- 3420 NOTES J. BACTERIOL. J. BACTERIOL. for helpful advice and discussion; and Wally Gilbert for the use of equipment. ry). We thank Dave Lane and Will Weisburg for providing PCR primers and advice; Sabrina Drill and Michael Eisen for skilled technical assistance; Rob Dorit, Barbara Bowen, and Adam Marsh REFERENCES q 16. Halanych, K. 1991. 5S ribosomal RNA sequences inappropriate for phylogenetic reconstruction. Mol. Biol. Evol. 8:249-253. 17. Jukes, T., and C. Cantor. 1969. Evolution of protein molecules, p. 21-132. In H. Munro (ed.), Mammalian protein metabolism. Academic Press, Inc., New York. 18. Krawiec, S., and M. Riley. 1990. Organization of the bacterial chromosome. Microbiol. Rev. 54:502-539. 19. Lane, D. L., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and N. R. Pace. 1985. Rapid determination of 16S rRNA sequences for phylogenetic analysis. Proc. Natl. Acad. Sci. USA 82:6955- 6959. 20. Maciukenas, M. 1991. Treetool computer program. University of Illinois Board of Trustees, Urbana. 21. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Nucleotide sequence accession number. The 16S rRNA sequence reported here has been submitted to GenBank and given accession number M90415. 22. Nomura, M., E. Morgan, and S. Jaskunas. 1977. Genetics of bacterial ribosomes. Annu. Rev. Genet. 11:297-347. 23. Olsen, G. 1988. Phylogenetic analysis using ribosomal RNA. Methods Enzymol. 164:793-812. 24. Olsen, G., N. Larsen, and C. Woese. 1991. The ribosomal RNA database project. Nucleic Acids Res. 19:2017-2021. We thank Dave Lane and Will Weisburg for providing PCR primers and advice; Sabrina Drill and Michael Eisen for skilled technical assistance; Rob Dorit, Barbara Bowen, and Adam Marsh 25. Rossau, R., L. Heyndrickx, and H. Van Heuverswyn. 1988. Nucleotide sequence of a 16S ribosomal RNA gene from Neis- NOTES 3421 NOTES 3421 VOL. 174, 1992 Campbell and R. Eikenbary (ed.), Aphid-plant genotype inter- actions. Elsevier Biomedical Press, Amsterdam. seria gonorrheae. Nucleic Acids Res. 16:6227. g 26. Rosson, R., and T. Schmidt. 1991. Unpublished data 33. Unterman, B. M., P. Baumann, and D. L. McLean. 1989. Pea-aphid symbiotic relationship established by analysis of 16S rRNAs. J. Bacteriol. 171:2970-2974. 27. Runnegar, B., and J. Pojeta, Jr. 1985. Origin and diversification of the mollusca, p. 1-58. In E. Trueman and M. Clarke (ed.), The mollusca, vol. 10. Evolution. Academic Press, Inc., Or- lando, Fla. 34. Weisburg, W. G., S. M. Barns, D. A. Pelletier, and D. J. Lane. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697-703. 28. Saiki, R. K. 1990. Amplification of genomic DNA, p. 13-20. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. A guide to methods and applications. REFERENCES Academic Press, New York. 35. Weisburg, W. G., M. E. Dobson, J. E. Samuel, G. A. Dasch, L. P. Mallavia, 0. Baca, L. Mandelco, J. E. Sechrest, E. Weiss, and C. R. Woese. 1989. Phylogenetic diversity of the rickettsiae. J. Bacteriol. 171:4202-4206. 198 , 29. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 36. Weisburg, W. G., Y. Oyaizu, H. Oyaizu, and C. R. Woese. 1985. Natural relationship between bacteroides and flavobacteria. J. Bacteriol. 164:230-236. i p g 30. Stackebrandt, E., R. G. E. Murray, and H. G. Truper. 1988. Proteobacteria classis nov., a name for the phylogenetic taxon that includes the "purple bacteria and their relatives." Int. J. Syst. Bacteriol. 38:321-325. 37. Winship, P. 1989. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 17:1266. y 31. Stahl, D. A., D. J. Lane, G. J. Olsen, and N. R. Pace. 1984. Analysis of hydrothermal vent-associated symbionts by rRNA sequences. Science 224:409-411. 38. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221- 271. 198 39. Yang, D., Y. Oyaizu, H. Oyaizu, G. Olsen, and C. Woese. 1985. Mitochondrial origins. Proc. Natl. Acad. Sci. USA 82:4443- 4447. 32. Unterman, B., and P. Baumann. 1990. Partial characterization of the ribosomal RNA operons of the pea-aphid endosymbionts: evolutionary and physiological implications, p. 329-350. In R.
https://openalex.org/W2041247709	https://discovery.ucl.ac.uk/id/eprint/1470789/1/journal.pone.0118899.pdf	English	null	Social Media and Internet Driven Study Recruitment: Evaluating a New Model for Promoting Collaborator Engagement and Participation	PloS one	2,015	cc-by	5,493	Methods Copyright: © 2015 Khatri 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. Interventions involved a new Twitter account, including weekly live question-and-answer sessions, a new Facebook group page, online YouTube presentations and an information page on a national association website. Link tracking analysis was undertaken using Goo- gle Analytics, which was then related to subsequent registration. Social influence was cal- culated using the proprietary Klout score. Chetan Khatri1, Stephen J. Chapman2, James Glasbey3, Michael Kelly4, Dmitri Nepogodiev5, Aneel Bhangu6, J. Edward Fitzgerald7, on behalf of the STARSurg Committee Chetan Khatri1, Stephen J. Chapman2, James Glasbey3, Michael Kelly4, Dmitri Nepogodiev5, Aneel Bhangu6, J. Edward Fitzgerald7, on behalf of the STARSurg Committee a1111 1 Imperial College London Medical School, London, United Kingdom, 2 University of Leeds, Leeds, United Kingdom, 3 Cardiff University Medical School, Cardiff, United Kingdom, 4 University of Liverpool Medical School, Liverpool, United Kingdom, 5 Norwich Academic Foundation Programme, Norwich, United Kingdom, 6 Academic Department of Surgery, 4th Floor, Old Queen Elizabeth Hospital, University of Birmingham, College of Medical and Dental Sciences, West Midlands, B15 2TH, Birmingham, United Kingdom, 7 University College London, London, United Kingdom * james.fitzgerald.11@ucl.ac.uk OPEN ACCESS Citation: Khatri C, Chapman SJ, Glasbey J, Kelly M, Nepogodiev D, Bhangu A, et al. (2015) Social Media and Internet Driven Study Recruitment: Evaluating a New Model for Promoting Collaborator Engagement and Participation. PLoS ONE 10(3): e0118899. doi:10.1371/journal.pone.0118899 Aims A substantial challenge facing multicentre audit and research projects is timely recruitment of collaborators and their study centres. Cost-effective strategies are required and fee-free social media has previously been identified as a potential conduit. We investigated and evaluated the effectiveness of a novel multi-format social media and Internet strategy for tar- geted recruitment to a national multicentre cohort study. Academic Editor: Patrick S Sullivan, Rollins School of Public Health, Emory University, UNITED STATES Received: March 8, 2014 Accepted: January 18, 2015 Published: March 16, 2015 * james.fitzgerald.11@ucl.ac.uk RESEARCH ARTICLE Social Media and Internet Driven Study Recruitment: Evaluating a New Model for Promoting Collaborator Engagement and Participation Chetan Khatri1, Stephen J. Chapman2, James Glasbey3, Michael Kelly4, Dmitri Nepogodiev5, Aneel Bhangu6, J. Edward Fitzgerald7*, on behalf of the STARSurg Committee Conclusions Targeted social media substantially aided study dissemination and collaborator recruitment. It acted as an adjunct to traditional methods, accounting for 18.2% of collaborator registra- tion in a short time period with no associated financial costs. We provide a practical model for designing future recruitment campaigns, and recommend Facebook, Twitter and tar- geted websites as the most effective adjuncts for maximising cost-effective study recruitment. Social Media and Internet Driven Study Recruitment the greatest rise in registration traffic and accounted for 312 (48%) of the total registrations within a 2-week period. A Twitter ‘social influence’ (Klout) score of 42/100 was obtained dur- ing this period. the greatest rise in registration traffic and accounted for 312 (48%) of the total registrations within a 2-week period. A Twitter ‘social influence’ (Klout) score of 42/100 was obtained dur- ing this period. Competing Interests: The authors have declared that no competing interests exist. Results Funding: The Royal College of Surgeons (England) provided the venue for the national collaborators’ meeting free of charge. A Regional Event Grant was received from the Association of Surgeons in Training (ASiT) as a contribution to the costs of the national collaborators’ meeting. No further funding was applied for or received. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Internet traffic analysis identified a total of 1562 unique registration site views, of which 285 originated from social media (18.2%). Some 528 unique registrations were received, with 96 via social media platforms (18.2%). Traffic source analysis identified a separate national association webpage as resulting in the majority of registration page views (15.8%), fol- lowed by Facebook (11.9%), Twitter (4.8%) and YouTube (1.5%). A combination of publicity through Facebook, Twitter and the dedicated national association webpage contributed to 1 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 Ethics According to regulations set out by the Health Resource Authority decision tool (http://www. hra-decisiontools.org.uk/research/), this evaluation of collaborator recruitment did not require ethical approval. The national cohort study protocol was reviewed by the National Research Ethics Service and a Research Ethics Committee, that the anonymous, observational nature of data collection meant that this did not require formal research ethics registration. STARSurg Collaborative Model The STudent Audit and Research in Surgery (STARSurg) research and audit collaborative net- work (http://www.starsurg.org/) undertook a national, multicentre cohort study using a proto- col-driven, medical student investigator-led approach to determine the safety profile of non- steroidal anti-inflammatory drugs (NSAIDs) following gastrointestinal (GI) surgery [14–16]. Collaborators were responsible for gaining local audit approval and undertaking prospective data collection. A structured hierarchy of collaborator support was provided, which included local clinical faculty, geographical regional coordinators and a dedicated steering committee. Collaborators were responsible for gaining local audit approval and undertaking prospective data collection. A structured hierarchy of collaborator support was provided, which included local clinical faculty, geographical regional coordinators and a dedicated steering committee. Subjects and Settings Junior doctors and medical students were targeted and invited to act as collaborators. An on- line registration via a dedicated Google form (http://www.google.com/google-d-s/createforms. html) was utilised for collaborator registration. A single web link to this registration form was created and used universally across all promotional interventions to facilitate link tracking and analytics. Introduction 2 / 11 Social Media and Internet Driven Study Recruitment The STudent Audit and Research in Surgery (STARSurg) group was formed based on a pre- viously described collaborative research model [13]. The group established a novel network of medical students, junior doctors, and supervising surgeons, to facilitate data collection for a na- tional, multi-centre cohort study. This paper reports and evaluates a multi-modal social media and Internet strategy to pro- mote and recruit collaborators for a national, multi-centre study. In addition, based on our ex- periences, we provide a practical model for future studies, for others who may wish to utilise this novel approach. Introduction Timely participant or collaborator recruitment is a major challenge in launching a large, multi- centre study. One eight-year review noted that only 31% of multi-centre randomised controlled trials achieved their original recruitment targets in a timely manner [1]. Non-completed studies can delay the introduction of new healthcare interventions or increase the time in which the population is exposed to a suboptimal treatment [2]. Delays in recruitment may also increase financial costs [3], which risks biasing investigators and funders towards smaller more rapidly delivered studies instead of longer term projects. Engaging with clinicians or patients to stimulate participant recruitment can be difficult. Clinician involvement can be hindered by issues such as time limitations, loss of professional autonomy, concern for patients, or a perceived lack of rewards and recognition [4,5]. Patients may be worried about the inconvenience, costs, or uncertainty of treatment, with some having a particular preference to receiving (or not receiving) the treatment [4,5]. These factors can be compounded in both groups by a lack of awareness to study opportunities. While patient re- cruitment has been the topic of extensive previous study, the area of clinician engagement is less well understood. Previous studies have proposed social media as a potential new modality for study promo- tion and recruitment [2,6–8] and have highlighted the reduced costs associated with this novel approach. With Facebook and Twitter having over one billion users each and YouTube having over 645 million users [9–11] many individuals access such platforms on a daily basis. Studies investigating social media uptake specifically within the medical profession have reported usage rates of between 13–47% amongst doctors [12] with 48% of all users logging on to use Facebook on any given day [10]. This has made social media a potentially valuable resource for promoting studies and collaborator recruitment, which could lead to more efficient patient recruitment. Social media is largely a free-to-use medium, which can be harnessed to advertise and re- cruit medical professionals for academic projects. It can potentially reach large, targeted popu- lations rapidly, providing recruitment opportunities to interested but unknown individuals who may not otherwise be engaged within standard professional or institutional contact net- works. Although existing studies have described the use of social media to recruit patients, there is a paucity of literature detailing its role in recruiting medical collaborators, and minimal guidance on how to practically undertake this. Research Question This study investigated the use of social media and online strategies as a cost-effective method of collaborator recruitment for a national cohort study, and evaluated the relative outcomes of these. We hypothesised that the use of these online platforms could potentially increase recruit- ment over traditional methods with no associated financial outlay. We designed a novel Inter- net and social media collaborator recruitment model to test this. YouTube A dedicated YouTube channel (YouTube, LLC, California, USA) was created at the start of the recruitment period with a single 7:53 minute video uploaded. This consisted of a narrated, step-by-step presentation, which aimed to explain the project and protocol to potential collabo- rators. To encourage participation, a link to the registration form was placed in the comments section. Twitter A dedicated Twitter account (Twitter, Inc., San Francisco, USA) was created at the start of the seven-week recruitment period. Tweets are short messages, which are limited to 140 characters, and can include links to external websites or images. Regular Tweets encouraged potential col- laborators to join our study with a specific web link leading to the registration page included in the message. We asked groups to ‘retweet’ (forward) these messages to their own followers and continued this throughout the recruitment period. Strategies to disseminate the study included targeting specific organisations with medical student and junior doctor interests. These groups included: student unions, student medical, surgical and academia societies, selected individuals and professional postgraduate bodies. These organisations were ‘followed’ leading to an organ- ic growth of reciprocal ‘followers’ that could see our posts. Recruitment strategies Five interventions were used to recruit collaborators. All social media interventions were used exclusively for study-related promotion, and were accessible on the Internet free of charge without the need for registration or subscription fees to access. 1. Targeted emails to medical student unions, university surgical and academic societies and professional bodies. 1. Targeted emails to medical student unions, university surgical and academic societies and professional bodies. 3 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 Social Media and Internet Driven Study Recruitment 2. A dedicated information webpage on the Association of Surgeons in Training (ASiT) web- site (http://www.asit.org/news/STARSurgUK1) 2. A dedicated information webpage on the Association of Surgeons in Training (ASiT) web- site (http://www.asit.org/news/STARSurgUK1) 2. A dedicated information webpage on the Association of Surgeons in Training (ASiT) web- site (http://www.asit.org/news/STARSurgUK1) 3. A dedicated Twitter feed targeting student unions, surgical and academic societies and post- graduate professional bodies (https://twitter.com/STARSurgUK). 3. A dedicated Twitter feed targeting student unions, surgical and academic societies and post- graduate professional bodies (https://twitter.com/STARSurgUK). 3. A dedicated Twitter feed targeting student unions, surgical and academic societies and post- graduate professional bodies (https://twitter.com/STARSurgUK). 4. A dedicated Facebook group page, linked to the Twitter feed, which could inform potential participants of recent developments (https://www.facebook.com/STARSurgUK). 4. A dedicated Facebook group page, linked to the Twitter feed, which could inform potential participants of recent developments (https://www.facebook.com/STARSurgUK). 4. A dedicated Facebook group page, linked to the Twitter feed, which could inform potential participants of recent developments (https://www.facebook.com/STARSurgUK). 5. A dedicated YouTube channel, including an introductory video with the purpose of explain- ing the project and protocol to potential collaborators (https://www.youtube.com/channel/ UCDewmFYVD1wfciYEwy8UHtQ?feature = watch). 5. A dedicated YouTube channel, including an introductory video with the purpose of explain- ing the project and protocol to potential collaborators (https://www.youtube.com/channel/ UCDewmFYVD1wfciYEwy8UHtQ?feature = watch). Facebook A dedicated Facebook group page (Facebook, Inc., California, USA) was created at the start of the recruitment period and linked to the Twitter feed, ensuring the same content was delivered through both social media platforms. Facebook pages can be ‘liked’ by users who wish to follow the content delivered through the group. Additional informative posts were added to other Facebook groups’ pages that were deemed to be of interest where members of the groups/pages would be able to see our post. Posts con- sisted of a short paragraph encouraging students and junior doctors to collaborate and includ- ed the same link to the registration page. For larger, professional organisations, private messages were sent to request they forward on our advert, which included a link to the online registration page. Collaborator Demographics A total of 476 collaborators collected data at 109 hospital centres across the UK. Analysis of the collaborator registration page showed a total of 1562 unique views, which resulted in 527 regis- trations to give a 33.7% (527/1562) conversion rate. Comparative Recruitment Between Different Platforms At the end of the registration period, using traffic source analysis from Google Analytics, the dedicated study webpage hosted on the national association website resulted in the greatest in- terest, accounting for 15.7% (245/1562) of views on the collaborator registration page, and gen- erating 83 registrations. Within the social media category, the Facebook group page attracted 160 ‘likes’ after 378 posts. This formed the majority of social media traffic, accounting for 11.9% views (186/1562) and 63 registrations. The Twitter page gained 236 followers from 378 tweets, resulting in 4.87% of views (76/1562) and resulting in 25 collaborator registrations. One video was published on our YouTube channel, which attracted 372 views. The link from this YouTube video page ac- counted for 1.47% (23/1562) of views to our registration page with 8 subsequent registrations. Traditional methods including emailing and organic spread accounted for the majority of collaborator registration site views, with 66.1% of views (1032/1562) eventually leading to 348 registrations. Data Analysis Descriptive analysis was performed using Google Analytics (http://www.google.com/analytics), an integral feature of Google shortened links. Social media influence was calculated using the ‘Klout Score’. Klout (Klout Inc, San Francisco, US) is a free online social media analytics tool 4 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 Social Media and Internet Driven Study Recruitment used to rank users according to their online social influence by means of their proprietary Klout Score. This is a numerical value between 1 and 100, measured using access- and analysis- related metrics from linked social media accounts in the preceding 90 days. These may include follower counts, retweets and the influence of the users who retweet your own messages. The average Klout score across all social media users is 40 [17]. Financial currency conversions are based on prevailing market rates on 29th November 2014 using the UK Forex exchange rate, rounded to the nearest whole unit of currency. Outcomes and Impact A flowchart detailing the comparative study recruitment through these different approaches is provided in Fig. 1. A Twitter impact ‘social influence’ (Klout) score of 42/100 was calculated during this seven-week time period, indicating an above average social media influence gained over the seven-week period. Discussion This study indicates that targeted use of free social media and internet resources substantially assisted study dissemination in a cost-effective manner, accounting for 18.2% of collaborator recruitment in a short time period. This resulted in a substantially higher click-through rate (33.7%) to recruit collaborators when compared to other studies recruiting members of the general public [8,18,19]. We have demonstrated that effective targeting of groups of interest can result in a high level of recruitment with no additional costs. This strategy is however time intensive and requires effective identification of groups of interest, together with approaches that harness and maximise the potential benefits of multi-modal social media usage. The broad use of social media for non-medical marketing has developed rapidly in recent years; however medical and scientific communities have been slower to adapt this new PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 5 / 11 Social Media and Internet Driven Study Recruitment Fig 1. Detailed comparative breakdown of collaborator recruitment by social media and standard methods. doi:10.1371/journal.pone.0118899.g001 Fig 1. Detailed comparative breakdown of collaborator recruitment by social media and standard methods. doi:10.1371/journal.pone.0118899.g001 doi:10.1371/journal.pone.0118899.g001 technology for their own needs. The potential advantages and disadvantages of using this new approach for study recruitment are summarised in Table 1. The current model of free social media use by the wider population is largely based on paid- for advertising by businesses seeking promotion. There have been previous reports of paid-for Facebook advertising to assist with study recruitment, with Kapp et al. reporting the use of ad- verts available to the general population [8]. Their promotion was ‘reached’ by 373,225 women of whom 250 clicked the advert. Out of those who clicked the advert, only 9 proceeded past the introduction page leading to a click through rate of 3.2%. Paid Facebook advertising to recruit patients to randomised controlled trials (RCTs) has been reported to cost between €Euros 12 ($USD 15.28) and $USD 20.14 (€Euros 15.81) per recruited participant [6,18]. Our study found a click-through rate of 33.7% without paid-for advertising, which is likely due to the highly targeted nature of our social media activities compared to general advertising. As such, although our readership was smaller, those viewing it may have been more likely to engage and follow through to registration. PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 Discussion Paid advertising may still be beneficial, as it also generates an in- crease in the associated Facebook or Twitter page visits, which can potentially enhance future recruitment and aid organic growth and awareness. Compared with alternative methods of trial recruitment, paid advertising via Facebook has been shown to be more cost-effective, with conventional methods costing between $USD 20 (€Euros 15.71) and $USD 500 (€Euros 392.63) [20,21]. Traditional methods such as face-to-face recruitment, which in our study consisted of lec- tures and talks at universities and hospitals, are also time intensive. This problem is further compounded when centres are located nationally or internationally. Bhatnager et al. described that whilst personally meeting with study sites increased recruitment by a modest mean of one 6 / 11 Social Media and Internet Driven Study Recruitment Table 1. Advantages and disadvantages of using targeted social media to aid study recruitment. Advantages Disadvantages Free to use other than the time spent establishing and maintaining a social media presence May introduce demographic selection bias by recruiting a younger, more internet-active population Easy and intuitive to use Requires working knowledge of relevant, active interest groups and inﬂuencers Rapid dissemination of information Overall potentially lower recruitment conversion rates compared to traditional methods Can be a useful adjunct to existing traditional models of study recruitment Requires time commitment to develop content and update this Used by up to 80.8% of medical students [30] May not be as effective for recruitment of established medical professionals (compared to recruitment of medical students) due to lower usage and penetration [12]; this is however increasing with time Potentially allows recruitment to extend across international borders Biased against countries or regions with restricted access to social media Facilitates two-way communication and effective engagement with the target population Social media platforms can be access-restricted by hospital IT departments, which may impede the ﬂow of communication. Provides an accessible medium by which to distribute electronic study resources including media, electronic protocols and information documents May bias against those internationally who lack internet or electronic resources to access these Offers the potential to engage collaborators who may not otherwise participate via traditional methods doi:10.1371/journal.pone.0118899.t001 doi:10.1371/journal.pone.0118899.t001 patient per month, this effect was only limited for 120 days, prompting suggestion that further interventions would be required to sustain this model [22]. PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 Social Media and Internet Driven Study Recruitment recruitment and in this study lead to an extra 23 clicks to our registration page, translating to 8 eventual collaborators. Depending on the nature of the study, social media may however introduce a bias into re- cruited participants. Previous studies have noted that an online advertisement paradigm re- sulted in recruitment of a younger population sample [28,29], which cannot necessarily be generalised to the rest of the population. In this study the target audience (study collaborators) fitted with the younger demographic profile of social media users, enhancing recruitment. However, social media may not be as effective when targeting more senior groups of clinicians. Whilst 80.8% of medical students within England use social media [12,30], this rate drops to much lower rates of between 12.8% to 46.7% within established medical professionals [12]. In addition, social media has little or no impact on the cohort of professionals who do not use it. This may imply that those wishing to recruit professionals rather than medical students or younger junior doctors as potential collaborators may experience lower rates of engagement. An additional general weakness of this recruitment method is that it introduces selection bias by potentially over-representing those individuals who spend the most amount of time on social media communities such as Facebook and Twitter. However, this bias is similar to many other forms of population sampling conducted over the Internet. The time spent on Facebook by an individual has been shown to be positively correlated to two personality traits: neuroti- cism and total loneliness, whilst a negative correlation was shown to be present for time spent and conscientiousness [31]. While unlikely to impact on our national cohort study, the impli- cations of these observations will need to be taken into account by others depending on the na- ture of the study. Although this study recruited a total of 528 investigators, only 476 ultimately collected data. This dropout rate is likely due to numerous factors. Firstly, there was often an oversubscription within many universities, with the number of collaborators wanting to collect data outnumbering the possible sites for data collection. Secondly there was a small cohort who entered their contact details incorrectly and therefore was lost to further contact. Lastly, there were collaborators who ex- pressed an interest in collecting data, but later withdrew due to other commitments. PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 Discussion However, it is notable that when specifically qualified and trained personal were used for site visits, site recruitment increased significantly, albeit with a greater cost and time intensity associated with this method [23]. Other methods for recruiting patients, like random digit phone calling, can result in both high staff time consumption as well as high recruitment costs per participant, potentially reach- ing up to $USD 1075 (€Euros 844.16) per patient [24]. Recruitment via social media, especially free-of-charge activities rather than paid adverts, potentially represents a cheaper, more time- effective method of recruitment. Once the appropriate social media accounts have been estab- lished and target groups identified, on-going promotion incurs little time-cost. These can also be re-purposed in future for other related studies and activities, maximising the return on the initial time investment required to set these up. The usefulness of Facebook and Twitter are not limited to paid adverts, although varying success has been previously reported by the use of free services in social media. Whilst a study by Godino et al. found that 12.2% of participants were recruited from social media [25], others have found social media to be the most successful method of recruiting participants to a study [26]. The relative success or otherwise reported to date is likely to be dictated by numerous fac- tors including a mix of the strategies used on these, the particular target audience and the na- ture of the study being recruited to. If carefully considered, a focused marketing strategy may allow Twitter to be used as the sole platform to collect data [27]. A novel feature in this study was an online presentation uploaded to YouTube. Researchers often struggle to provide the same high-quality message to all participants [19]. This method is far less time intensive than face-to-face interactions as well providing a standardised message. For those engaged by the protocol, a link placed at the bottom of this video helped enhance 7 / 11 Such dropout rates need to be considered when setting target numbers for recruitment. Unfortunately the data does not enable us to link those ultimately collecting data with their mode of initial engagement (social media, website, or email), and these confounding factors influencing the dropout means that such linkage would not add insight. However, in future studies it would be interesting to inves- tigate whether the mode of initial engagement influenced the likelihood of eventual participation. Although this study was limited to national participation, the ease with which social media reaches across borders potentially provides particularly cost-effective methods for recruitment to international studies. However, targeting this may be more difficult in countries where the influencing groups and individuals are less well known, and may also be biased towards coun- tries with adequate and unrestricted use of social media. Additionally, this study relates to tar- geting healthcare professionals and medical students; the degree to which these findings can be extrapolated to direct patient recruitment for studies is unknown. Future corroborating research in this area is needed to investigate the overall effectiveness of social media on the recruitment of medical professionals including a comparison of different strategies and approaches to facilitate this. Within this, it is important that future research should specifically determine variation of productivity and reliability of collaborators recruited by social media versus recruited via other methods. Finally, there is a need for an increased awareness of the usefulness of social media amongst the wider scientific community as a poten- tial conduit to disseminate study protocols and to recruit collaborators. 8 / 11 Social Media and Internet Driven Study Recruitment Fig 2. Suggested practical model recruitment strategies for utilising social media. Fig 2. Suggested practical model recruitment strategies for utilising social media. doi:10.1371/journal.pone.0118899.g002 Proposed Practical Model for Social Media Driven Collaborator Recruitment Based on our experiences using targeted social media for collaborator recruitment, we have de- veloped a proposed practical model for developing future approaches to this (Fig. 2). Our model outlines the practical steps that need to be considered and put in place in order to con- struct such a targeted campaign and how to go about deploying this. In particular, care should be taken in selecting groups to be targeted, as irrelevant groups will lead to ineffective uptake. PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 References 1. McDonald AM, Knight RC, Campbell MK, Entwistle VA, Grant AM, et al. (2006) What influences recruit- ment to randomised controlled trials? A review of trials funded by two UK funding agencies. Trials 7: 9. PMID: 16603070 1. McDonald AM, Knight RC, Campbell MK, Entwistle VA, Grant AM, et al. (2006) What influences recruit- ment to randomised controlled trials? A review of trials funded by two UK funding agencies. Trials 7: 9. PMID: 16603070 2. Watson JM, Torgerson DJ (2006) Increasing recruitment to randomised trials: a review of randomised controlled trials. BMC Med Res Methodol 6: 34. PMID: 16854229 3. Treweek S, Wilkie E, Craigie AM, Caswell S, Thompson J, et al. (2013) Meeting the challenges of re- cruitment to multicentre, community-based, lifestyle-change trials: a case study of the BeWEL trial. Tri- als 14: 436. doi: 10.1186/1745-6215-14-436 PMID: 24351063 4. Mapstone J, Elbourne D, Roberts I (2007) Strategies to improve recruitment to research studies. Cochrane database Syst Rev: MR000013. PMID: 17443634 5. Treweek S, Mitchell E, Pitkethly M, Cook J, Kjeldstrøm M, et al. (2010) Strategies to improve recruit- ment to randomised controlled trials. Cochrane database Syst Rev: MR000013. doi: 10.1002/ 14651858.MR000013.pub5 PMID: 20393971 6. Fenner Y, Garland SM, Moore EE, Jayasinghe Y, Fletcher A, et al. (2012) Web-based recruiting for health research using a social networking site: an exploratory study. J Med Internet Res 14: e20 doi: 10.2196/jmir.1978 PMID: 22297093 7. Ryan GS (2013) Online social networks for patient involvement and recruitment in clinical research. Nurse Res 21: 35–39. doi: 10.7748/nr2013.09.21.1.35.e302 PMID: 24004430 8. Kapp JM, Peters C, Oliver DP (2013) Research recruitment using Facebook advertising: big potential, big challenges. J Cancer Educ 28: 134–137. doi: 10.1007/s13187-012-0443-z PMID: 23292877 9. Statistics—YouTube (n.d.). Available: https://www.youtube.com/yt/press/en-GB/statistics.html. 10. Facebook Statistics \| Statistic Brain (n.d.). Available: http://www.statisticbrain.com/facebook-statistics/. 11. Twitter Statistics \| Statistic Brain (n.d.). Available: http://www.statisticbrain.com/twitter-statistics/. 12. Von Muhlen M, Ohno-Machado L (2012) Reviewing social media use by clinicians. J Am Med Inform Assoc 19: 777–781. doi: 10.1136/amiajnl-2012-000990 PMID: 22759618 13. Bhangu A, Kolias AG, Pinkney T, Hall NJ, Fitzgerald JE (2013) Surgical research collaboratives in the UK. Lancet 382: 1091–1092. doi: 10.1016/S0140-6736(13)62013-9 PMID: 24075040 14. Nepogodiev D, Chapman SJ, Glasbey JCD, Kelly M, Khatri C, et al. (2014) Multicentre observational cohort study of NSAIDs as risk factors for postoperative adverse events in gastrointestinal surgery. BMJ Open 4: e005164. doi: 10.1136/bmjopen-2014-005164 PMID: 24972607 15. Conclusions Targeted social media substantially aided study dissemination and collaborator recruitment as an adjunct to traditional methods, accounting for 18.2% of collaborator registration in a short time period with no associated financial outlay. We propose a practical model for designing fu- ture recruitment campaigns and recommend Facebook, Twitter and targeted websites as the most effective approach to achieving this, this novel method of communication can supplement more traditional methods, maximising rapid study recruitment in a cost-effective manner. 9 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 Social Media and Internet Driven Study Recruitment Author Contributions Conceived and designed the experiments: CK SJC JG MK DN AB JEF. Performed the experi- ments: CK SJC JG MK DN AB JEF. Analyzed the data: CK SJC JG MK DN AB JEF. Contribut- ed reagents/materials/analysis tools: CK SJC JG MK DN AB JEF. Wrote the paper: CK SJC JG MK DN AB JEF. PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 References STARSurg Collaborative (2014) Impact of postoperative non-steroidal anti-inflammatory drugs on ad- verse events after gastrointestinal surgery. 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Control Clin Trials 23: 157–171. PMID: 11943442 25. Godino L, Turchetti D, Skirton H (2013) Knowledge of genetics and the role of the nurse in genetic health care: a survey of Italian nurses. J Adv Nurs 69: 1125–1135. doi: 10.1111/j.1365-2648.2012. 06103.x PMID: 22853043 26. Leonard A, Hutchesson M, Patterson A, Chalmers K, Collins C (2014) Recruitment and retention of young women into nutrition research studies: practical considerations. Trials 15: 23. doi: 10.1186/ 1745-6215-15-23 PMID: 24433229 27. O’Connor A, Jackson L, Goldsmith L, Skirton H (2014) Can I get a retweet please? Health research re- cruitment and the Twittersphere. J Adv Nurs 70: 599–609. PLOS ONE \| DOI:10.1371/journal.pone.0118899 March 16, 2015 References doi: 10.1111/jan.12222 PMID: 23909740 28. Frandsen M, Walters J, Ferguson SG (2014) Exploring the viability of using online social media adver- tising as a recruitment method for smoking cessation clinical trials. 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https://openalex.org/W2612428807	http://revistaseletronicas.pucrs.br/ojs/index.php/civitas/article/download/24706/15545	Portuguese	null	Pontos de inflexão para a análise do pensamento vianniano: as posições de Oliveira Vianna sobre a entrada de 3.000 chineses no Brasil e o seu trabalho de consultor jurídico (1932-1940)	Civitas - Revista de Ciências Sociais	2,017	cc-by	9,152	http://dx.doi.org/10.15448/1984-7289.2017.1.24706 http://dx.doi.org/10.15448/1984-7289.2017.1.24706 http://dx.doi.org/10.15448/1984-7289.2017.1.24706 Artigos Artigos Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 Exceto onde especificado diferentemente, a matéria publicada neste periódico é licenciada sob forma de uma licença Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License Pontos de inflexão para a análise do pensamento vianniano As posições de Oliveira Vianna sobre a entrada de 3.000 chineses no Brasil e o seu trabalho de consultor jurídico (1932-1940) Inflection points for the analysis of Oliveira Vianna thought His position concerning the entrance of 3,000 Chinese in Brazil and his work as legal adviser (1932-1940) He exposed them in a technical report sent by him, through the Ministry of Labor, Industry and Commerce, to the Ministry of Foreign Affairs. This work helps us: 1) to amplify our understanding of Vianna's thought; 2) to highlight the specificity of his technical, juridical and political engagement. Finally, we find in this report a clear intersection between Oliveira Vianna as an intellectual and as a legal advisor. In this article, I will present, punctually, Vianna's positions about the introduction of 3,000 Chinese in Brazil. He exposed them in a technical report sent by him, through the Ministry of Labor, Industry and Commerce, to the Ministry of Foreign Affairs. This work helps us: 1) to amplify our understanding of Vianna's thought; 2) to highlight the specificity of his technical, juridical and political engagement. Finally, we find in this report a clear intersection between Oliveira Vianna as an intellectual and as a legal advisor. Keywords: Oliveira Vianna. Ministry of Labor, Industry and Commerce. First government of Getúlio Vargas. Immigration in Brazil. Oliveira Vianna's thought. Pontos de inflexão para a análise do pensamento vianniano As posições de Oliveira Vianna sobre a entrada de 3.000 chineses no Brasil e o seu trabalho de consultor jurídico (1932-1940) Inflection points for the analysis of Oliveira Vianna thought His position concerning the entrance of 3,000 Chinese in Brazil and his work as legal adviser (1932-1940) Pontos de inflexão para a análise do pensamento vianniano As posições de Oliveira Vianna sobre a entrada de 3.000 chineses no Brasil e o seu trabalho de consultor jurídico (1932-1940) Inflection points for the analysis of Oliveira Vianna thought His position concerning the entrance of 3,000 Chinese in Brazil and his work as legal adviser (1932-1940) Felipe Fontana* Resumo: Oliveira Vianna é um relevante expoente de nosso pensamento social e político e é justamente por isso que encontramos muitos estudos que buscam esmiuçar determinadas questões de seu pensamento ou compreender as raízes do mesmo. No entanto, se há certa facilidade em localizar trabalhos dedicados à revista do autor e de sua teoria, o mesmo não ocorre quando o interesse é investigar, por meio dos relatórios/pareceres técnicos produzidos por Vianna, a especificidade do trabalho político desenvolvido por ele entre os anos de 1932 e 1940. Nessa direção, este artigo apresentará pontualmente as posições de Vianna acerca da introdução de 3.000 chineses no Brasil. Elas estão explicitadas em um parecer técnico encaminhado por ele, através do Ministério do Trabalho, Indústria e Comércio, ao Ministério das Relações Exteriores. A realização de tal tarefa nos auxilia tanto na complexificação de algumas interpretações sobre o pensamento vianniano, quanto para dimensionarmos parte da especificidade de seu trabalho político como statemaker. Por fim, encontramos nesse parecer uma clara intersecção entre o Oliveira Vianna intelectual e o Oliveira Vianna consultor jurídico. Palavras-chave: Oliveira Vianna. Ministério do Trabalho, Indústria e Comércio. Primeiro Governo Vargas. Imigração no Brasil. Pensamento vianniano. Abstract: Oliveira Vianna is a relevant exponent of Brazilian social and political thought and it is precisely for this reason that many studies seek to analyze the questions of his thinking or the roots of it. However, if it is easy to locate works dedicated to the analysis of the author and his theory, the same does not occur when our interest is researching the specificity of Vianna’s political activities between 1932 and 1940. * Doutorando no PPG em Ciência Política da Universidade Federal de São Carlos (UFSCar, São Carlos, SP, Brasil) <buthjaum@yahoo.com.br>. Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 178 Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 In this article, I will present, punctually, Vianna's positions about the introduction of 3,000 Chinese in Brazil. Introdução Oliveira Vianna fez inúmeras incursões ao ambiente mais estrito do estado. Uma delas liga-se com a participação do intelectual na construção do anteprojeto de Constuinte, de 1933, denominada de Comissão do Itamaraty. E outra ao cargo de Ministro do Tribunal de Contas da União exercido brevemente por ele em 1940. Além disso, como será analisado neste artigo, a ação de Vianna como consultor jurídico do Ministério do Trabalho, Indústria e Comércio, desenvolvida entre os anos de1932 e 1940. A atividade de consultor é relevante na trajetória do autor em função do contexto histórico em que se abriga: a etapa de consolidação das regras de regulamentação e organização do mundo do trabalho no Brasil. De modo geral, Oliveira Vianna já foi estudado por meio de sua interpretação acerca da sociedade brasileira (Carvalho, 1991; Dória, 1993; Paim, 1993; Madeira, 1993; Ianni, 1993; Bastos, 1993; Iglesias, 1993; Queiroz, 1975); são basilares as análises acerca da dimensão do pensamento vianniano que ratifica o caráter “plástico” e inorgânico do Brasil e de seu povo (Odália, 1993; Brandão, 2007; Queiroz, 1975); foi revisitado no sentido de compreender as raízes de seu pensamento (Lippi Oliveira, 1993; Bresciani, 2007; Carvalho, 2004; Bittencourt, 2011; Fontana, 2013); já foi um autor estudado como um dos principais defensores de um estado forte e interventor no Brasil (Vieira, 1976; Gomes, 1993; Moraes, 1993; Odália, 1997; Gomes, 2009; Brasil Jr., 2010; Queiroz, 1975); já foi problematizado no sentido de ser alinhado a uma tradição/corrente ibérica de pensamento no Brasil (Vianna, 1993; Vianna, 2004); e, por fim, verificamos que já foi pesquisado tanto na chave longa (revisitado no conjunto de sua obra) (Faria, 1993; Bresciani, 2007) quanto na chave curta (examinado por meio de estudos específicos de sua autoria) (Wehling, 1993; Bittencourt, 2011; Fontana, 2013). Todavia, há uma questão importante na trajetória de Oliveira Vianna que o indica, assim F. Fontana – Pontos de inflexão para a análise do pensamento vianniano 179 como outros expoentes de nosso pensamento social e político, como um intelectual híbrido que, para além de analisar, compreender e interpretar o Brasil, foi um statemaker. Considerando o intercâmbio de Oliveira Vianna entre a intelectualidade e a vida pública brasileira, a inquietação que se impõe é a seguinte: qual o nível de unidade entre o intelectual que pensou o Brasil e o homem de estado que propôs soluções legais e pragmáticas para se transformar o país? 1 Essa informação foi obtida por meio de um trabalho de campo – digitalização e posterior catalogação documental – realizado pelo autor no Museu Casa de Oliveira Vianna. 2 Ver Mannheim e o conceito de intelligentsia (1976) e Gomes (1993) com a noção de statemaker. 3 As lições de Karl Mannheim presentes na obra Ideologia e utopia (1976) auxiliam em grande medida na compreensão destes dois movimentos, quais sejam: 1) o vínculo profundo entre as condições materiais da vida e a emergência de um dado conhecimento; 2) o modo como um conhecimento determinado retorna à realidade que o inspirou, ensejando assim, mudanças sociais e políticas. Introdução A resposta para essa indagação é complexa e exige estudo longo e detido; todavia, buscaremos neste artigo trazer algumas pistas para dinamizar ainda mais o debate intrínseco a essa questão. Para isso, lançaremos mão de um único parecer, dos milhares produzidos por Vianna,1 que trata do exame do autor acerca da vinda de 3.000 trabalhadores chineses ao nosso país. 1 Essa informação foi obtida por meio de um trabalho de campo – digitalização e posterio catalogação documental – realizado pelo autor no Museu Casa de Oliveira Vianna. 2 V M h i i d i lli i (1976) G (1993) ã d k O momento no qual Vianna edificou seu trabalho político O contexto referente ao Primeiro Governo Vargas é extremamente importante para compreendermos em quais condições sociais e políticas estava imerso Oliveira Vianna no momento em que ele desenvolvia sua atividade política. Não por acaso, esse período da história brasileira é colocado como aquele que possibilitou a consolidação de um pensamento de cunho autoritário em nosso país (Lamounier, 1985; Santos, 1978; Vieira, 1976; Fausto, 2001). Dessa forma, revisitar esse momento de nosso passado pode auxiliar na compreensão das questões existenciais e extrateóricas que alimentaram a emergência e a estruturação de um conjunto de influentes intelectuais2 formadores de conhecimentos determinados que tinham o propósito de modificar a realidade social e política brasileira desse período.3 Oliveira Vianna, especificamente, é entendido por nós, assim como por muitos outros estudiosos, como um destes intelectuais que pensaram o Brasil de acordo com suas especificidades e que propuseram medidas que requeriam a existência de um tipo de estado forte e interventor. O período circunscrito ao Primeiro Governo Vargas inicia-se com fortes transformações originadas pela Revolução de 1930 e com a complexa tarefa de lidar com a ascensão das classes, a agudização da questão social, as tensões políticas e ideológicas (vide organização do campo comunista, do integralismo, 180 Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 da radicalização de experiências corporativistas) e a conformação de uma relação nova entre sociedade e estado baseada no protagonismo deste último (Sallum, 2003). Nesse período, houve o fortalecimento do papel e da função a serem exercidas pelas políticas públicas – em especial aquelas dirigidas à reorganização da vida pública no amplo arco das ações da matriz centralizadora associada ao modelo do Governo Vargas (Draibe, 1985; Lamounier, 1985). Assim, um projeto minimamente conformado se delineava tendo como amparo o arranjo do idealismo orgânico, ora pautado na ideia da inorganicidade da sociedade brasileira, como na tese de Vianna em O idealismo da Constituição, ora na percepção de que a sociedade civil não estaria apta, pela experiência oligárquica da Primeira República e/ou pelo surgimento de uma teoria mais sólida sobre o atraso com bases no modelo econômico primário-exportador como obstáculo ao desenvolvimento nacional, a gerir a construção do futuro. 4 O MTIC – criado em 26 de novembro de 1930 – tinha, como primordial função, regular os conflitos inerentes à relação entre o capital e o trabalho. Observamos, com sua fundação: 1) a regulamentação dos sindicatos; 2) a criação das Comissões de Conciliação (futura Justiça do Trabalho – 1941); 3) a constituição de importantes avanços vinculados à legislação e aos direitos trabalhistas; 4) a constituição de uma hermenêutica jurídica capaz de orientar as relações profissionais (Garcia Neto, 2010, p. 248). O momento no qual Vianna edificou seu trabalho político Desta configuração e deste panorama contextual ressaltarão três consequências: a primeira é sobre a tarefa do estado de orientar a mudança social, lembrando que as ferramentas mais estratégicas são dadas pela fixação de normas, leis e diretrizes; a segunda, de fazer essa reorganização da vida social com base em algum tipo de diagnóstico e, simultaneamente, de prognóstico (balizas inevitá- veis às mudanças sociais); terceiro, o enfrentamento de questões percebidas como centrais nesse processo de transformação e modernização, fixada em um projeto nacional. Profundamente ligado a isso, destacamos que na produção de diagnósticos e de prognósticos surgiriam dois atores políticos importantes: os intelectuais (entendidos como intelligentsia) e os policymakers (atores dentro do aparelho do estado, capazes de utilizar os capitais estatais para mudança social). O forte nexo entre intelectuais e vida pública no Brasil, destacado por autores como Pécaut (1990) e Brandão (2001), teria aí sua justificação. Assim, nesse contexto surgiria, também, o enfrentamento das questões sociais e da regulação do mundo do trabalho, essenciais na passagem e na edificação de uma sociedade com feições industriais e urbanas (Gomes, 1978; Vianna, 1978). De 1930 até 1934, Getúlio Vargas governou por decreto como Chefe do Governo Provisório. Nesse período, mesmo sob o exercício do poder quase ilimitado do Presidente e a pouca autonomia dos estados brasileiros, criou-se no Brasil novos ministérios, como, por exemplo, o Ministério do Trabalho, Indústria e Comércio (MTIC)4 e o Ministério da Educação e F. Fontana – Pontos de inflexão para a análise do pensamento vianniano 181 Saúde. Além disso, nesse momento de nossa história, edificou-se a Lei da Sindicalização e importantes leis de trabalho (vistas como avanços, mas também como mecanismos fundamentais e germinais para a construção de um populismo no Brasil [Vianna, 1978, p. 141]). Mesmo diante da centralização exacerbada do poder exercida por Getúlio Vargas e da utilização de medidas consideradas antidemocráticas, o Primeiro Governo Vargas legou ao Brasil importantes avanços, principalmente quando pensamos na construção de um Brasil moderno. É inegável que, ao passo que desmantelava poderes estaduais e fortalecia o governo central, o Presidente Getúlio Vargas e seu staff enfraqueciam as elites e as oligarquias locais existentes no Brasil. Na medida em que colocava o estado como o principal propulsor do desenvolvimento econômico e industrial, o governo de Getúlio Vargas promoveu no país um crescimento econômico e industrial nunca antes experimentado pela nação brasileira. O momento no qual Vianna edificou seu trabalho político Além disso, o sufrágio universal e a consolidação de importantes leis trabalhistas são exemplos de avanços no âmbito dos direitos sociais e políticos alcançados no período em que Vargas comandou o país. Essa mescla entre ações e intervenções antidemocráticas de um lado, e conquistas sociais, políticas e econômicas de outro é uma característica ímpar desse momento da história brasileira (Sola, 1968, p. 282). ( p ) Considerando este panorama contextual, faz sentido propor a análise da produção intelectual de Vianna que esteve diretamente ligada ao cerne das novas tarefas do estado: a regulamentação da esfera do Direito do Trabalho e a regulação sindical (tema que contrapôs duas “escolas” como a de Vianna – sindicalismo único – e a de Evaristo de Moraes Filho – sindicalismo livre [Brasil Júnior, 2007, p. 93-94]). Pesa também o fato de Vianna possuir mais que uma visão jurídica formal; ao contrário, ele foi um dos mais importantes intelectuais do período que lidaram com a interpretação da formação nacional, possuindo uma teoria social e política de fôlego sobre as características particulares da trajetória brasileira com base na análise do território, povo, atores, cultura, economia e instituições. Sendo assim, pode-se perguntar: quais os nexos que caracterizam o momento de encontro entre o intelectual e o consultor jurídico? Lidando com a percepção de forte vínculo entre texto e contexto histórico, entre autor e ator, que liames – enquanto continuidades e mudanças – aparecem no “ponto” consultor jurídico do Ministério do Trabalho e no “sistema” de interpretação do intelectual fluminense? Embora a leitura e o tratamento do legado intelectual de Vianna sejam vastos, não se sabe precisamente quais as características da produção do autor no exercício da função de consultor jurídico e, principalmente, quais os nexos entre a postura adotada por ele e o conjunto das matrizes de seu pensamento. Considerando esse Considerando este panorama contextual, faz sentido propor a análise da produção intelectual de Vianna que esteve diretamente ligada ao cerne das novas tarefas do estado: a regulamentação da esfera do Direito do Trabalho e a regulação sindical (tema que contrapôs duas “escolas” como a de Vianna Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. O momento no qual Vianna edificou seu trabalho político 2017 182 dado pouco explorado, este artigo propõe investigar um parecer específico de Oliveira Vianna com fins de dimensionar, exploratoriamente, a especificidade de seu trabalho como consultor jurídico, contribuindo parcialmente assim, para a análise de nossa trajetória estadocêntrica ou de estadania (Santos, 1998; Carvalho, 2007). O Brasil de Vianna: insolidarismo e raça e assimilação Uma valiosa questão presente no pensamento vianniano vincula-se com a crítica que o intelectual estabelece em relação aos legados negativos de nosso passado colonial e os meios pelos quais seria possível transformar/ modernizar o Brasil. Vinculado a este debate são articulados e teorizados por ele alguns relevantes temas e questões, como por exemplo: a particularidade da formação do Brasil enquanto nação; a especificidade do período monárquico no Brasil; a peculiaridade e a relevância do corporativismo na nação brasileira; a questão racial brasileira; os atributos distintivos de um direito do trabalho, de uma legislação trabalhista e de um direito corporativo para o Brasil; as inconsistências da Constituição Brasileira de 1891; as características constitutivas de nossas instituições políticas; e a especificidade e a tipologia do poder central (estado) necessário e congruente à modernização brasileira. Para a análise do parecer em questão, utilizaremos dois temas relevantes intrínsecos ao pensamento vianniano: a noção de insolidarismo cunhada pelo autor (interpretação sobre a formação do Brasil) e suas ideias acerca da questão racial em nosso país. Populações meridionais do Brasil é o estudo clássico de Oliveira Vianna que edifica uma interpretação acerca da formação da sociedade brasileira e, consequentemente, dos caracteres que a marcaram indelevelmente como um povo sui generis. A análise do Brasil Colônia feita pelo sociólogo brasileiro nos ajuda a perceber uma relação de continuidade existente entre as formas morfológicas brasileiras e o tipo de atividade econômica presente na colônia (Vianna, 1938, p. 148). Dessa forma, para o autor, cria-se no Brasil um tipo específico de sociedade, a qual tem como eixo condutor o latifúndio (Vianna, 1938, p. 150). Pode-se dizer, segundo Vianna, que o absenteísmo urbano é uma lógica da formação social brasileira. No Brasil Colonial, houve uma autonomia exagerada do latifúndio que, por sua vez, impediu que o país caminhasse rumo à urbanização e modernidade. Aqui, em um dado momento da colonização, a retirada de riquezas feita pela metrópole era efetivada através da exploração da terra, dessa maneira, os investimentos nacionais ligavam-se exclusivamente com o desenvolvimento dos latifúndios e das atividades rurais. Assim, a sociedade colonial brasileira é caracterizada por ter profundas F. Fontana – Pontos de inflexão para a análise do pensamento vianniano 183 raízes rurais, as quais dificultaram a edificação de nossos conglomerados citadinos, zonas urbanas ou cidades. O Brasil de Vianna: insolidarismo e raça e assimilação Desta forma, adverte o autor, os grupos sociais presentes nas cidades estariam presos ao poder dos latifundiários, não possuindo assim, um “espírito corporativo”, o que constitui um problema, pois não há a construção de corporações com “solidariedade moral”. No Brasil de Vianna, a insolidariedade ou o insolidarismo é um traço marcante que, por conta de nossas especificidades formativas, atinge tanto dimensões sociais, quanto culturais, econômicas e políticas. Em relação à questão racial no pensamento vianniano e acerca das teorizações presentes na obra Raça e assimilação (1932) observamos que, se por um lado, o enclave da raça não é de fundamental importância para se compreender as principais ideias e interpretações de Vianna sobre o Brasil, por outro, ao passo que o intelectual fluminense ainda em 1932 perpetuava e ratificava paradigmas racistas e eugênicos, ele contribuía em muito para o atraso da ciência e, em especial, das humanidades em nosso país. Além disso, um ponto central de nossa argumentação liga-se com a percepção de que as ideais do autor sobre a dinâmica racial brasileira eram um produto do momento histórico no qual ele estava imerso e, sobretudo, do ambiente intelectual e do contexto de produção do conhecimento no qual o pensador fluminense se localizava. Raça e assimilação é um estudo síntese, produto da junção de dois trabalhos que Oliveira Vianna pretendia publicar, quais sejam: O ariano no Brasil e Antropologia social. Nesta obra, ele buscou, levando em consideração a sua percepção do que é antropologia social, realizar um estudo de cunho antropológico no Brasil. Dentre as muitas posições de Oliveira Vianna defendidas nessa obra, destaca-se a percepção de que a América Latina é um excepcional laboratório de investigação para os cientistas sociais interessados no estudo das raças justamente porque nessa região houve uma efetiva mistura de distintas matrizes raciais. Adicionado a isso, o intelectual brasileiro ratifica, ao passo que investiga uma vasta e, à época, canônica bibliografia sobre as tipologias raciais, psicofisiologia, psicometria e nosologia, uma série de ideias ligadas com a percepção de que é possível determinar através do entendimento das raças e dos tipos físicos dos indivíduos, “quais as suas predisposições patológicas, quais as modalidades prováveis de seu temperamento e da sua inteligência” e quais tipos sociais e humanos seriam mais evoluídos, inteligentes e aptos para a realização de determinadas atividades, tarefas, trabalhos e funções (Ramos, 2003, p. 586). O Brasil de Vianna: insolidarismo e raça e assimilação Segundo Vianna, a concretizada visão de que no Brasil só haviam brancos, caboclos, negros e pardos escondia um conjunto heterogêneo de raças. Somado a isso, ele também afirma que o branco Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 184 brasileiro se difere daqueles presentes na Europa, afinal, o clima tropical e as condições geográficas do Brasil requereram adaptações profundas. Oliveira Vianna também engendra às suas discussões o “problema dos imigrantes” como um fator de complexificação da questão racial no Brasil e refuta a tese de que a miscigenação tenderia à constituição de um tipo único racial brasileiro. Para ele, a miscigenação levaria à maior complexificação das raças na nação, consequentemente, aumentaria o número de indivíduos degradados e inferiores em nosso país. Os pressupostos eugenistas e raciais de Oliveira Vianna ligavam-se, no ambiente nacional, às ideias de Oswaldo Cruz e Carlos Chagas; já no cenário internacional, relacionava-se às contribuições de Gunther, Lapouge, Gobineau, Amon, Woltmann, Sigaud, Pende, Colajanni, entre outros. Os principais autores que contribuíram com Vianna em sua leitura acerca do vínculo entre a raça e o meio físico foram os ecologistas e antropogeógrafos Glenn Trewartha, Emory Ross, Griffith Taylor, Mark Jefferson, Dunlop e Joyeux (Ramos, 2003, p. 590). É relevante notarmos que todos esses pressupostos teórico-metodológicos voltados à compreensão dos, até então, dilemas raciais e eugênicos estudados por Vianna em Raça e assimilação não aparecem, segundo nossa percepção, de modo contundente na argumentação geral do autor sobre nossa formação como povo em suas principais obras (não aparecem enfaticamente nas obras anteriores à publicação de Raça e assimilação e muito menos em obras posteriores à 1932). Por exemplo, Vianna não faz, em Populações meridionais do Brasil, uma sistematização profunda das tipologias raciais presentes no Brasil e as consequências das misturas raciais aqui existentes. Para o autor, a edificação do latifúndio e, sobretudo, os aspectos geográficos, morfológicos e estruturais da sociedade brasileira – condição de colônia, autossuficiência, características do latifúndio e economia agroexportadora – foram os condicionantes primordiais dos nossos problemas formativos. Raça e assimilação, segundo nossa visão, foi publicada em um mo- mento ímpar da intelectualidade brasileira que se preocupava com o tema da raça no país e com as dinâmicas raciais aqui estabelecidas. O Brasil de Vianna: insolidarismo e raça e assimilação De modo geral, os trabalhos sobre a formação do Brasil publicados após 1932 que, dentre outras cosias, discutiam a questão racial no país tinham um viés altamente culturalista e realizavam enfáticas críticas às teorias racistas e eugênicas disseminadas até então, promovendo assim incisivas inflexões nos estudos, nas interpretações e nas teorias sobre a formação do Brasil. Um emblemático exemplo dessa mudança de perspectiva adotada pela intelectualidade brasileira é a publicação de Raça e assimilação em 1932 (ratificação de uma série de F. Fontana – Pontos de inflexão para a análise do pensamento vianniano 185 teses racistas e eugenistas) e Casa grande & senzala, de Gilberto Freyre, em 1933 (obra que, através de um viés claramente culturalista, exprimia um afastamento face às teses eugênicas e raciais). Segundo Jair de Souza Ramos (2003, p. 599): O racismo científico vinha perdendo terreno também nas interpretações histórico-sociológicas sobre o desenvolvimento nacional tanto a partir do surgimento de interpretações como a de Gilberto Freyre, que consolidavam a imagem de cooperação entre as raças, quanto em movimentos como o sanitarista, que davam destaque à doença, e não só na origem racial, para explicar a situação de atraso do trabalhador nacional. O racismo científico vinha perdendo terreno também nas interpretações histórico-sociológicas sobre o desenvolvimento nacional tanto a partir do surgimento de interpretações como a de Gilberto Freyre, que consolidavam a imagem de cooperação entre as raças, quanto em movimentos como o sanitarista, que davam destaque à doença, e não só na origem racial, para explicar a situação de atraso do trabalhador nacional. Após a publicação de Raça e assimilação, a questão racial não desaparece completamente do pensamento vianniano. No estudo Instituições políticas brasileiras, lançado em 1949, o intelectual chega a afirmar que para ele, após seus muitos anos de pesquisa, o método mais eficiente e avançado capaz de ensejar estudos no campo do direito público no Brasil deve considerar três relevantes dimensões de uma sociedade: sua cultura, suas determinações físicas (meio) e os seus aspectos raciais. No entanto, o tom no qual se insere a questão da raça é outro – e ela está subordinada ou equalizada, enquanto fonte de explicação entendimento da realidade social, às condições materiais (morfológicas e geográficas), culturais, sociais, econômicas e políticas nas quais se insere um determinado povo: Em suma, o quadro clássico dos ‘fatores da civilização e da história’ se está restaurando. O Brasil de Vianna: insolidarismo e raça e assimilação Em vez de uma causa única – meio só (Buckle), ou raça só (Lapouge), ou cultura só (Spengler, Frobenius, Boas) – a ciência confessa que tudo se encaminha para uma explicação múltipla, eclética, conciliadora: raça + meio + cultura. Com estes elementos é que ela está recompondo o quadro moderno dos ‘fatores da civilização’ [1949] (Vianna, 1999, p. 90). Adicionado a isso, notamos que Vianna, neste trabalho de 1949, não faz explicações profundas sobre as particularidades de nossas instituições políticas, ou ainda, acerca das especificidades do povo brasileiro, tendo como fonte principal determinados pressupostos raciais ou eugênicos. Longe disso, notamos que boa parte da argumentação do intelectual fluminense está amparada em seu diagnóstico sobre a formação do Brasil edificado em Populações meridionais do Brasil e, em determinados momentos, na sua leitura, mesmo que indireta, de específicos períodos da história brasileira, como é o caso de nosso período monárquico (em O ocaso império – 1925) e da nossa Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 186 Primeira República (em O idealismo da Constituição – 1927). Outro aspecto que podemos apreender através de Instituições políticas brasileiras para compreendermos como se deu a continuidade da questão racial no pensamento vianniano após a publicação de Raça e assimilação é a ambiguidade que o tema da raça ganha nas ideias de Vianna. Tal imprecisão, distante de evidenciar uma contradição interna aos escritos e ao pensamento do autor, expressa, antes de tudo, o próprio momento em que ele estava inserido e os novos paradigmas científicos amplamente aceitos com os quais ele foi obrigado a dialogar. Em 1949, o descrédito com relação às teses raciais era imenso, assim como era marginal e contraproducente a utilização de argumentos eugênicos para o entendimento das especificidades de uma nação ou de um povo (cf. Vianna, 1999, p. 77 [1949]). Nesse movimento complexo de abandonos, resquícios e permanências de temas, de conceitos e de paradigmas é que a questão racial se insere no pensamento vianniano pós-publicação de Raça e assimilação, revelando assim, algumas ambiguidades e imprecisões. Por exemplo, se por um lado Oliveira Vianna (1999, p. 404-405 [1932]) continuaria a afirmar que a questão racial é importantíssima para ele entender “nossa civilização e em nossa história”, por outro ele já havia reconhecido em 1934 que a raça não tinha assumido um papel crucial em sua produção bibliográfica. O Brasil de Vianna: insolidarismo e raça e assimilação Segundo Oliveira Vianna, citado por Vasconcelos Tôrres, no Prefácio à segunda edição da obra A evolução do povo brasileiro (1923): Dando uma explicação definitiva aos opositores apressados, comenta o sociólogo: ‘Tendo concentrado a sua análise exclusivamente sobre este tema (cujo desdobramento no livro, ocupa menos de meia dúzia de páginas), os críticos acabaram dando a impressão, aos que acostuma ler críticas dos livros, mas não os livros criticados, de que todo o volume de Evolução do povo brasileiro – 1923 – havia sido exclusivamente consagrado à sustentação dessa tese temerária. Em certo momento, acabei mesmo passando por ter escrito uma obra volumosa para expor e defender, no Brasil, a tese da superioridade da raça germânica... Pura obra crítica insincera ou desonesta, como se vê. Daí uma viva reação – e a atoarda foi grande. O único, entretanto, que não deu quase nenhuma significação à crítica feita fui eu mesmo; porque sempre considerei este tema como um ponto secundário e insignificante, que não valia aos meus adversários perderem tanto tempo em combatê-lo, nem a mim em defendê-lo’ (Vianna apud Tôrres, 1956, p. 82-83). Ou ainda, já sentindo que nadava contra a corrente, no Prefácio à primeira edição de Raça e assimilação, Vianna faz uma afirmação que, no mínimo, traduz uma preocupação em resguardar a legitimidade de sua pesquisa, ideias F. Fontana – Pontos de inflexão para a análise do pensamento vianniano 187 e interpretações em um contexto de produção intelectual arisco às hipóteses que ali perpetuaria: Formulo apenas algumas hipóteses – e as pesquisas dos técnicos vão dizer se são ou não verdadeiras. Pertencem, na maioria, ao grupo daqueles working hypothesis, de que fala Handdon, isto é, sujeitas ainda a referendum dos investigadores. Estas hipóteses, aliás, não causam mal nenhum à ciência: são estímulos para o trabalho, são sugestões para a pesquisa. Mesmo que se verifiquem erradas, a ciência lucrará com elas (Vianna, 1959, p. 7). Formulo apenas algumas hipóteses – e as pesquisas dos técnicos vão dizer se são ou não verdadeiras. Pertencem, na maioria, ao grupo daqueles working hypothesis, de que fala Handdon, isto é, sujeitas ainda a referendum dos investigadores. Estas hipóteses, aliás, não causam mal nenhum à ciência: são estímulos para o trabalho, são sugestões para a pesquisa. Mesmo que se verifiquem erradas, a ciência lucrará com elas (Vianna, 1959, p. 7). A questão racial no pensamento vianniano é extremamente complexa e difícil de ser acessada. O Brasil de Vianna: insolidarismo e raça e assimilação Aqui, tentamos fazer uma breve aproximação no sentido de apresentar este tema e problematizá-lo, para assim verificar o quanto ele está afeito a influências decorrentes de mudanças históricas e alterações no contexto de produção no qual o autor estava inserido. Nesse sentido, tivemos a oportunidade de verificar o dilema circunscrito à transitoriedade de ideias, de conceitos e de paradigmas que influi no pensamento do intelectual niteroiense, provocando dessa forma, descontinuidades, ambiguidades e descompassos. Além disso, ao passo que apresentamos as percepções do autor sobre a questão racial no Brasil, verificamos como tais incursões analíticas não afetaram de modo efetivo e profundo algumas canônicas ideias e interpretações do autor sobre o Brasil, suas instituições políticas e o povo brasileiro. A imigração de 3.000 chineses sob a ótica de Oliveira Vianna Passaremos agora à análise do parecer técnico encaminhado por Oliveira Vianna, através do Ministério do Trabalho, Indústria e Comércio, ao Ministério das Relações Exteriores que, por sua vez, trata da integração de 3.000 chineses ao território brasileiro. O parecer não possui data, mas certamente foi produzido entre os anos de 1932 e 1940. De modo geral, temas vinculados à noção de insolidariedade e à questão racial no Brasil aparecerem no parecer e nos ajudam a dinamizar e a complexificar ideias, paradigmas e posições presentes no pensamento vianniano. Seguem alguns trechos desse parecer que, por sua vez, serão reproduzidos de modo a manter e respeitar a linguagem e a gramática adotada à época de Oliveira Vianna: O que propõe o Sr. Consul de China sobre a introdução em nosso paiz de 3.000 chinezes pode ser encarado sob vários aspectos: O legal; b) O biológico; c) O econômico e social; d) O ethnograhico e politico. Do ponto de vista legal, é forçoso confessar que a nossa legislação não nos arma de meios para repellir a immigração, nem de 188 Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 chinezes, nem de qualquer raça. [...] Em relação a China, ha mesmo um decreto espacial autorisando a introdução dos seus nacioneses aqui – e é justamente o invocado pelo capitalista Sr. Chin-Dong- Tai. De modo que, em boa verdade, não temos meios para impedir a entrada destes chinezes aqui (Vianna, s. d., p. 1). Verificamos, nessas primeiras palavras, que o fator preponderante considerando por Oliveira Vianna liga-se à dimensão política, ratificando assim, a atual posição e função que ocupa: consultor jurídico do Ministério do Trabalho, Indústria e Comércio. Nessa direção, a lei, sua viabilidade e sua aplicabilidade são os paradigmas centrais da avaliação construídos por Vianna nesse parecer. Dessa forma, o jurista claramente busca se afastar da posição que sempre assumiu: a de intelectual. Sendo a lei falha ou inexistente, não há impedimentos ou imposições à entrada dos chineses em questão ao Brasil. Todavia, o restante do parecer mostra que esse afastamento inicial não é efetivo e muito da sua leitura sobre o Brasil pode ser identificada em suas palavras. A imigração de 3.000 chineses sob a ótica de Oliveira Vianna Quanto aos aspectos biológicos latentes ao tema da imigração chinesa, Vianna dirá que: Do ponto de vista da aclimatação, não ha que duvidar de adaptabilidade do chinez ao nosso meio nordestino: o mongol, é sabido, é de todos os typos humanos o que offerece uma capacidade maior de adaptação a todos os climas, vivendo no equador tão bem como vive no polo ártico. Quanto a questão do eugenismo ou cacogenismo dos cruzamentos entre elles e os nossos typos nordestinos, não creio que possam decerrer inconvenientes destes cruzas, dada a grande afinidade anthropologica entre mongol e o nosso caboclo. [...] Por este lado, pois, não me parece que possamos encontrar grande base para refugar a immigração chineza (Vianna, s. d., p. 2). Nas palavras acima, verificamos uma análise de Vianna pautada em pressupostos raciais e antropogeográficos (morfológicos) que ratificam leituras eugênicas sobre os nordestinos brasileiros e acerca dos chineses que para o Brasil poderiam imigrar. Para o jurista, os tipos raciais, nordestinos e chineses, não se diferenciam significativamente, assim, uma negativa para a imigração não poderia se pautar em tais paradigmas. Por outro lado, não creio que a repulsa à immigração chineza possa ser collocada, como foi pelo Sr. Director do Departamento e o Sr. Director de Secção, sobre a base do padrão de vida, principalmente tratando-se de núcleos ruraes. [...] Somos num paiz de baixo padrão de vida e onde o homem do campo – o nosso Geca – não tem um F. Fontana – Pontos de inflexão para a análise do pensamento vianniano 189 modo de existencia melhor e mais alto do que o japonez ou o chinez. Caipiras, matutos, sertanejos, todos são uma raça de homens sobrios até o maximo das possibilidades humanas – e os baixos, os baixíssimos salarios do nosso operario agricola, especialmente no Norte, bem demonstram a limitação das exigencias de vida do nosso trabalhador. O chinez sob esta aspecto, não víria trazer-lhe nenhuma concorrencia desfavoravel. [...] O inconveniente dos asiáticos como colonos está senão unicamente, ao menos principalmente nisto: na sua infusibilidade (Vianna, s. d., p. 2-3). Em termos de padrões socioeconômicos, Vianna também não realiza um parecer desfavorável à vinda destes trabalhadores chineses ao Brasil. Segundo ele, o padrão médio de vida do trabalhador rural brasileiro não estaria distante daquele experimentado pelos chineses. A imigração de 3.000 chineses sob a ótica de Oliveira Vianna Todavia, sua grande preocupação articula-se à predisposição à infusibilidade que alguns povos, assim como os orientais, apresentam no momento que migram para outros territórios. Disse naquelle livro acima citado [Raça e assimilação] que o japonez é como o enxofre: insoluvel. O mesmo podemos dizer do chinez: é tambem insoluvel. O proprio syrio-arabe, mais sociavel e diffusivo, não deixa de ser tambem infusível – e os dados estatísticos e, sinão elles, a observação vulgar bem o comprovam esta infusibilidade. [...] Dahi a tendencia dos enthnios semites ou mongolicas, no sentido da ‘ilha ethnica’, isto é, para o ‘enkistamento’, com todos os inconvenientes sociais e políticos dahi decorrentes. [...] O problema da desintegração das ethnias asiaticas e da sua assimilação não é, entretanto, o mesmo quando o encaramos sob o aspectos das colonias agrícolas o do seu estabelecimento rural. Nossa politica tem sido, neste ponto, imprevidente – e temos commettido a leviandade de adoptarmos o methodo da colonisação homogenea, como já fizemos como os allemães em Santa Catharina e como estamos fazendo, ainda mais levianamente, com os japonezes na Amazonia, onde estamos concentrando uma massa enorme de colonos exclusivamente niponicos numa região inteiramente despovoada, sem possibilidade nenhum de qualquer especie de contacto com os nucleos de população nacional, que alli, no caso, praticamente, é como se não existissem. Tendo errado como os allemães ao sul, estando a errar com os japonezes ao centro e ao norte do paiz, seria absurdo que continuassemos a errar com os chinezes, que se nos offerecem. É preciso que, aceitando-os, deixando-os entratarem e fixarem-se no nosso territorio, tomemos as providencias que a nossa experiencia secular nos está insinando. [...] O problema, pois, será este: impedir ou corrigir a infusibilidade dos futuros colonos (Vianna, s. d., p. 3-4). 190 Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 Nas palavras acima, podemos verificar uma preocupação de Vianna que não está distante da interpretação mais indelével impressa em seu pensamento: seu diagnóstico sobre a formação do Brasil e o consequente problema do insolidarismo. Não por acaso, o autor ressalta a dimensão desagregativa ainda existente na nação brasileira, em especial nas regiões localizadas ao Norte de nosso país. A imigração de 3.000 chineses sob a ótica de Oliveira Vianna Nesse sentido, a integração de 3.000 mil chineses ao território nacional poderia ampliar, assim como já ocorreu na Amazônia com os japoneses e em Santa Catarina com os alemães, o caráter insolidário, amorfo e difuso que marca a construção do Brasil. Tais preocupações de Vianna pautam-se em visões culturais e sociológicas – tanto sobre a população brasileira, quanto em relação ao povo chinês – que são conservadas pelo jurista e veiculadas densamente neste parecer. Como? Para mim, por este meio: condicionando a concessão a uma clausula, que impeça a formação homogenia da população colonial, isto é, obrigando á empreza a não admitir mais de 50% de elementos chinezes em cada colonia. Os outros 50% restantes da população colonial seriam formados de elementos nacionaes, de preferencia, ou então, de colonos europeus. Para maior garantia do processo assimilador, poderíamos estabelecer tambem uma outra clausula – e é: que, na distribuição dos lotes ou das habitações, à empreza se obrigaria a intercalar, tanto quanto fosse possivel, entre os lotes ou habitações que deveriam obrigatoriamente ser concedidas a nacionaes ou, as falta destes, a colonos europeus, especialmente portuguezes, hespanhões ou italianos. Estas duas medidas obrigariam, penso, facilmente esta tradicional inassimilabilidade dos colonos chinezes. Sujeitos assim a esta assimilação forçada e intensiva, elles poderiam trazer uma contribuição apreciavel a nossa actividade economica, sem os inconvenientes dos “enkistamentos”. [...] Estes elementos vindos dos centros urbanos, não servem – e, ai aqui forem introduzidos, serão, dentro em pouco, um peso morto, embaraçante do nosso progresso. Só os chinezes de pura formação agricola, o chinez vindo dos campos, é que nos serviriam. O chinez é um dos maiores e mais perfeitos agricultores do mundo: na cultura dos cereses, especialmente arroz, são exímios e tem disso uma tradicção do milennios. Em nosso meio tropical, poderiam trazer a sua experiencia antiga e ser excellentes cultivadores de milho, de arroz o de algodão, culturas, proprias ao meio nordestino, nos seus sertões e nos seus brejaes (Vianna, s. d., p. 4-6). Como? Para mim, por este meio: condicionando a concessão a uma clausula, que impeça a formação homogenia da população colonial, isto é, obrigando á empreza a não admitir mais de 50% de elementos chinezes em cada colonia. Os outros 50% restantes da população colonial seriam formados de elementos nacionaes, de preferencia, ou então, de colonos europeus. A imigração de 3.000 chineses sob a ótica de Oliveira Vianna Para maior garantia do processo assimilador, poderíamos estabelecer tambem uma outra clausula – e é: que, na distribuição dos lotes ou das habitações, à empreza se obrigaria a intercalar, tanto quanto fosse possivel, entre os lotes ou habitações que deveriam obrigatoriamente ser concedidas a nacionaes ou, as falta destes, a colonos europeus, especialmente portuguezes, hespanhões ou italianos. Estas duas medidas obrigariam, penso, facilmente esta tradicional inassimilabilidade dos colonos chinezes. Sujeitos assim a esta assimilação forçada e intensiva, elles poderiam trazer uma contribuição apreciavel a nossa actividade economica, sem os inconvenientes dos “enkistamentos”. [...] Estes elementos vindos dos centros urbanos, não servem – e, ai aqui forem introduzidos, serão, dentro em pouco, um peso morto, embaraçante do nosso progresso. Só os chinezes de pura formação agricola, o chinez vindo dos campos, é que nos serviriam. O chinez é um dos maiores e mais perfeitos agricultores do mundo: na cultura dos cereses, especialmente arroz, são exímios e tem disso uma tradicção do milennios. Em nosso meio tropical, poderiam trazer a sua experiencia antiga e ser excellentes cultivadores de milho, de arroz o de algodão, culturas, proprias ao meio nordestino, nos seus sertões e nos seus brejaes (Vianna, s. d., p. 4-6). Na passagem acima, observamos algumas proposições de Oliveira Vianna quanto à integração destes trabalhadores chineses à realidade brasileira. Sendo assim, destacam-se, segundo o jurista: 1) a necessidade de os mesmos serem “misturados” ou integrados a diferentes tipos culturais (nacionais, português, F. Fontana – Pontos de inflexão para a análise do pensamento vianniano 191 italiano ou espanhol) considerando medidas específicas (integração/mistura laboral entre brasileiros e nordestinos, assim como intercalação de moradias/ habitações entre os diferentes tipos raciais); 2) a importância dos imigrantes chineses conservarem aptidões agrícolas. Mais uma vez, as recomen- dações dadas por Vianna coadunam-se com a necessidade de se evitar enquistamento e de se promover a assimilação destes imigrantes chineses em nosso país. Esta, pois, a primeira condição de seleção immigratoria: que os immigrantes, futuros colonos, seja agricultores e exclusivamente isto. Onde, porém, buscar estes agricultores? Na China, na região de Hong-Kong, ou na America de Norte, como suggere a representação de Sr. Consul da China? [...] O centro unico do rechutamente que nos serve seria os Estados Unidos. Ha alli, na costa californianas, uma densa população do chinezes, toda de agricultores, prospera, rica, progressiva – e já afeiçoada ás condições e praxes de nossa civilasação. seguidas. Por fim, Vianna faz um parecer favorável à integração destes trabalhadores ao território nacional mediante algumas exigências/cláusulas. seguidas. Por fim, Vianna faz um parecer favorável à integração destes trabalhadores ao território nacional mediante algumas exigências/cláusulas. A imigração de 3.000 chineses sob a ótica de Oliveira Vianna Estes agriculteres, que dalli nos viessem, estariam em condições de assimilação mais favoraveis do que outros, vindos de centros chinezes. [...] O que o Consul da China pede é a concessão para a introducção e a localisação de 3.000 chinezes. Notem bem: 3.000 chinezes – e não 3 mil famílias. Ora 3.000 chinezes devem corresponder a umas 600 ou 500 familias. Não vejo, como, desde que sejam, como suggerimos, sufficientemente misturados com os nossos sertanejos nos mesmos nucleos, possam vir elles e constituir o problema ethnico. [...] É uma experiencia que penso podermos tentar sem receio, tanto mais que a empreza que financia a obra colonisadora é poderosamente dotada de recursos pecuniarios – e a introducção de vastas capitaes numa região carecente delles, como o Nordeste, não é causa que se possa desdenhar. Opinaria, pois, para que atendesse a proposta do Sr. Consul Chines, mediante as condições seguintes, satisfeitas, está claro, as exigencias dos nossos regulamentos de imigração e colonização [vide as recomendações dadas anteriormente por Oliveira Vianna] (Vianna, s. d., p. 6-7). Acima, Oliveira Vianna debate sobre a procedência dos chineses que aqui se fixariam. Nessa direção, ele ressalta a necessidade destes chineses conservarem aptidões agrícolas, sendo assim, as regiões de origem mais propícias destes imigrantes deveriam ser aquelas nas quais eles melhormente desenvolveram suas predisposições agrárias. Somado a essa questão, Vianna ressalta a relevância da integração de imigrantes ao nordeste brasileiro, principalmente quando se dimensiona os recursos ali aplicados (originados desse processo de imigração). Além disso, o jurista desataca que o número de chineses é relativamente pequeno, viabilizando assim, um processo de assimilação bem sucedido desde que as recomendações dadas por ele fossem Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 192 seguidas. Por fim, Vianna faz um parecer favorável à integração destes trabalhadores ao território nacional mediante algumas exigências/cláusulas. Conclusão Fontana – Pontos de inflexão para a análise do pensamento vianniano 193 de nosso povo. Contudo, vale destacar, se em um primeiro momento essa questão aparece nas obras de Vianna, neste documento, constatamos que o uso de paradigmas eugênicos e raciais – mesmo que tenha sido feito de modo enfático e constante pelo autor – não conduziu, pelo menos neste caso específico, a um parecer desfavorável à imigração chinesa em nosso país. A visão de Oliveira Vianna acerca da questão racial neste documento, de acordo com nossa avaliação, é tangencial quando temos em mente o seu impacto interpretativo e/ou político-decisório. De fato, as preocupações mais relevantes do jurista brasileiro articulam-se à necessidade de se promover, por meio de medidas e recomendações específicas, a assimilação e a integração do imigrante chinês no Brasil, diminuindo assim, a probabilidade de se acentuar a desagregação, a desintegração e a insolidariedade em nosso país. Ora, combater desagregação, a desintegração e a insolidariedade no país não é um dos motes mais significativos no pensamento vianniano? Conclusão Considerando as análises acima, observa-se que há uma “complexa” relação interseccional entre o intelectual Oliveira Vianna e o consultor jurídico do Ministério do Trabalho, Indústria e Comercial. Nota-se que há uma transposição de ideias circunscritas ao pensamento vianniano ao conteúdo do parecer por nós investigado. Por mais que Vianna tente, logo no início do documento, salientar que as bases legais são as principais condutoras de suas posições, o conteúdo do mesmo, inclusive a divisão temática a que ele obedece, mostra que as preocupações do jurista extrapolam os limites da legislação brasileira vigente que regulava a imigração em nosso país. Nessa transposição do intelectual/pensador para o jurista diretamente ligado à vida pública e política brasileira, notamos que determinadas posições viannianas se colocam de modo mais enfático. Dessa maneira, a visão conservada por ele em relação à questão racial no Brasil, assim como sua leitura sobre o insolidarismo intrínseco à formação brasileira, aparecem de forma mais proeminente, contudo, com desígnios e resultados distintos. Ainda no parecer analisado, deve-se destacar que as posições raciais de Vianna se mostram concatenadas ao momento intelectual de transição pelo qual um conjunto de ideias e ideologias raciais estava passando em nosso país. Ou seja, a década de 1930 constituiu-se como um momento ímpar no qual pressupostos eugênicos perderam força significativa na tarefa de explicar e compreender o Brasil desde a sua formação como povo até a sua condição de nação preocupada com os dilemas da modernização. Se em Raça e assimilação (1932) a questão da miscigenação continuaria sendo um problema e um enclave explicativo importante para Oliveira Vianna, no documento que analisamos ela passa a ser condição necessária à integração de estrangeiros ao território nacional. Essa percepção fica clara ao passo que o jurista fluminense ressalta a necessidade de, diferentemente do que fizeram com os alemães em Santa Catarina e com os japoneses na Amazônia, “fundir” ou promover a “fusibilidade” destes chineses ao povo brasileiro, agenciando assim, um processo distinto – e talvez mais moderno – de assimilação. Para nós, a lógica circunscrita à visão vianniana sobre a raça no Brasil parece obedecer àquela vinculada aos seus estudos. Ou seja, assim como em seus principais trabalhos que não tratam especificamente da questão racial no Brasil, há a projetação de uma leitura diagnóstica sobre Brasil em detrimento da utilização de pressupostos raciais e eugênicos ligados à especificidade F. Referências BASTOS, Elide Rugai. Oliveira Vianna e a Sociologia no Brasil (um debate sobre a formação do povo). In: Élide Rugai Bastos; João Quartim Moraes (orgs.). O pensamento de Oliveira Vianna. Campinas: Unicamp, 1993. p. 405-428. BITTENCOURT, André Veiga. 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VIANNA, Oliveira. Instituições políticas brasileiras. Brasília: Senado Federal, 1999. VIANNA, Oliveira. Populações meridionais do Brasil. 4. ed. São Paulo: Nacional, 1938. VIANNA, Oliveira. Raça e assimilação. 4. ed. Rio de Janeiro: José Olympio, 1959. 196 Civitas, Porto Alegre, v. 17, n. 1, p. 177-196, jan.-abr. 2017 VIEIRA, Evaldo. Oliveira Vianna e o estado corporativo. São Paulo: Grijalbo, 1976. WEHLING, Arno. O estado colonial na obra de Oliveira Vianna. In: Élide Rugai Bastos; João Quartim Moraes (orgs.). O pensamento de Oliveira Vianna. Campinas: Unicamp, 1993. p. 63-86. VIEIRA, Evaldo. Oliveira Vianna e o estado corporativo. São Paulo: Grijalbo, 1976. WEHLING, Arno. O estado colonial na obra de Oliveira Vianna. In: Élide Rugai Bastos; João Quartim Moraes (orgs.). O pensamento de Oliveira Vianna. Campinas: Unicamp, 1993. p. 63-86. WEHLING, Arno. O estado colonial na obra de Oliveira Vianna. In: Élide Ruga Bastos; João Quartim Moraes (orgs.). O pensamento de Oliveira Vianna. Campinas Unicamp, 1993. p. 63-86. Recebido em: 26 jul. 2016 Aprovado em: 17 fev. 2017 Autor correspondente: Felipe Fontana Rua Pioneiro Lourenço Favoreto, 140 – Parque das Grevíleas II 87025-030 Maringá, PR, Brasil Autor correspondente: Felipe Fontana Rua Pioneiro Lourenço Favoreto, 140 – Parque das Grevíleas II 87025-030 Maringá, PR, Brasil
https://openalex.org/W2801330756	https://researchonline.lshtm.ac.uk/id/eprint/4652919/1/Shorter%20treatment%20for%20minimal%20tuberculosis%20%28TB%29%20in%20children%20%28SHINE%29%20a%20study%20protocol%20for%20a%20randomised%20controlled%20trial.pdf	English	null	Shorter treatment for minimal tuberculosis (TB) in children (SHINE): a study protocol for a randomised controlled trial	Trials	2,018	cc-by	10,991	Chabala et al. Trials (2018) 19:237 https://doi.org/10.1186/s13063-018-2608-5 Chabala et al. Trials (2018) 19:237 https://doi.org/10.1186/s13063-018-2608-5 STUDY PROTOCOL Open Access Shorter treatment for minimal tuberculosis (TB) in children (SHINE): a study protocol for a randomised controlled trial Chishala Chabala1* , Anna Turkova2, Margaret J. Thomason2, Eric Wobudeya3, Syed Hissar4, Vidya Mave5, Marieke van der Zalm6, Megan Palmer6, Monica Kapasa1, Perumal K. Bhavani4, Sarath Balaji7, Priyanka A. Raichur5, Anne-Marie Demers6, Graeme Hoddinott6, Ellen Owen-Powell2, Aarti Kinikar5, Philippa Musoke3, Veronica Mulenga1, Rob Aarnoutse8, Helen McIlleron9, Anneke Hesseling6, Angela M. Crook2, Mark Cotton10, Diana M. Gibb2 and on behalf of the SHINE trial team Open Access Shorter treatment for minimal tuberculosis (TB) in children (SHINE): a study protocol for a randomised controlled trial Chishala Chabala1* , Anna Turkova2, Margaret J. Thomason2, Eric Wobudeya3, Syed Hissar4, Vidya Mave5, Marieke van der Zalm6, Megan Palmer6, Monica Kapasa1, Perumal K. Bhavani4, Sarath Balaji7, Priyanka A. Raichur5, Anne-Marie Demers6, Graeme Hoddinott6, Ellen Owen-Powell2, Aarti Kinikar5, Philippa Musoke3, Veronica Mulenga1, Rob Aarnoutse8, Helen McIlleron9, Anneke Hesseling6, Angela M. Crook2, Mark Cotton10, Diana M. Gibb2 and on behalf of the SHINE trial team * Correspondence: cchabala@gmail.com; angela.crook@ucl.ac.uk; diana.gibb@ucl.ac.uk 1University Teaching Hospital, Children’s Hospital, Private Bag RW IX, Ridgeway, Lusaka, Zambia 2Medical Research Council Clinical Trials Unit at University College London, Institute of Clinical Trials and Methodology, High Holborn, London WC1V 6LJ, UK Full list of author information is available at the end of the article Disease setting and context in the study Disease setting and context in the study Of the estimated 10.4 million new tuberculosis (TB) cases globally per annum, approximately one million occur in children in Africa and South East Asia [1]. In Africa, where the TB and HIV epidemics are closely linked, children contribute approximately 30% of inci- dent TB cases [2–4]. Furthermore, in countries with high HIV prevalence, the peak age-prevalence of TB has shifted towards younger adults. These adults are often parents of young children, increasing the exposure of children to TB. Among both HIV-infected children and adults, TB remains a leading cause of death and is the most common opportunistic infection, despite improved access to antiretroviral therapy (ART) [5–7]. Microbiological confirmation of TB in younger children, who have the highest risk of TB disease, is challenging as they cannot spontaneously produce sputum. In addition, most children with pulmonary (intrathoracic) TB typically have paucibacillary disease. Of the 75% of children with in- trathoracic disease, more than 85% have negative-smear mi- croscopy on respiratory samples [11, 12]. Confirmation by culture (the diagnostic ‘gold standard’) is typically made only in approximately 30% of children [11, 14]. Of note, positive culture can also occasionally be obtained in children with normal chest radiographs and a positive tuberculin skin test or history of Mycobacterium tuberculosis exposure [15–17]. Therefore, the diagnosis of intrathoracic tuberculosis in chil- dren frequently relies on non-specific clinical symptoms and signs, supported by evidence of TB contact and/or TB infec- tion and changes on chest radiography [18]. Historically, studies of anti-tuberculosis treatment in chil- dren have lagged substantially behind adult studies and data regarding TB treatment extrapolated from adults. Children have also been seen to pose little risk of onward transmis- sion. In combination with the challenges in confirming a TB diagnosis in children and the resource constraints, the focus has been on adult trials. The approaches used to treat childhood TB have relied on extrapolation to children of the evidence from clinical trials in adults who have a differ- ent spectrum of TB disease and lower bacillary burden [8]. Data on objective measures of TB treatment response in children are limited. Response to anti-tuberculosis treatment can be evaluated by resolution of clinical symptoms, radiological features and smear and culture conversion in bacteriologically confirmed cases. Radio- logical resolution has limitations as some features may temporarily worsen and residual changes may persist at the end of treatment [19–22]. Abstract Background: Tuberculosis (TB) in children is frequently paucibacillary and non-severe forms of pulmonary TB are common. Evidence for tuberculosis treatment in children is largely extrapolated from adult studies. Trials in adults with smear-negative tuberculosis suggest that treatment can be effectively shortened from 6 to 4 months. New paediatric, fixed-dose combination anti-tuberculosis treatments have recently been introduced in many countries, making the implementation of World Health Organisation (WHO)-revised dosing recommendations feasible. The safety and efficacy of these higher drug doses has not been systematically assessed in large studies in children, and the pharmacokinetics across children representing the range of weights and ages should be confirmed. Methods/design: SHINE is a multicentre, open-label, parallel-group, non-inferiority, randomised controlled, two-arm trial comparing a 4-month vs the standard 6-month regimen using revised WHO paediatric anti-tuberculosis drug doses. We aim to recruit 1200 African and Indian children aged below 16 years with non-severe TB, with or without HIV infection. The primary efficacy and safety endpoints are TB disease-free survival 72 weeks post randomisation and grade 3 or 4 adverse events. Nested pharmacokinetic studies will evaluate anti-tuberculosis drug concentrations, providing model-based predictions for optimal dosing, and measure antiretroviral exposures in order to describe the drug-drug interactions in a subset of HIV-infected children. Socioeconomic analyses will evaluate the cost-effectiveness of the intervention and social science studies will further explore the acceptability and palatability of these new paediatric drug formulations. (Continued on next page) * Correspondence: cchabala@gmail.com; angela.crook@ucl.ac.uk; diana.gibb@ucl.ac.uk 1University Teaching Hospital, Children’s Hospital, Private Bag RW IX, Ridgeway, Lusaka, Zambia 2Medical Research Council Clinical Trials Unit at University College London, Institute of Clinical Trials and Methodology, High Holborn, London WC1V 6LJ, UK Full list of author information is available at the end of the article Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 12 Chabala et al. The spectrum of TB disease in children The TB disease spectrum observed in children is differ- ent compared to adults [9, 10]. Whereas a higher pro- portion of young children (below 3 years) have disseminated forms of TB, including miliary TB and TB meningitis, [9] primary childhood TB is typically more benign than adult TB. Three-quarters of children have intrathoracic TB, mainly with non-severe forms of intra- thoracic lymph node TB [10–12]. Adult forms with pleural effusions and cavitation commonly emerge dur- ing adolescence [13]. Abstract Trials (2018) 19:237 (Continued from previous page) Discussion: Although recent trials of TB treatment-shortening in adults with sputum-positivity have not been successful, the question has never been addressed in children, who have mainly paucibacillary, non-severe smear- negative disease. SHINE should inform whether treatment-shortening of drug-susceptible TB in children, regardless of HIV status, is efficacious and safe. The trial will also fill existing gaps in knowledge on dosing and acceptability of new anti-tuberculosis formulations and commonly used HIV drugs in settings with a high burden of TB. A positive result from this trial could simplify and shorten treatment, improve adherence and be cost-saving for many children with TB. Recruitment to the SHINE trial begun in July 2016; results are expected in 2020. Trial registration: International Standard Randomised Controlled Trials Number: ISRCTN63579542, 14 October 2014. Pan African Clinical Trials Registry Number: PACTR201505001141379, 14 May 2015. Clinical Trial Registry-India, registration number: CTRI/2017/07/009119, 27 July 2017. Trial registration: International Standard Randomised Controlled Trials Number: ISRCTN635 Pan African Clinical Trials Registry Number: PACTR201505001141379, 14 May 2015. Clinical Trial Registry-India, registration number: CTRI/2017/07/009119, 27 July 2017. Keywords: Tuberculosis, Efficacy, HIV, Shorter course, Child, Keywords: Tuberculosis, Efficacy, HIV, Shorter course, Child, Disease setting and context in the study Repeat microbiological sampling during therapy may also not be feasible in the majority of children who stop coughing early in treat- ment. Although well-defined clinical case definitions for research are useful for TB diagnosis and evaluation of treatment response in paediatric studies, [8, 23] there are no published data on the gold standard of TB treat- ment response, i.e. long-term relapse-free survival. Rationale for the trial The implementation of standard regimens for both adults and children is attractive for TB programmes. The needs of children with TB, who potentially benefit from shorter treatment and who form a considerable proportion of the global disease burden, should be ad- dressed. The costs to children and their families, burden to health services and added toxicity associated with po- tentially overly long treatment regimens should be also considered. In addition, risks of suboptimal HIV control from drug-drug interactions, the increased pill-burden and its effect on adherence are significant issues for HIV-co-infected children [47] which could be amelio- rated by shortening TB treatment. WHO-recommended increased drug doses were diffi- cult to implement in children using the original fixed- dose combination (FDC) RHZ 60/30/150 mg which, in particular, delivered low isoniazid doses [33]. New co- formulated RHZ 75/50/150 mg FDC tablets were launched in 2015 with rapid and wide uptake globally [34]. The new FDC, which we use in the SHINE trial, fa- cilitates achieving the recommended doses [35]. Treatment outcomes appear considerably better in children than adults with multi-drug resistant (MDR) TB [48], likely because most children have paucibacillary disease. It therefore follows that TB outcomes for shorter first-line regimens could be as good as the longer regimens currently used in adults and children [49, 50]. In addition, there are similarities between minimal TB disease in children and smear-negative, paucibacillary TB in adults, in whom trials of shorter regimens have suggested success. This makes the implementation of tri- als of shorter treatment of standard first-line regimens conducted in children all the more important, and in different countries where the metabolism of anti- tuberculosis drugs may differ (India/Africa). Finally, re- vised WHO dosing guidelines for TB drugs aim to achieve substantially higher TB drug exposures in children which should be more efficacious, providing add- itional rationale to perform this treatment-shortening trial at the time that these dosing guidelines are being imple- mented globally. Drug doses and fixed-dose formulations of anti- tuberculosis drugs in children The doses of first-line anti-tuberculosis drugs recom- mended for children have been based on the same dose Chabala et al. Trials (2018) 19:237 Page 3 of 12 per kilogram of body weight that is recommended for adults [8]. In 2010, the WHO revised the doses of the first-line TB drugs in children, increasing daily doses of isoniazid (H) from 5 to 10 mg/kg, rifampicin (R) from 10 to 15 mg/kg, pyrazinamide (Z) from 25 to 35 mg/kg and recommending ethambutol (E) at a dose of 20 mg/kg/ day [24]. The change was triggered by a review of the aca- demic literature including pharmacokinetic (PK) studies showing that doses extrapolated from adults do not achieve comparable drug exposures in children [25–29]. With the revised doses it was thought to be possible to at- tain similar rifampicin, isoniazid and pyrazinamide con- centrations to those in adults on standard doses [30–32]. WHO-recommended increased drug doses were diffi- cult to implement in children using the original fixed- dose combination (FDC) RHZ 60/30/150 mg which, in particular, delivered low isoniazid doses [33]. New co- formulated RHZ 75/50/150 mg FDC tablets were launched in 2015 with rapid and wide uptake globally [34]. The new FDC, which we use in the SHINE trial, fa- cilitates achieving the recommended doses [35]. no trial evidence about optimum regimen duration for children. per kilogram of body weight that is recommended for adults [8]. In 2010, the WHO revised the doses of the first-line TB drugs in children, increasing daily doses of isoniazid (H) from 5 to 10 mg/kg, rifampicin (R) from 10 to 15 mg/kg, pyrazinamide (Z) from 25 to 35 mg/kg and recommending ethambutol (E) at a dose of 20 mg/kg/ day [24]. The change was triggered by a review of the aca- demic literature including pharmacokinetic (PK) studies showing that doses extrapolated from adults do not achieve comparable drug exposures in children [25–29]. With the revised doses it was thought to be possible to at- tain similar rifampicin, isoniazid and pyrazinamide con- centrations to those in adults on standard doses [30–32]. Aims The primary aim of the trial is to determine whether the standard 6-month regimen (8 weeks’ HRZE followed by 16 weeks’ HR) can be reduced with similar efficacy, to 4 months’ (8 weeks’ HRZE followed by 8 weeks’ HR), in African and Indian children with non-severe TB and re- gardless of HIV status, using revised WHO dosing guidelines and recently introduced new paediatric fixed- dose combination tablets. Anti-TB and antiretroviral treatment interactions in HIV co-infected children Rifampicin interacts with many antiretroviral drugs necessitating ART adjustment for children. For children ≥3 years, efavirenz-based ART is preferred during co- administration of anti-tuberculosis drugs as efavirenz has minimal drug interactions with rifampicin. For chil- dren < 3 years on lopinavir/ritonavir-based ART, two op- tions are available: first, super boosting with additional ritonavir to overcome the rifampicin-inducing effect of liver enzymes, and second, changing to three nucleoside reverse transcriptase inhibitors (NRTIs) [36]. However, neither option is optimal. Super boosting with separate liquid ritonavir is frequently not feasible due to its un- availability, complexity of administration, short shelf-life and poor tolerability [37], and triple NRTI ART is less efficacious in viral suppression [38]. Alternative solu- tions for child-friendly dosing are urgently required for young children [36, 39]. Setting and population The safety endpoint will be grade 3 and 4 adverse events. The study recruits children aged < 16 years diagnosed with minimal non-severe TB from diverse populations in three sites in Africa (Cape Town, South Africa; Kampala, Uganda; Lusaka Zambia) and two sites in India (Pune and Chennai). The trial sites were selected based on the expertise of treating children with TB, sufficient clinical trial experience and the potential for recruiting the re- quired number of suitable participants within the agreed recruitment period. Trial design SHINE is an open-label, phase II/III, non-inferiority trial. Participants are randomised in a 1:1 ratio to the experi- mental 4-month or standard 6-month treatment arm. Both arms have an 8-week intensive phase with rifampi- cin, isoniazid, pyrazinamide and ethambutol followed by a continuation phase with rifampicin and isoniazid given for 8 weeks in the shorter arm vs 16 weeks in the stand- ard arm (Fig. 1). 1. Favourable outcome; TB-free survival at week 72 post randomisation 2. Unfavourable outcomes; a composite of death, reinfection or recurrence 3. Not assessable; will include unknown outcomes at trial completion such as losses to follow-up after completing treatment and clinically well when last seen. Secondary outcomes 1. Mortality 1. Mortality 2. Adverse drug reactions within 30 days of completing treatment 3. Relapse/re-infection-free survival including only cases adjudicated to be TB by an independent Endpoint Review Committee The patient inclusion and exclusion criteria are de- tailed in Table 1. 4. HIV viral suppression at weeks 24 and 48 in HIV-infected children TB treatment-shortening trials in adults Early trials in HIV-negative adults with smear-negative TB in Hong Kong and Singapore showed that, in principle, reductions of treatment duration from 6 to 4 months have resulted in relapse rates of only 4% [40– 42]. WHO guidelines subsequently recommended 4- month treatment duration for smear-negative TB in 1993, although this decision was soon reversed [43]. Re- cent trials of treatment-shortening in adults with smear positive TB have demonstrated poorer outcomes associ- ated with shorter regimens [44–46]. However, there is The other objectives are: 1. To determine whether the higher WHO- recommended doses of daily first-line anti-TB drugs, given as fixed-dose combination dispersible tablets, and prescribed according to weight bands, give appropriate drug exposures when compared Chabala et al. Trials (2018) 19:237 Page 4 of 12 with historical paediatric and adult pharmacokinetic (PK) data with historical paediatric and adult pharmacokinetic (PK) data extrathoracic lymph node TB, intrathoracic uncompli- cated lymph node TB and other non-severe forms of pul- monary TB as per an established classification system [51]. 2. To determine, among HIV-infected children, whether currently recommended adjusted strategies and doses of antiretroviral drugs can appropriately overcome the effect of rifampicin at the new higher doses 2. To determine, among HIV-infected children, whether currently recommended adjusted strategies and doses of antiretroviral drugs can appropriately overcome the effect of rifampicin at the new higher doses Primary outcome The main trial outcome for efficacy is categorised as follows: The main trial outcome for efficacy is categorised as follows: Definition of minimal (non-severe) TB Informed written consent from the parent/legal caregiver(s) and assent in children as per local Ethics 1. Smear-positive respiratory sample 2. Premature (< 37 weeks) and aged under 3 months 3. Miliary tuberculosis (TB), spinal TB, TB meningitis, osteoarticular TB, abdominal TB, congenital TB 4. Pre-existing non-tuberculous disease likely to prejudice the response to, or assessment of, treat- ment, e.g. liver or kidney disease, peripheral neuropathy, cavitation 5. Any known contraindication to taking anti-TB drugs 6. Known contact with drug-resistant adult source case (including mono- resistant TB) 7. Known drug resistance in the child 8. Severely ill 9. Pregnancy where given as part of standard care treatment) for an 8- week intensive phase followed by isoniazid and rifampicin for a continuation phase of a further 8 weeks. In the 6- month arm, children receive the same intensive phase as the other group followed by a continuation phase of 16 weeks. Table 1 Patient inclusion and exc Inclusion criteria E 1. Age 0–16 years 2. Weight ≥3 kg 3. Clinician has decided to treat with standard first-line regimen (intensive phase of 4 drugs or 3 drugs as per local practice) 4. Symptomatic but non-severe TB including: a. extrathoracic lymph node TB; intrathoracic uncomplicated (hilar) lymph node TB b. minimal or no parenchymal abnormality on CXR c. smear-negative on gastric aspirate/other respiratory sample Note: GeneXpert may be positive or negative and a negative GeneXpert can be used as a substitute for a negative smear; culture of respiratory sample may be positive or negative; lymph node aspirate may be smear/culture/ GeneXpert positive or negative) 5. Not treated for previous TB unless successfully treated > 2 years since last completed treatment 6. Known (or pending confirmation of)HIV status; HIV-infected or HIV- uninfected 7. Willing and likely to adhere to 72 weeks’ follow-up 8. Informed written consent from the parent/legal caregiver(s) and assent in children, as per local Ethics Committee guidance 9. Home address accessible for visiting and intending to remain within the recruitment area for follow-up. 1 2 u 3 4 5 6 7 8 9 1. Smear-positive respiratory sample 2. Premature (< 37 weeks) and aged under 3 months 1. Smear-positive respiratory sample 2. Premature (< 37 weeks) and aged under 3 months 3. Miliary tuberculosis (TB), spinal TB, TB meningitis, osteoarticular TB, abdominal TB, congenital TB Protocol treatment discontinuation 8. Informed written consent from the parent/legal caregiver(s) and assent in children, as per local Ethics Committee guidance Protocol treatment discontinuation is considered under any of the following circumstances: 9. Home address accessible for visiting and intending to remain within the recruitment area for follow-up. 1. Unacceptable toxicity or adverse event 2. Disease progression necessitating change of regimen (e.g. with drug-resistant TB or complication of disease) recruitment area for follow-up. Non-trial treatment At enrolment, a clinical history and physical examin- ation is performed including measurements of weight, height, mid-upper arm circumference, paediatric WHO staging for HIV and signs/symptoms suggestive of TB. HIV-infected children enrolled in the trial are provided with ART in accordance with local country programmes including alternative ART drugs regimens indicated for concomitant use with rifampicin during the duration of the TB treatment. All HIV-infected children with TB re- ceive co-trimoxazole prophylaxis as per local guidelines. Screening and enrolment procedures 3. Withdrawal of consent for treatment or participa- tion by the participant’s carer/guardian The standard screening procedures include: a chest radio- graph (CXR) and at least two consecutive respiratory sam- ples including gastric aspirate, expectorated or induced sputum for smear microscopy, Xpert MTB/RIF© (Xpert; Cepheid, Sunnydale, CA, USA), culture and drug- susceptibility testing (DST). In children with extrathoracic cervical lymphadenopathy, lymph node needle aspiration will be undertaken. A Mantoux test or IGRA (interferon- gamma release assay) will be done as available. 4. Participant’s DST-identifying drug resistance, in- cluding isoniazid mono-resistance The children whose protocol treatment is discontinued remain in the trial for the purpose of follow-up and data analysis (unless the carer withdraws consent). Data are kept and included for participants who stop follow-up early, up to the point of exit. The children whose protocol treatment is discontinued remain in the trial for the purpose of follow-up and data analysis (unless the carer withdraws consent). Data are kept and included for participants who stop follow-up early, up to the point of exit. Randomisation and allocation Randomisation is undertaken at each site using a web-based system controlled through an authorised username and password. The method of minimisation with a random element is employed. To reduce potential predictability of the randomisation in this open-label study, the minimisation factors are not disclosed to the investigators. TB treatment products, dosing schedule and treatment supervision Anti-TB drugs are administered according the WHO dosing recommendations using new FDCs and new weight bands for children weighing < 25 kg (Table 2). Children weighing ≥25 kg are treated according to adult guidelines (Table 3). Treatment is implemented in accordance with each site’s local practice. Typically this involves daily adminis- tration by the child’s carer(s) and documenting ingestion on TB treatment cards provided by the trial. p p g 5. Not treated for previous TB unless successfully treated > 2 years since last completed treatment Adherence to trial medication is assessed by pill counts, specific adherence questions at each 4-weekly visit, as well as the number of doses recorded on the TB treatment card. 6. Known (or pending confirmation of)HIV status; HIV-infected or HIV- uninfected 7. Willing and likely to adhere to 72 weeks’ follow-up Definition of minimal (non-severe) TB 5. Adherence and acceptability 5. Adherence and acceptability Minimal TB in this protocol refers to a form of non- severe, symptomatic, smear-negative TB including y 6. Bacterial infections requiring hospitalisation Fig. 1 Trial schema Fig. 1 Trial schema Chabala et al. Trials (2018) 19:237 Page 5 of 12 Table 1 Patient inclusion and exclusion criteria Inclusion criteria Exclusion criteria 1. Age 0–16 years 2. Weight ≥3 kg 3. Clinician has decided to treat with standard first-line regimen (intensive phase of 4 drugs or 3 drugs as per local practice) 4. Symptomatic but non-severe TB including: a. extrathoracic lymph node TB; intrathoracic uncomplicated (hilar) lymph node TB b. minimal or no parenchymal abnormality on CXR c. smear-negative on gastric aspirate/other respiratory sample Note: GeneXpert may be positive or negative and a negative GeneXpert can be used as a substitute for a negative smear; culture of respiratory sample may be positive or negative; lymph node 1. Smear-positive respiratory sample 2. Premature (< 37 weeks) and aged under 3 months 3. Miliary tuberculosis (TB), spinal TB, TB meningitis, osteoarticular TB, abdominal TB, congenital TB 4. Pre-existing non-tuberculous disease likely to prejudice the response to, or assessment of, treat- ment, e.g. liver or kidney disease, peripheral neuropathy, cavitation 5. Any known contraindication to taking anti-TB drugs 6. Known contact with drug-resistant adult source case (including mono- resistant TB) 7. Known drug resistance in the child 8. Severely ill 9. Pregnancy Table 1 Patient inclusion and exclusion criteria Inclusion criteria Exclusion criteria Table 1 Patient inclusion and exclusion criteria Inclusion criteria Exclusion criteria 1. Age 0–16 years 2. Weight ≥3 kg 3. Clinician has decided to treat with standard first-line regimen (intensive phase of 4 drugs or 3 drugs as per local practice) 4. Symptomatic but non-severe TB including: a. extrathoracic lymph node TB; intrathoracic uncomplicated (hilar) lymph node TB b. minimal or no parenchymal abnormality on CXR c. smear-negative on gastric aspirate/other respiratory sample Note: GeneXpert may be positive or negative and a negative GeneXpert can be used as a substitute for a negative smear; culture of respiratory sample may be positive or negative; lymph node aspirate may be smear/culture/ GeneXpert positive or negative) 5. Not treated for previous TB unless successfully treated > 2 years since last completed treatment 6. Known (or pending confirmation of)HIV status; HIV-infected or HIV- uninfected 7. Willing and likely to adhere to 72 weeks’ follow-up 8. Trial assessments and follow-up p All participants are seen at screening, enrolment/randomisa- tion and then at weeks 2, 4, 8, 12, 16, 20, 24, 28, 36, 48, 60 and finally at week 72, when they will exit the trial (Fig. 2). A symptom checklist is performed at each visit to de- tect inter-current illnesses, new TB exposure including DR-TB, TB treatment failure/recurrence or relapse or adverse events to TB drugs (and ART if applicable). All participants are seen at screening, enrolment/randomisa- tion and then at weeks 2, 4, 8, 12, 16, 20, 24, 28, 36, 48, 60 and finally at week 72, when they will exit the trial (Fig. 2). A symptom checklist is performed at each visit to de- tect inter-current illnesses, new TB exposure including DR-TB, TB treatment failure/recurrence or relapse or adverse events to TB drugs (and ART if applicable). 7. Known drug resistance in the child 8. Severely ill 9. Pregnancy 6. Known contact with drug-resistant adult source case (including mono- resistant TB) Trial interventions Children randomised to the 4-month arm receive rifam- picin, isoniazid and pyrazinamide (plus ethambutol Page 6 of 12 Chabala et al. Trials (2018) 19:237 Table 2 Number of tablets to be taken once daily based on WHO guidelinesa Weight bands Intensive phase Continuation phase 50H, 75R, 150Z 100Eb 50H, 75R 3.0–3.9 kg 0.75 0.75 0.75 4.0–7.9 kg 1 1 1 8.0–11.9 kg 2 2 2 12.0–15.9 kg 3 3 3 16.0–24.9 kg 4 4 4 ≥25.0 kg Adult formulation and doses Adult formulation and doses Adult formulation and doses R rifampicin, H isoniazid, Z pyrazinamide, E ethambutol aH: 10 mg/kg (range 7–15 mg/kg) to a maximum dose 300 mg/day; R: 15 mg/kg (range 10–20 mg/kg) to a maximum dose 600 mg/day; Z: 35 mg/kg (30–40 mg/ kg); E: 20 mg/kg (15-25 mg/kg) b100E: additional, separate, single-dose E 100-mg tablets, where this is given as part of local practice, are provided for the intensive phase Table 2 Number of tablets to be taken once daily based on WHO guidelinesa R rifampicin, H isoniazid, Z pyrazinamide, E ethambutol aH: 10 mg/kg (range 7–15 mg/kg) to a maximum dose 300 mg/day; R: 15 mg/kg (range 10–20 mg/kg) to a maximum dose 600 mg/day; Z: 35 mg/kg (30–40 mg/ kg); E: 20 mg/kg (15-25 mg/kg) b100E: additional, separate, single-dose E 100-mg tablets, where this is given as part of local practice, are provided for the intensive phase Respiratory samples for TB assessment and blood sam- ples for biochemistry and haematology, as well as lymphocyte subsets and HIV1 viral load in HIV-infected children, are obtained as summarised in Fig. 2. Relevant samples are stored for pharmacogenomics, biomarkers and hair PK substudies at pre-specified time points (Fig. 2). A quality of life questionnaire, adapted for younger children from a standardised EQ-5D questionnaire [52], is administered at weeks 0, 16, 24, 48 and 72. to check eligibility for enrolment and retrospectively reviewed centrally by independent experts blind to treat- ment allocation using a standardised approach. Where available, Mantoux/IGRA is repeated at week 12 or 16 if the initial test was negative or missed at screening. At each visit a clinical examination for TB signs and symptoms and new TB exposure is conducted to identify children with possible treatment failure or recurrence. Repeat specimens for smear microscopy and culture for M. tuberculosis are collected if clinically indicated. Trial interventions For children with culture-positive respiratory specimens, re- peat TB microscopy and culture are attempted every 2 months until culture conversion. Lymph node needle as- piration is not routinely repeated during follow-up. Participants are expected to attend all scheduled visits unless agreed in advance with the study team. Additional study visits are arranged when a participant misses a clinic visit or whenever clinical care is required. Procedures for assessing safety TB diagnosis and treatment response will be adjudicated by an Endpoint Review Committee (ERC) with independ- ent chair and membership. The ERC will review the end- points (treatment failure or recurrence) and cause of death against standard criteria [23], blinded to treatment allocation, based on all available clinical, microbiological and molecular data obtained from the follow-up assess- ments and procedures. TB disease severity at diagnosis will be ascertained using Wiseman criteria [51]. Symptoms checklists and laboratory investigations are performed as part of safety monitoring at predetermined intervals (Fig. 2). In the case of adverse events, add- itional tests are done as clinically indicated. To monitor for the potential ocular toxicity with etham- butol use, colour-vision testing is done at screening and at week 8 for children aged 3 years and older, with repeat testing at 16 weeks only in those patients with abnormal results. Children with obvious symptoms at any time within the intensive phase of treatment are referred to an ophthalmologist for further testing as appropriate. In the event of confirmed ocular toxicity, ethambutol is stopped. CXRs are repeated at week 24, for suspected treatment failure or TB recurrence, and if clinically indicated. All CXRs will be prospectively read by the study site clinician For girls of reproductive age, or who have started men- ses, a pregnancy test is performed at screening. Any girl found to be pregnant is not eligible for the trial. Girls who become pregnant during the trial will be closely moni- tored and followed for treatment and pregnancy outcome. Table 3 Number of tablets to be taken by children weighing ≥ 25 kg based on WHO adult tuberculosis (TB) guidelinesa Weight bands Intensive phase Continuation phase 75H, 150R, 400Z, 275Eb 75H, 150R 25–36.9 kg 2 2 37–54.9 kg 3 3 55–70.0 kg 4 4 aH: 5 mg/kg (range 4–6 mg/kg) to a maximum dose 300 mg/day; R: 10 mg/kg (range 8–12 mg/kg) to a maximum dose 600 mg/day; Z: 25 mg/kg (20–30 mg/ kg); E: range 15–20 mg/kg; bEither HRZE or HR fixed-dose combinations with additional Z Table 3 Number of tablets to be taken by children weighing ≥ 25 kg based on WHO adult tuberculosis (TB) guidelinesa Pharmacokinetic (PK) studies The trial includes two PK substudies. The first includes 65 children and describes the PK parameters of rifampi- cin, isoniazid and pyrazinamide when administered in the new FDC according to WHO weight-band doses in aH: 5 mg/kg (range 4–6 mg/kg) to a maximum dose 300 mg/day; R: 10 mg/kg (range 8–12 mg/kg) to a maximum dose 600 mg/day; Z: 25 mg/kg (20–30 mg/ kg); E: range 15–20 mg/kg; bEither HRZE or HR fixed-dose combinations with additional Z Page 7 of 12 Chabala et al. Trials (2018) 19:237 Fig. 2 SHINE trial assessment and follow-up schedules children weighing < 25 kg, or the adult formulation for children 25 to < 37 kg [18]. Ethambutol will be measured in the subset of children treated with this fourth drug. from a health sector perspective will be made using na- tionally representative unit costs combined with resource use data. Health outcomes will be estimated in terms of life years gained (LYGs), quality-adjusted life years (QALYs) and disability-adjusted life years (DALYs). The adapted quality of life questionnaire [52] will be combined with existing tariffs to estimate QALYs. Existing DALY weights for TB and TB-free survival will be used. The second substudy evaluates PK interactions between anti-TB and antiretroviral drugs (efavirenz, lopinavir, rito- navir, nevirapine, abacavir and zidovudine) in up to 60 HIV-infected children on ART. Children have cross-over intensive PK undertaken during the continuation phase and 4 weeks after stopping anti-TB treatment. The nature of the economic analyses will largely de- pend upon the trial results. If the shortened regimen arm is non-inferior, and is not associated with any add- itional costs, this will indicate its cost-effectiveness. The cost savings from adopting this strategy at the national level will then be estimated based upon national esti- mates of childhood TB incidence. If the longer regimen arm is shown to be more effective, a cost-effectiveness analysis will be undertaken to investigate whether this represents ‘value for money’ to each of the health care systems in terms of the cost per QALY/DALY/LYG, and estimates of the opportunity costs of resources required to fund the intervention. For both studies, seven blood samples (0.8 ml each) are collected before drug intake and then at 1, 2, 4, 6, 8 and 12 h after drug intake for intensive PK. Social science The substudy evaluates palatability and acceptability of the new FDC using acceptability questionnaires and semi-structured interviews in health facilities and at the homes of a subset of the South African trial population. Challenges with administration of the anti-TB drugs and differences between clinical advice and practice, as well as changes in administration and acceptability over the period of treatment, will be described. Hair PK sub-study Novel assays to measure concentrations of two TB drugs, isoniazid and pyrazinamide, in small hair samples have been developed and validated. These assays have the poten- tial to monitor adequate drug adherence and exposure and need further investigation. In the SHINE study, small hair samples are being collected in around 600 consenting par- ticipants to evaluate hair concentrations of isoniazid and pyrazinamide as surrogates of adherence and predictors of TB treatment outcomes (e.g. treatment success, recurrence and death) among HIV-infected and uninfected children. Non-inferiority margin In discussion with study investigators, 6% was consid- ered a reasonable margin of non-inferiority for the ex- perimental arm for the endpoint of relapse or re- infection-free survival. This has been used in previous treatment-shortening phase III trials in adult smear- positive TB [45, 46]. PK substudies analysis plan It is expected that a proportion of enrolled children treated for TB will not actually have TB, thus reducing the recurrence rate and increasing the chance of demon- strating non-inferiority. For this reason, although the primary analysis will include all randomised children (with assessable outcomes), a key secondary analysis will exclude children (assumed 20%) considered by the inde- pendent Blinded-endpoint Review Committee not to have TB at enrolment. Non-linear mixed effects (NLME) models will be used to describe the population PK of the TB and ART drugs taking account of age, weight, race (African/Asian) and actual dose. In addition to describing the PK for each drug, these models would account for any non-linearity in the PK related to dose. Differences in the Area Under the Curve (AUC) by age, HIV status, and between Asian vs African would be determined using model-based individual post-hoc Bayesian estimates. The geometric mean ratios of the post-hoc estimates of the AUCs for the TB drugs in the FDC compared to single drugs in regulatory approved products will be computed and evaluated against bio- equivalence criteria. Due to the importance of this analysis the trial will be powered to demonstrate non-inferiority also in this sub- population, resulting in a total of 1200 children to be enrolled. Statistical considerations Sample size and power necessarily need to satisfy the strict definition of non- inferiority. It is expected that TB recurrence (relapse or re- infection) and mortality rates will differ by HIV status and co-administration of ART. Based on estimates from the ARROW trial (Uganda and Zimbabwe) [53] and rou- tine data from paediatric HIV clinics in South Africa, we estimate mortality in children HIV-negative, HIV- infected children on and not on ART to be 2%, 5% and 7%, respectively, over 18 months. Corresponding % esti- mates for TB recurrence are 3%, 4% and 6%, respect- ively, in these three groups. We assume that the proportion of HIV-infected children will be approxi- mately 35% maximum (averaged across sites), of whom the majority will be on ART at the start of TB therapy. Using these estimates, the overall rate of TB recurrence or death was estimated at approximately 8%. The MITT analysis will include all randomised partici- pants excluding those withdrawn from treatment be- cause of a protocol violation at enrolment (late exclusions from the study, based on data collected prior to randomisation). The PP analysis will be restricted to participants who have received an adequate amount of study treatment. Any further exclusions in both MITT and PP populations will be detailed in the statistical ana- lysis plan. Primary efficacy analysis The primary efficacy analysis will be the difference in pro- portions of the unfavourable outcomes (proportion with unfavourable outcome on the 4-month regimen arm less the proportion with unfavourable outcome on the 6- month regimen) adjusted for the minimisation factors. Based on these assumptions, the total number of evalu- able children required to demonstrate non-inferiority be- tween the short-regimen arm and the control arm (90% power, one-sided 97.5% confidence intervals (CIs)), would be 860 across two arms. Based on experience from other phase III TB trials, 10% loss-to-follow-up was considered reasonable, resulting in a total of 956 children. Non-inferiority will be assessed using the upper bound of the one-sided 97.5% CI for this difference. If the upper bound of this CI is less than 6% (the margin of non-inferiority) the 4-month regimen will be considered non-inferior to the 6-month regimen. Health economics Economic evaluation of the alternative interventions will be undertaken. Resource use data are collected in the trial on health care visits, hospitalisations, TB and HIV medica- tions, concomitant medications, and diagnostic tests. Esti- mates of the total costs of the two treatment strategies Page 8 of 12 Page 8 of 12 Chabala et al. Trials (2018) 19:237 Definition of adequate treatment The definition of adequate treatment sets limits both for the amount of treatment missed in each treatment phase and the amount of treatment missed overall. This defin- ition of adequate treatment has been used in adult TB trials and is based on receiving at least 80% of the pre- scribed doses within a pre-specified time frame [45]. Analysis plan There will be two main analysis populations: the modi- fied intention-to-treat (MITT) and per-protocol (PP) analysis populations. While both of these will be consid- ered primary in order to declare non-inferiority, consist- ent results should be obtained from key subgroup analyses (confirmed TB cases, both MITT and PP) and sensitivity analyses, although not all of these will y Adverse events and adverse reactions The definitions of adverse events (AE) and adverse reac- tions (AR) outlined in the EU Directive 2001/20/EC Art- icle 2 based on the principles of ICH GCP apply to this trial protocol (Table 4) [54]. Page 9 of 12 Chabala et al. Trials (2018) 19:237 Page 9 of 12 Table 4 Safety reporting definitions Term Definition Adverse event (AE)a Any untoward medical occurrence in a patient or clinical trial subject to whom a medicinal product has been administered including occurrences that are not necessarily caused by or related to that product Adverse reaction (AR) Any untoward and unintended response to an investigational medicinal product related to any dose administered Unexpected adverse reaction (UAR) An adverse reaction, the nature or severity of which is not consistent with the information about the medicinal product in question set out in the Summary of Product Characteristics (SPC) or Investigator Brochure (IB) for that product Serious adverse event (SAE) or serious adverse reaction (SAR) or suspected unexpected serious adverse reaction (SUSAR) Respectively, any adverse event, adverse reaction or unexpected adverse reaction that: Results in death Is life-threatening Requires hospitalisation or prolongation of existing hospitalisation Results in persistent or significant disability or incapacity Consists of a congenital anomaly or birth defect Is another important medical condition infections requiring hospitalisation) are notified to the Medical Research Council Clinical Trials Unit (MRC CTU) within one working day of the investigator be- coming aware of the event. Other reportable events are reported within seven working days. The investigators at each site are also responsible for the onward reporting of adverse events to their ethics and regulatory bodies, as per their local reporting requirements. MRC CTU is responsible for annual onward reporting of a line listing of events to the trial sponsor, University College London (UCL). Investigators are responsible for reporting all SAEs, notable events and other adverse events meeting their reporting requirements that occur from the time of randomisation until 30 days after the last protocol treat- ment administration. Quality assurance, data management and monitoring UCL is responsible for trial oversight and ensuring that arrangements are in place for adequate management, monitoring, analysis and reporting of the trial. Quality assurance and quality control are based on a formal risk assessment which leads to the development of the trial quality management plan implemented by the sponsor. Serious adverse event (SAE) or serious adverse reaction (SAR) or suspected unexpected serious adverse reaction (SUSAR) y Adverse events and adverse reactions On-site monitoring and data validation and assessment of adherence to International Conference on Harmonisation-Good Clinical Practice (ICH-GCP) prin- ciples is regularly conducted by independent local moni- tors at each site and periodically directly by MRC CTU on behalf of the sponsor. Each site is responsible for its own data entry and local trial management. aAEs will include; an exacerbation of a pre-existing illness, an increase in frequency or intensity of a pre-existing episodic event or condition, a condition (even though it may have been present prior to the start of the trial) detected after trial drug administration or continuous persistent disease (or a symptom present at baseline) that worsens following administration of the study treatment Conditions exempted from AEs will include; medical or surgical procedures (the condition that leads to the procedure is the adverse event), pre-existing disease or a condition present before treatment that does not worsen, hospitalisations where no untoward or unintended response has occurred (e.g. elective cosmetic surgery, social admissions) or overdose of medication without signs or symptoms Trial status The first child was enrolled to SHINE in July 2016 and recruitment is due to finish by the end of June 2018. p y g TB studies in children have long been neglected owing to the perception that paediatric TB is not a public health priority. Paediatric TB trials, in particular, face challenges because of difficulties in confirming TB bac- teriological conversion as a study endpoint [8]. While acknowledging the limitations associated with the estab- lishment of paediatric TB diagnosis and evaluating end- points, both clinically diagnosed and bacteriologically confirmed cases of non-severe TB will be included, reflecting the pragmatic approaches utilised in routine clinical settings where children are diagnosed, treated and monitored predominantly using clinical and radio- logical parameters. The TB diagnoses and primary end- points will be adjudicated by an independent ERC, blinded to the study arms, against the standard paediat- ric TB research consensus criteria [23, 51, 56]. The trial will utilise a centralised imaging review by independent paediatric TB radiology experts to assist with decisions on the endpoints. Although the main primary analysis will include all randomised children, it is anticipated that some children might not have TB, thus contributing to a non-inferiority outcome. To overcome this, the study was powered for demonstration of non-inferiority in a key subpopulation considered to have TB by the ERC. Abbreviations 3TC: Lamivudine; ABC: Abacavir; AE: Adverse event; ALT: Alanine aminotransferase; AR: Adverse reaction; ART: Antiretroviral therapy; ARV: Antiretroviral; AUC: Area under the curve; CTU: Clinical trials unit; CXR: Chest x-ray; DALY: Disability-adjusted life years; DST: Drug-susceptibility test; E: Ethambutol; EFV: Efavirenz; ERC: Endpoint Review Committee; FDC: Fixed-dose combination; GCP: Good clinical practice; H: Isoniazid; Hb: Haemoglobin; HIV: Human immunodeficiency virus; ICH: International Conference on Harmonisation; IDMC: Independent Data Monitoring Committee; IGRA: Interferon-gamma release assay; IRB: Institutional review board; ISRCTN: International standard randomised controlled trial number; LPV: Lopinavir; LPV/r: Lopinavir with ritonavir; LYG: Life years gained; MDR: Multiple drug resistant tuberculosis; MITT: Modified intention-to-treat; MRC: Medical Research Council; NNRTI: Non-nucleoside reverse transcriptase inhibitor; NRTI: Nucleoside reverse transcriptase inhibitor; NVP: Nevirapine; PK: Pharmacokinetics; PP: Per protocol; QALY: Quality-adjusted life year; R: Rifampicin; SAE: Serious adverse event; TB: Tuberculosis; TSC: Trial Steering Committee; UCL: University College London; WHO: World Health Organisation; ZDV: Zidovudine Discussion approaches in routine clinical practice for TB treatment in children, regardless of HIV status. SHINE is the first large-scale paediatric trial that will test whether 4-month therapy is as effective as the standard 6-month course in children with non-severe TB. Recently, three large multicentre trials to determine whether fluoroquinolone-containing regimens can shorten treatment from 6 to 4 months did not demon- strate non-inferiority of the 4-month regimens in adults with drug susceptible TB [44–46]. In SHINE, unlike in these adult trials, smear-negative children with non- severe paucibacillary disease will use revised doses of first-line anti-TB drugs. In addition, the trial will also demonstrate whether the PK parameters and safety pro- file of the new dosing recommendations for rifampicin, isoniazid, pyrazinamide and ethambutol across popula- tions of Indian and African children are adequate. As TB often occurs alongside HIV infection, SHINE will provide answers for optimal anti-TB drug dosing in HIV co-infected children on concomitant ART. The nested economic and social science substudies enable SHINE to rapidly translate findings into budget relevant, patient- oriented, and context-sensitive policy guidance. Economic analyses will assess the cost-effectiveness of reducing treatment from 6 months to 4 months while the social science analyses will evaluate the palatability and acceptability of using the recently implemented paediatric anti-tuberculosis FDCs. SHINE will confirm whether or not treatment- shortening of drug susceptible TB in children is effica- cious and safe. It will also fill existing gaps in knowledge on dosing of the new anti-TB formulations and com- monly used HIV drugs in settings with a high burden of childhood TB. A positive result from this trial (i.e. if 4 months is shown to be non-inferior to 6 months) will be to simplify treatment, to potentially improve adher- ence and to reduce treatment costs for a large propor- tion of children with TB (Additional file 1). Acknowledgements Th SHINE ld The SHINE team would like to thank all the staff in all the participating sites and the children and families that have participated in the trial to date. The trial includes HIV-infected children as current evidence suggests no association between treatment duration and outcomes in patients on ART receiving rifampicin-containing regimens [57]. According to WHO guidance and existing programmatic approaches, HIV-infected and uninfected children should be treated for the same duration [18, 36]. Ethically, the risk profile of the trial will be similar to existing recommended Trial governance Th i l i The trial is overseen by a number of committees each with distinct functions and membership. The Trial Steer- ing Committee (TSC) consists of the chief investigator, site principal investigators and independent members. The TSC provides overall supervision for the trial and advice through its independent chair. The ultimate deci- sion for the continuation of the trial lies with the TSC. The investigational medicinal products in this trial will be rifampicin, isoniazid, pyrazinamide and ethambutol. ARs include any untoward or unintended response to any of these drugs. The Independent Data Monitoring Committee (IDMC) reviews confidential, accumulating data for the trial produced by the trial statisticians. The functions and frequency of its meeting are dictated by the DMC charter. The IDMC can recommend premature closure or reporting of the trial. All AEs meeting the standard definitions above are re- ported, regardless of their relationship to TB. The ser- iousness of the events is assessed using the ICH-GCP definitions, and severity is determined in accordance with the 2014 Division of AIDS toxicity grading scale [55]. The causality and expectedness of all serious events or reactions in relation to the trial drugs is categorised as unrelated, unlikely, possibly, probably and definitely related, and for the latter three categories as expected and unexpected based on the Summary of Product Characteristics. The Endpoint Review Committee (ERC), blinded to the trial arm, will determine the primary endpoint classi- fication for all participants as favourable, unfavourable or unassessable. In addition, the ERC will examine evi- dence and adjudicate causes of death based on all avail- able sources of data and determine the TB diagnosis at enrolment across sites for the key secondary analysis of those with definitive TB [23]. All SAEs, grade 3/4 adverse reactions and notable events (ocular toxicities, pregnancies and bacterial Chabala et al. Trials (2018) 19:237 Page 10 of 12 Page 10 of 12 Additional file Additional file 1: SPIRIT 2013 Checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 125 kb) Additional file 1: SPIRIT 2013 Checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 125 kb) Competing interests The authors declare that they have no competing interests. 17. Schaaf HS, Beyers N, Gie RP, Nel ED, Smuts NA, Scott FE, Donald PR, Fourie PB. Respiratory tuberculosis in childhood: the diagnostic value of clinical features and special investigations. Pediatr Infect Dis J. 1995;14(3):189–94. Funding S f g SHINE is funded by the Joint Global Health Trials Scheme of the Department for International Development, UK (DFID), the Wellcome Trust and the Medical Research Council (MRC UK), Grant number MR/L004445/1; and TB Alliance. This work was supported by core support to the UK Medical Research Council [MC_UU_12023/26]. Trial drugs are manufactured by Macleods Pharmaceuticals Ltd. The funders, the sponsor (University College London) and the drug manufacturer had no role in the study design or report writing. g SHINE is funded by the Joint Global Health Trials Scheme of the Department for International Development, UK (DFID), the Wellcome Trust and the Medical Research Council (MRC UK), Grant number MR/L004445/1; and TB Alliance. This work was supported by core support to the UK Medical Research Council [MC_UU_12023/26]. Trial drugs are manufactured by Macleods Pharmaceuticals Ltd. The funders, the sponsor (University College London) and the drug manufacturer had no role in the study design or report writing. Page 11 of 12 Page 11 of 12 Page 11 of 12 Chabala et al. Trials (2018) 19:237 Chabala et al. Trials (2018) 19:237 Authors’ contributions 6. Oliwa JN, Karumbi JM, Marais BJ, Madhi SA, Graham SM. Tuberculosis as a cause or comorbidity of childhood pneumonia in tuberculosis-endemic areas: a systematic review. Lancet Respir Med. 2015;3:235–43. CC drafted the first version of this manuscript. DG, MC, AC and AT conceived and designed the overall study with contributions from MT, AH, EW, CC, VMa, SH, MvdZ, PM, VMu, AK, PKB, RA, HM, AMD and MP. HM, RA and VMa led the design and implementation of the pharmacokinetic substudies and GH of the social science studies. AMD leads the TB laboratory components of the trial and MP is responsible for clinical radiology aspects. AT, EW, PAR, MvdZ, MK, SB, PKB and AK are responsible for patient recruitment, care and follow-up. MT and EOP are responsible for the study management, co-ordination and governance. AC developed the statistical methods of the study. All authors reviewed the manuscript for intellectual content and approved the final version of the report. and designed the overall study with contributions from MT, AH, EW, CC, VMa, SH, MvdZ, PM, VMu, AK, PKB, RA, HM, AMD and MP. HM, RA and VMa led the design and implementation of the pharmacokinetic substudies and GH of the social science studies. AMD leads the TB laboratory components of the trial and MP is responsible for clinical radiology aspects. AT, EW, PAR, MvdZ, MK, SB, PKB and AK are responsible for patient recruitment, care and follow-up. MT and EOP are responsible for the study management, 7. Venturini E, Turkova A, Chiappini E, Galli L, de Martino M, Thorne C. Tuberculosis and HIV co-infection in children. BMC Infect Dis. 2014;14(Suppl 1):S5. 8. Burman WJ, Cotton MF, Gibb DM, Walker AS, Vernon AA, Donald PR. Ensuring the involvement of children in the evaluation of new tuberculosis treatment regimens. PLoS Med. 2008;5(8):e176. 9. Marais BJ, Gie RP, Schaaf HS, Hesseling AC, Obihara CC, Starke JJ, Enarson DA, Donald PR, Beyers N. The natural history of childhood intra-thoracic tuberculosis: a critical review of literature from the pre-chemotherapy era. Int J Tubercul Lung Dis. 2004;8(4):392–402. co-ordination and governance. AC developed the statistical methods of the study. All authors reviewed the manuscript for intellectual content and approved the final version of the report. 10. Lincoln EM, Harris LC, Bovornkitti S, Carretero RW. Endobronchial tuberculosis in children, a study of 156 patients. Am Rev Tubercul. 1958; 77(1):39–61. Author details 1 1University Teaching Hospital, Children’s Hospital, Private Bag RW IX, Ridgeway, Lusaka, Zambia. 2Medical Research Council Clinical Trials Unit at University College London, Institute of Clinical Trials and Methodology, High Holborn, London WC1V 6LJ, UK. 3Makerere University-John Hopkins University Care Ltd, Kampala, Uganda. 4India Council of Medical Research, National Institute for Research in Tuberculosis, Chennai, India. 5Byramjee Jeejeebhoy Government Medical College, Pune, India. 6Desmond Tutu TB Centre, Stellenbosch University, Cape Town, South Africa. 7India Institute of Child Health and Hospital for Children, Chennai, India. 8Radbound University Medical Center, Nijmegen, The Netherlands. 9University of Cape Town, Cape Town, South Africa. 10Family Infectious Diseases Clinical Research Unit, Stellensbosch University, Cape Town, South Africa. 20. Al-Dossary FS, Ong LT, Correa AG, Starke JR. Treatment of childhood tuberculosis with a six month directly observed regimen of only two weeks of daily therapy. Pediatr Infect Dis J. 2002;21(2):91–7. 21. Ramachandran P, Kripasankar AS, Duraipandian M. Short-course chemotherapy for pulmonary tuberculosis in children. Indian J Tuber. 1998; 45(2):83–87. 22. Swaminathan S, Raghavan A, Duraipandian M, Kripasankar AS, Ramachandran P. Short-course chemotherapy for paediatric respiratory tuberculosis: 5-year report. Int J Tubercul Lung Dis. 2005;9(6):693–6. 23. Graham SM, Cuevas LE, Jean-Philippe P, Browning R, Casenghi M, Detjen AK, Gnanashanmugam D, Hesseling AC, Kampmann B, Mandalakas A, et al. Clinical case definitions for classification of intrathoracic tuberculosis in children: an update. Clin Infect Dis. 2015;61(Suppl 3):S179–87. References 25. McIlleron H, Willemse M, Werely CJ, Hussey GD, Schaaf HS, Smith PJ, Donald PR. 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https://openalex.org/W2002506740	https://europepmc.org/articles/pmc3996962?pdf=render	English	null	Angiographic and Clinical Impact of Successful Manual Thrombus Aspiration in Diabetic Patients Undergoing Primary PCI	International journal of vascular medicine	2,014	cc-by	6,509	Hindawi Publishing Corporation International Journal of Vascular Medicine Volume 2014, Article ID 263926, 8 pages http://dx.doi.org/10.1155/2014/263926 Hindawi Publishing Corporation International Journal of Vascular Medicine Volume 2014, Article ID 263926, 8 pages http://dx.doi.org/10.1155/2014/263926 Hindawi Publishing Corporation International Journal of Vascular Medicine Volume 2014, Article ID 263926, 8 pages http://dx.doi.org/10.1155/2014/263926 Mohamed Shehata Department of Cardiology, Faculty of Medicine, Ain Shams University Hospital, Abbasia Square, P.O. Box 11381, Cairo, Egypt Department of Cardiology, Faculty of Medicine, Ain Shams University Hospital, Abbasia Square, Correspondence should be addressed to Mohamed Shehata; smarttmann@hotmail.com Received 5 December 2013; Accepted 4 March 2014; Published 2 April 2014 Academic Editor: Robert S. Dieter Copyright © 2014 Mohamed Shehata. 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. Background. Diabetes mellitus is associated with worse angiographic and clinical outcomes after percutaneous coronary intervention (PCI). Aim. To investigate the impact of manual thrombus aspiration on in-stent restenosis (ISR) and clinical outcome in patients treated by bare-metal stent (BMS) implantation for ST-segment elevation myocardial infarction (STEMI). Methods. 100 diabetic patients were prospectively enrolled. They were randomly assigned to undergo either standard primary PCI (group A, 50 patients) or PCI with thrombus aspiration using Export catheter (group B, 50 patients). The primary endpoint was the rate of eight- month ISR. The secondary endpoint included follow-up for major adverse cardiac events (MACE). Results. Mean age of the study cohort was 59.86 ± 8.3 years, with 64 (64%) being males. Baseline characteristics did not differ between both groups. Eight-month angiogram showed that group B patients had significantly less late lumen loss (0.17 ± 0.35 versus 0.60 ± 0.42 mm, 𝑃< 0.001), with lower incidence of ISR (4% versus 16.6%, 𝑃< 0.001). There was a trend towards lower rate of MACE in the same group of patients. Conclusion. In diabetic patients undergoing primary PCI, manual thrombus aspiration (compared with standard PCI) was associated with better ISR rate after BMS implantation. 2. Methods 2.1. Study Design and Data Collection. 100 consecutive dia- betic patients suffering from acute STEMI were prospec- tively enrolled in this study. They were referred to the catheterization laboratory on emergency basis, after being presented to the emergency department (ED), in the period between March 2011 and January 2013. All included patients suffered from insulin-dependent diabetes mellitus (IDDM). Other inclusion criteria included symptoms suggesting acute myocardial ischemia lasting >30 minutes, the onset of symp- toms <12 hours before presentation to ED, and ST-segment elevation of >0.1 mV in two or more leads on the elec- trocardiogram (ECG). Exclusion criteria included patients undergoing rescue PCI after thrombolysis, patients with prior history of unstable angina or MI, those with prior PCI or coronary artery bypass graft (CABG) surgery, and those with congenital heart disease or any myocardial disease apart from ischemia. Patients with limited life expectancy due to coexistent disease, for example, malignancy, were excluded. After enrollment and before coronary angiography, patients were randomly assigned in 1 : 1 fashion to undergo either standard PCI (group A) or PCI with thrombus aspiration (group B), according to a computer-generated random series of numbers. Randomization was performed by block ran- domization (blocks of 10 patients). Physicians participating in PCI procedures were unaware of block randomization. Before inclusion, informed written consent was obtained from each patient and the study protocol was reviewed and approved by our local institutional human research committee, as it conforms to the ethical guidelines of the 1975 Declaration of Helsinki, as revised in 2008. The primary endpoint was the rate of in-stent restenosis after eight months of follow-up, defined as angiographic luminal diameter stenosis by >50% using quantitative coro- nary angiography (QCA). The secondary endpoint included follow-up for major adverse cardiac events (MACE), that is, a composite of death due to cardiac cause, nonfatal myocardial infarction, and target lesion revascularization (TLR). 2.4. Quantitative Coronary Angiography (QCA). Intracoro- nary nitroglycerine was administered before the initial, final, and follow-up angiograms to achieve maximal vasodilatation. Thrombolysis in myocardial infarction (TIMI) flow grade was recorded individually when flow restoration attempts were finalized. Contrast-filled guide catheters were used as the reference standard in QCA. Matched end-diastolic frames of the angiograms before and after PCI and at eight-month follow-up were analyzed using a contour detection minimum cost algorithm (QCA-CMS Version 3.0, MEDIS, Leiden, The Netherlands). International Journal of Vascular Medicine International Journal of Vascular Medicine 2 2 calcification. In patients in the conventional PCI group (group A), this step was followed by balloon dilation to estab- lish antegrade flow. Concerning patients in the thrombus aspiration group (group B), this step was followed by the advancing of the 6F Export Aspiration Catheter (Medtronic, Minneapolis, MN; crossing profile, 0.068 in.) into the target coronary segment during continuous aspiration. Aspiration was started proximal to the occluded site, gently pushing the catheter through the occlusion and then pulling it in a proximal direction, keeping negative pressure even when the occlusion was crossed or when there was no longer back bleeding in the syringe. Withdrawal of the catheter from the artery and from the guiding catheter was performed with permanent negative pressure. When necessary for stent deliv- ery, balloon dilation was performed before stenting. In all patients, after the restoration of antegrade flow, intracoronary nitrates were given to ensure maximal epicardial vasodi- latation, to determine the size and length of the required stent, and to facilitate stent placement. All placed stents were bare-metal stents. Pharmacological treatment before PCI included the administration of aspirin (a loading dose of 500 mg), heparin (70 IU/kg), and clopidogrel (a loading dose of 600 mg). All patients also received the glycoprotein IIb/IIIa inhibitor abciximab with an intravenous procedural bolus of 0.25 mg/kg followed by a continuous intravenous infusion of 0.125 𝜇g/kg/min for 12 hours and postprocedural infusion without heparin.h implantation in these patients [30]. In the current study, the author sought to explore the impact of upfront manual thrombus aspiration on angiographic (in-stent restenosis) and clinical outcomes, in diabetic patients treated by bare- metal stent implantation for acute ST-segment elevation myocardial infarction (STEMI). 1. Introduction resulted in the development of various devices to protect the microcirculation [16–24]. Other proposed mechanisms contributing to microvascular obstruction and dysfunction include reperfusion injury, production of oxygen free radi- cals, neutrophil activation, endothelial and myocyte edema, loss of antioxidant enzymes, cardiomyocyte apoptosis, loss of endothelial mediated vasomotion, alteration of sympathetic innervation, plugging of platelets and neutrophils, epicardial coronary vasoconstriction, and increased myocardial cell calcium level [25]. It is proposed that the resulting cellular dysfunction, apoptosis, and necrosis mediate myocardial stunning, no-reflow, reperfusion arrhythmias, and additional loss of myocardium (lethal reperfusion injury) [26]. Acute myocardial infarction (MI) with ST-segment elevation is caused by rupture or erosion of an atherosclerotic plaque, initiating intraluminal thrombosis resulting in occlusion of a coronary artery [1–3]. Primary percutaneous coronary intervention (PCI) is the preferred treatment for MI with ST-segment elevation and is effective in opening the infarct- related artery [4–6]. However, microvascular obstruction with diminished myocardial perfusion occurs in a large proportion of patients with patent epicardial vessels after primary PCI, and this event is associated with an increased infarct size, reduced recovery of ventricular function, and increased mortality [7–11]. Microvascular obstruction is related to the embolization of plaque or thrombotic material downstream in the infarct-related artery [12, 13]. Emboliza- tion can occur spontaneously or by means of mechanical fragmentation during PCI [12–15]. The high frequency of suboptimal myocardial reperfusion after primary PCI has Diabetes mellitus is an important risk factor for poor outcome after PCI using bare-metal stents [27–29]. A more diffused and accelerated form of atherosclerosis in diabetic patients, accompanied by small vessel size, long lesions, and greater plaque burden, may contribute to the well-documented increased risk of restenosis after stent 2. Methods Table 1: Baseline characteristics of the two study groups. CAD: coronary artery disease. Age, HbA1c, serum creatinine, and door to balloon time are presented as mean ± standard deviation. Other variables are presented as number (percentage). ∗𝑡-test and Pearson’s Chi-square test. door to balloon time are presented as mean ± standard deviation. Other variables are presented as number (percentage). st. more patients in the same group showed TIMI III flow grade after culprit lesion stenting (𝑃< 0.05). No serious periprocedural complications were recorded in both study groups. 𝑡-test. For comparing categorical data, Pearson’s Chi-square test was performed. 𝑃values were used to describe signifi- cance. All statistical calculations were done using Statistical Package for Social Sciences (SPSS for Windows) software (version 15.0, SPSS Inc., Chicago, IL, USA). 3.3. Quantitative Angiographic Outcomes. Angiographic measures are shown in Table 3. Angiographic follow-up was obtained for 98 patients (98%), at a mean of 247.1 ± 27.4 days. Late luminal loss was found to be less in the group B (0.17 ± 0.35 mm versus 0.60 ± 0.42; 𝑃< 0.001). Also, the percentage of stenosis (in-stent restenosis) at follow-up was less in the same group (22.2 ± 9.7 in group B versus 35.37 ± 14.46 in group A; 𝑃< 0.001). The rate of in-stent restenosis (primary endpoint), defined as luminal diameter stenosis of >50 percent, was 4% (𝑛= 2) in group B versus 16.6% (𝑛= 8) in group A (𝑃< 0.001). 3. Results 3.1. Baseline Clinical Characteristics. A total of 100 con- secutive IDDM patients suffering from acute STEMI were prospectively enrolled in the current study, which comprises 50 patients randomly assigned to undergo conventional PCI (group A) and 50 others randomly assigned to undergo PCI with thrombus aspiration (group B). The mean age of the whole study cohort was 59.86±8.3 years, with 64 (64%) being male patients. The two groups were matched regarding age, gender, and risk factors of coronary artery disease (CAD). No significant difference was recorded between the two groups; concerning baseline mean serum HbA1c levels. Table 1 shows baseline clinical characteristics of the two study groups. All patients underwent follow-up coronary angiography after eight months, except for six patients. Five (group A: three patients, group B: two patients) patients had definite late (>30 days after primary PCI) stent thrombosis and target lesion revascularization, while one patient belonging to group A died after 220 days, and thus a possible stent thrombosis is considered. So, follow-up angiograms were available in 94 (94%) patients (group A: 𝑛= 46, group B: 𝑛= 48). Blood samples were drawn for HbA1c quantitation at the day of follow-up of coronary angiography and revealed almost the same mean results (group A: 7.02 ± 0.3%, group B: 7.07 ± 0.4%; 𝑃> 0.05). 3.4. Clinical Events. Clinical follow-up data revealed that 11 patients experienced major adverse cardiac events (MACE) as shown in Table 4, with no statistical difference between both study groups but a trend towards lower incidence in group B. Five patients suffered from definite stent thrombosis and TRL was done. Only one patient (belonging to group A) died during the follow-up period with possible stent thrombosis. 2. Methods Restenosis was defined as a stent stenosis >50% in diameter anywhere within the stent and/or within the 5 mm borders proximal or distal to the stent. Late lumen loss was defined as the difference between minimal lumen diameters (MLD) immediately after PCI and on eight-month follow-up angiography. QCA analyses were performed by an experienced technician who was blinded to patients’ assignment. 2.2. Definition of Risk Factors of Coronary Artery Disease. The presence of hypertension was defined as systolic blood pres- sure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg, previously recorded by repeated noninvasive office measure- ments, which led to life-style modification and/or intake of antihypertensive drug therapy [31]. The presence of diabetes mellitus was defined as fasting plasma glucose ≥126 mg/dL and/or two-hour postglucose load ≥200 mg/dL or spe- cific antidiabetic drug therapy intake [32]. Dyslipidemia was defined as LDL cholesterol >100 mg/dL and/or serum triglycerides >150 mg/dL and/or HDL cholesterol <40 mg/dL (<50 mg/dL in women) [33]. 2.3. PCI and Medications. Coronary angioplasty and stent implantation were performed according to institutional stan- dards. For all patients, the first procedural step was the passing of a floppy, steerable guide wire through the target culprit lesion; direct stenting was left to operator’s discretion and usually performed in patent vessel with no or mild 2.5. Statistics. All continuous variables were statistically described in terms of mean ± standard deviation (±SD). Categorical variables were described with absolute and rel- ative (percentage) frequencies. Comparison of continuous variables between the study groups was done using Student’s International Journal of Vascular Medicine 3 Table 1: Baseline characteristics of the two study groups. Table 1: Baseline characteristics of the two study groups. Item Group A (𝑛= 50) Group B (𝑛= 50) 𝑃value∗ Age (years) 59.4 ± 7.4 60.32 ± 9.2 >0.05 Males 33 (66) 31 (62) >0.05 Hypertension 32 (64) 30 (60) >0.05 Smoking 9 (18) 12 (24) >0.05 Dyslipidemia 18 (36) 16 (32) >0.05 Family history of CAD 8 (16) 6 (12) >0.05 HbA1c (%) 7.1 ± 1.3 7.2 ± 1.6 >0.05 Serum creatinine (mg%) 1.1 ± 0.3 1.0 ± 0.4 >0.05 Door to balloon time (minutes) 73.7 ± 39.2 77.7 ± 36.4 >0.05 Killip class ≥3 4 (8) 6 (12) >0.05 CAD: coronary artery disease. Age, HbA1c, serum creatinine, and door to balloon time are presented as mean ± standard deviation. Other variables are presented as number (percentage). ∗𝑡-test and Pearson’s Chi-square test. 4. Discussion The clinical importance of embolization of atherothrombotic materials from unstable plaques in patients with myocardial infarction with ST-segment elevation has been recognized [12, 15], and the use of thrombectomy aspiration devices to reduce distal embolization, preserving tissue-level perfusion, has been tested in several studies with conflicting results [16, 17, 20–24, 34–38]. Results achieved using different techniques to explore different aspects of microvascular integrity further enhance the strength of the tested hypothesis that distal embolization during primary PCI plays a significant role in the pathogenesis of microvascular obstruction. This was 3.2. Lesion and Procedure Characteristics. Table 2 shows lesion and procedure characteristics. No statistically sig- nificant difference was recorded between the two study groups regarding lesion length, stent length, need for stent postdilatation, and frequency of stenting of each coronary artery. However, incidence of angiographic no-reflow was significantly less in group B patients (𝑃< 0.05). Moreover, International Journal of Vascular Medicine 4 Table 2: Procedural characteristics of the two study groups. Table 2: Procedural characteristics of the two study groups. Item Group A (𝑛= 50) Group B (𝑛= 50) 𝑃value∗ Lesion length (mm) 14.4 ± 3.2 13.5 ± 4.2 >0.05 Stent length (mm) 16.6 ± 5.3 16.7 ± 5.8 >0.05 Stent diameter 2.77 ± 0.5 2.8 ± 0.5 >0.05 Stent postdilatation 21 (42) 18 (36) >0.05 TIMI 3 flow (after PCI) 20 (40) 38 (76) <0.05 Angiographic no-reflow 10 (20) 2 (4) <0.05 Infarct-related artery LAD 25 (50) 29 (58) >0.05 LCx 10 (20) 8 (16) >0.05 RCA 15 (30) 13 (26) >0.05 TIMI: thrombolysis in myocardial infarction; LAD: left anterior descending; LCx: left circumflex; RCA: right coronary artery. Lesion length and stent length are presented as mean ± standard deviation. Other variables are presented as number (percentage). ∗𝑡-test and Pearson’s Chi-square test. ocardial infarction; LAD: left anterior descending; LCx: left circumflex; RCA: right coronary artery. ngth are presented as mean ± standard deviation. Other variables are presented as number (percentage). TIMI: thrombolysis in myocardial infarction; LAD: left anterior descending; LCx: left circumflex; RCA: right coronary artery. Lesion length and stent length are presented as mean ± standard deviation. Other variables are presented as number (percentage). ∗𝑡-test and Pearson’s Chi-square test Table 3: Quantitative coronary angiography results: baseline and 8-month follow-up. 4. Discussion Item Group A Group B 𝑃value∗ After primary PCI† Reference vessel diameter (mm) 2.85 ± 0.60 3.11 ± 0.55 >0.05 Minimal lumen diameter (mm) 2.55 ± 0.40 2.73 ± 0.69 >0.05 Diameter stenosis (residual in-stent stenosis) (%) 9.5 ± 0.3 10.2 ± 0.54 >0.05 After follow-up coronary angiography‡ (8 months) Reference vessel diameter (mm) 2.82 ± 0.58 2.95 ± 0.35 >0.05 Minimal lumen diameter (mm) 1.95 ± 0.55 2.56 ± 0.33 <0.001 Diameter stenosis (ISR) (%) 35.37 ± 14.46 22.2 ± 9.7 <0.001 Late lumen loss (mm) 0.60 ± 0.42 0.17 ± 0.35 <0.001 PCI: percutaneous coronary intervention; ISR: in-stent restenosis. All variables are presented as mean ± standard deviation. ∗𝑡-test; †100 patients (group A: 50, group B: 50); ‡94 patients (group A: 46, group B: 48). Table 3: Quantitative coronary angiography results: baseline and 8-month follow-up. Table 4: Clinical events at 8-month follow-up. Item Group A (𝑛= 50) Group B (𝑛= 50) 𝑃value∗ Cardiac death 1 0 Stent thrombosis Definite 3 2 Probable 0 0 Possible 1 0 Myocardial infarction STEMI 3 2 NSTEMI 3 2 Target lesion revascularization 3 2 MACE 7 4 >0.05 STEMI: ST-segment elevation myocardial infarction; NSTEMI: non-ST- segment elevation myocardial infarction; MACE: major adverse cardiac events. ∗Pearson’s Chi-square test. Table 4: Clinical events at 8-month follow-up. mortality rates for STEMI continue to be high. Thus, further improvement of the initial therapy is necessary to improve patient outcome. The pivotal TAPAS (Thrombus Aspira- tion during Percutaneous coronary intervention in Acute myocardial infarction Study) trial of patients with STEMI, which compared a strategy with thrombectomy with a strat- egy without thrombectomy, showed a reduction in cardiac mortality at one year [38]. By contrast, other randomized, controlled studies did not observe superiority of thrombec- tomy over standard PCI with respect to surrogate endpoints of reperfusion success [40, 41]. However, meta-analyses in patients with STEMI showed a mortality-related benefit after thrombectomy compared with PCI alone [42, 43]. Therefore, the current guidelines of the European Society of Cardiology (ESC) on revascularization therapy in patients with STEMI reperfused by primary PCI have increased the recommenda- tion class for manual thrombectomy from the previous class IIb, level of evidence B, to class IIa, level of evidence A [44]. In line with the ESC guidelines, thrombectomy is strongly recommended by the American Heart Association/American College of Cardiology (AHA/ACC) guidelines (class IIa, level of evidence B) [45]. 4. Discussion Although the favorable clinical impact of thrombus aspiration did not reach a statistical significance (when compared to the angiographic impact), numerical values still show that adopting thrombus aspiration strategy was associated with less incidence of MACE. A larger number of patients for a longer follow- up period might magnify the obtained follow-up clinical results. The author hypothesized that adopting thrombus aspiration strategy in this clinical setting could improve both angiographic and clinical outcomes encountered in diabetic patients, especially those who are managed using bare-metal stents. 4.2. Clinical Implications. Diabetes mellitus was proven to be associated with worse angiographic (in-stent restenosis, target lesion revascularization, and stent thrombosis) and clinical (MACE) outcomes after PCI, regardless of the type of the used stent [53]. That is why interventionalists are always attempting to optimize the procedural circumstances, in order to achieve satisfactory short- and long-term results in diabetic patients. The current study highlighted the favorable impact of thrombus aspiration on late angiographic and clinical outcomes in diabetic patients undergoing primary PCI, using bare-metal stents. The author recommends adopt- ing thrombus aspiration strategy, whenever possible, in the aforementioned clinical setting. 4.3. Limitations of the Study. The data presented in our study only apply for patients defined by inclusion and exclusion criteria. Moreover, this is a single-centre study with a rel- atively small sample size of the cohort. 2D-QCA was used for angiographic assessment in the present study. Subsequent studies are needed to verify the obtained results using 3D- QCA and/or intravascular ultrasound (IVUS). Follow-up period in the current study was limited to eight months. A longer follow-up period is warranted for confirmation of the current study results, perhaps with the use of DESs. Assessment of successful tissue reperfusion and infarct size was not included in the current study, as these parameters were outside the scope of the study protocol. 4.1. Comparison with Other Studies. Previous studies demon- strated safety and merits of using thrombus aspiration devices in primary PCI prior to coronary stenting, especially concerning myocardial salvage and improved myocardial reperfusion [20, 22, 46–49]. However, this was not always associated with reduction of infarct size [42, 47]. The current study results showed better early angiographic outcomes upon using thrombus aspiration device in diabetic patients in the setting of primary PCI, in the form of higher frequency of TIMI III flow and less frequent no-reflow phenomenon. 4. Discussion The current study sought to evaluate the clinical and angiographic outcome of thrombus aspiration during primary PCI in diabetic patients presented with because thrombus aspiration significantly reduces the sever- ity and extent of the phenomenon [39]. Despite early invasive strategies and optimal medical treatment, morbidity and 5 International Journal of Vascular Medicine STEMI. Eligible patients were randomly assigned to either primary PCI with thrombus aspiration or standard PCI. Bare- metal stents were used in all patients. Bare-metal stents are still the only available option (especially on emergency basis) for percutaneous revascularization in most of centers located in developing countries. The current study was accomplished in one of these countries, where there is still a consider- able cost burden concerning the use of drug-eluting stents (DESs). Patients who underwent primary PCI with thrombus aspiration showed significantly higher incidence of final TIMI III flow and less incidence of angiographic no-reflow phenomenon. Eight-month follow-up coronary angiography showed favorable QCA results concerning the same group of patients with statistically significant less incidence of in-stent restenosis, less percentage of diameter stenosis, and less late lumen loss. It is worth mentioning that mean culprit lesion length and mean used stent length did not differ between patients belonging to thrombus aspiration strategy and others belonging to standard PCI strategy. No recorded significant difference between both study groups concerning follow-up for MACE after the same period of time, that is, eight months. However, there was a recorded case of cardiac mortality, pos- sibly stent thrombosis, and a case of definite stent thrombosis mounting to target lesion revascularization in the standard PCI group of patients. The presented results highlighted the favorable impact of thrombus aspiration, during primary PCI in diabetic patients, on incidence of in-stent restenosis and consequently the other QCA parameters. Although the favorable clinical impact of thrombus aspiration did not reach a statistical significance (when compared to the angiographic impact), numerical values still show that adopting thrombus aspiration strategy was associated with less incidence of MACE. A larger number of patients for a longer follow- up period might magnify the obtained follow-up clinical results. The author hypothesized that adopting thrombus aspiration strategy in this clinical setting could improve both angiographic and clinical outcomes encountered in diabetic patients, especially those who are managed using bare-metal stents. thrombus aspiration as was previously proven to be effective and safe in primary PCI [41, 49, 51]. 4. Discussion In agreement with the results of the current study, prior trials reported satisfactory intermediate and long-term follow-up clinical outcomes, favoring adopting thrombectomy strategy in primary PCI settings [49, 51]. It is worth mentioning that all patients in both current study groups were assumed to exhibit more or less controlled blood glucose during the follow-up period as shown in mean HbA1c levels tested shortly before follow-up angiograms. So, quality of glycemic control during the follow- up period was not a determining factor for incidence of in- stent restenosis in the present study. A prior study stated that proper glycemic control was associated with lower incidence of in-stent restenosis at six-month follow-up, after acute STEMI [52]. However, thrombectomy aspiration procedure was not included in the study protocol. STEMI. Eligible patients were randomly assigned to either primary PCI with thrombus aspiration or standard PCI. Bare- metal stents were used in all patients. Bare-metal stents are still the only available option (especially on emergency basis) for percutaneous revascularization in most of centers located in developing countries. The current study was accomplished in one of these countries, where there is still a consider- able cost burden concerning the use of drug-eluting stents (DESs). Patients who underwent primary PCI with thrombus aspiration showed significantly higher incidence of final TIMI III flow and less incidence of angiographic no-reflow phenomenon. Eight-month follow-up coronary angiography showed favorable QCA results concerning the same group of patients with statistically significant less incidence of in-stent restenosis, less percentage of diameter stenosis, and less late lumen loss. It is worth mentioning that mean culprit lesion length and mean used stent length did not differ between patients belonging to thrombus aspiration strategy and others belonging to standard PCI strategy. No recorded significant difference between both study groups concerning follow-up for MACE after the same period of time, that is, eight months. However, there was a recorded case of cardiac mortality, pos- sibly stent thrombosis, and a case of definite stent thrombosis mounting to target lesion revascularization in the standard PCI group of patients. The presented results highlighted the favorable impact of thrombus aspiration, during primary PCI in diabetic patients, on incidence of in-stent restenosis and consequently the other QCA parameters. 5. Conclusion Successful upfront manual thrombus aspiration (in diabetic patients on insulin therapy) during primary PCI showed beneficial effects on the reduction of in-stent restenosis after bare-metal stent implantation compared with standard PCI. 4. Discussion This was also demonstrated by previous trials [41, 49, 50], not specifically targeting diabetic patients. A previous trial reported the favorable impact of using thrombus aspiration (in primary PCI) on incidence of in-stent restenosis [51]. However, this trial also was not addressing diabetic patients in particular. To the best of the author’s knowledge, the current study is a unique one, exploring the impact of aspiration thrombectomy on in-stent restenosis in diabetic patients. The current study utilized export aspiration catheter for upfront References The Task Force for Percutaneous Coronary Interventions of the European Society of Cardiology,” European Heart Journal, vol. 26, no. 8, pp. 804–847, 2005. [7] A. 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https://openalex.org/W1975758565	https://europepmc.org/articles/pmc4068063?pdf=render	English	null	Recent Advancements in Computer & Software Technology	The scientific world journal/TheScientificWorldjournal	2,014	cc-by	563	K. K. Mishra,1 A. K. Misra,1 Peter Mueller,2 Gregorio Martinez Perez,3 Sanjiv K. Bhatia,4 and Yong Wang5 1 Department of Computer Science, MNNIT, Allahabad, India 2 IBM Zurich Research Laboratory, Saeumerstraße 4, 8803 Rueschlikon, Switzerland 3 University of Murcia, Murcia, Spain 4University of Missouri-St. Louis, MO, USA 5 School of Information Science and Engineering Central South University Changsha China 1 Department of Computer Science, MNNIT, Allahabad, India 2 IBM Zurich Research Laboratory, Saeumerstraße 4, 8803 Rueschlikon, Switzerland 3 University of Murcia, Murcia, Spain 4University of Missouri-St. Louis, MO, USA 5 School of Information Science and Engineering, Central South University, Changsha, China 1 Department of Computer Science, MNNIT, Allahabad, India 4University of Missouri-St. Louis, MO, USA 5 School of Information Science and Engineering, Central South University, Changsha, China 5 School of Information Science and Engineering, Central South University, Changsha, China Correspondence should be addressed to K. K. Mishra; mishrakrishn@gmail.com Correspondence should be addressed to K. K. Mishra; mishrakrishn@gmail.com Received 18 May 2014; Accepted 18 May 2014; Published 5 June 2014 Copyright © 2014 K. K. Mishra 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. Over the last few decades, advancements in computer and software technologies have reached an impressive level. These technologies improve not only very common areas of our daily life, but also areas of education, health, production industries, and so on. Thus, recent advancements in computer and software technologies are the base for the society of tomorrow.h software testing, vulnerability and video quality assessment, artificial immune systems, improvements in file systems, and component models for distributed simulation, amongst others. Due to the space limitation we will not reiterate the contents here. We hope the reader will find this special issue informative and stimulating. g We would like to thank all the contributors who have submitted their high quality papers. The feasibility of future developments strongly relies on the existence of key technologies and their deployments. Analyzing global trends in cloud computing including all its services reveals cornerstone fields, such as distributed parallel processing, advanced software engineering, image processing, and security solutions. These fields require dif- ferent sets of resources like computing hardware, Internet, software and hardware tools, mobility technologies, storage, system management, and security technology.h K. K. Mishra A. K. Misra Peter Mueller Gregorio Martinez Perez Sanjiv K. Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 609512, 1 page http://dx.doi.org/10.1155/2014/609512 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 609512, 1 page http://dx.doi.org/10.1155/2014/609512 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 609512, 1 page http://dx.doi.org/10.1155/2014/609512 K. K. Mishra,1 A. K. Misra,1 Peter Mueller,2 Gregorio Martinez Perez,3 Sanjiv K. Bhatia,4 and Yong Wang5 Bhatia Yong Wang The objective of this special issue is to present a col- lection of articles that cover recent research results and comprehensive reviews on relevant computer and software technologies. In particular, it aims to present highly technical papers describing the areas mentioned above: distributed parallel processing, advanced software engineering, image processing, and security solutions.h This special issue received an overwhelming response from the community. Due to the limited space, only 10 papers from the initial 109 manuscripts have been selected. These papers represent the most up-to-date research work covering topics such as software process improvement and
https://openalex.org/W1979815078	http://www.mdpi.com/1424-2818/5/2/409/pdf/	English	null	Up Against The Wall: The Effects of Climate Warming on Soil Microbial Diversity and The Potential for Feedbacks to The Carbon Cycle	Diversity	2,013	cc-by	8,658	Diversity 2013, 5, 409-425; doi:10.3390/d5020409 Diversity 2013, 5, 409-425; doi:10.3390/d5020409 diversity ISSN 1424-2818 www.mdpi.com/journal/diversity OPEN ACCESS diversity ISSN 1424-2818 www.mdpi.com/journal/diversity OPEN ACCESS 1. Introduction Soil is one of the most diverse habitats on Earth, but also one of the least characterized in terms of the identification and ecological roles of the microbiota. Soils also contain the largest repository of organic carbon (C) in the terrestrial biosphere, and the activities of heterotrophic soil organisms are responsible for large portions of the annual CO2 flux to the atmosphere. A substantial fraction of soil C occurs in relatively complex organic compounds, which tend to be resistant to decomposition under current environmental conditions. It is likely that soil organic matter (SOM) decay will change under future climate, and of the approximately 3100 Pg of carbon stored in soils, an estimated 5% will be lost in the next decade due to warming [1,2]. These estimates illustrate the vulnerability of this stored C, though the mechanisms of how the C will be lost are not well understood. Microorganisms catalyze key processes related to greenhouse gas fluxes between soils and the atmosphere [3,4], but the role that microbes will play in the evolution of Earth’s climate over decades to centuries is undefined. A more complete understanding of how global warming will affect carbon-cycle feedbacks to the climate system is central to model projections of future climate [5]. Preliminary data from the longest-running soil warming study at Harvard Forest suggests that over 20 years of warming results in a loss of labile, microbial available C that puts the soil microbes ―up against the wall‖ metaphorically, forcing adaptation that will affect global C cycling [6–8]. Advances in molecular biological methods, including high-throughput sequencing [9,10] combined with manipulative field experiments such as in situ soil warming [11,12] make it possible to identify and model relationships between microbial communities and climate system feedbacks. This paper focuses on understanding how climate warming affects soil microbial community diversity, and its impacts on ecosystem function. Two non-mutually exclusive diversity hypotheses developed for macroecology [13] can be applied to describe observed changes in carbon cycling with climate warming. The first community diversity hypothesis suggests that an overall increase in diversity has resulted in communities more resistant to higher and more extreme temperatures. This hypothesis is based on evidence that in general, more diverse communities are more resistant to stress and more efficient [13–15], possibly because taxonomic and functional richness often go side by side [16,17]. Microbiology Department, University of Massachusetts Amherst, Amherst, MA 01003, USA; E-Mail: apold@microbio.umass.edu Microbiology Department, University of Massachusetts Amherst, Amherst, MA 01003, USA; E-Mail: apold@microbio.umass.edu * Author to whom correspondence should be addressed; E-Mail: deangelis@microbio.umass.edu; Tel.: +1-413-577-4669; Fax: +1-413-545-1578. Received: 15 April 2013; in revised form: 27 April 2013 / Accepted: 17 May 2013/ Published: 3 June 2013 Abstract: Earth’s climate is warming, and there is evidence that increased temperature alters soil C cycling, which may result in a self-reinforcing (positive), microbial mediated feedback to the climate system. Though soil microbes are major drivers of soil C cycling, we lack an understanding of how temperature affects SOM decomposition. Numerous studies have explored, to differing degrees, the extent to which climate change may affect biodiversity. While there is ample evidence that community diversity begets ecosystem stability and resilience, we know of keystone species that perform functions whose effects far outweigh their relative abundance. In this paper, we first review the meaning of microbial diversity and how it relates to ecosystem function, then conduct a literature review of field-based climate warming studies that have made some measure of microbial diversity. Finally, we explore how measures of diversity may yield a larger, more complete picture of climate warming effects on microbial communities, and how this may translate to altered carbon cycling and greenhouse gas emissions. While warming effects seem to be ecosystem-specific, the lack of observable consistency between measures is due in some part to the diversity in measures of microbial diversity. Keywords: climate warming; keystone species; long-term field study; metagenomics; microbial community diversity; niche theory; richness; soil; stress response Diversity 2013, 5 Diversity 2013, 5 410 1. Introduction The alternative hypothesis is somewhat complementary: the keystone species hypothesis suggests that the increase in populations specifically associated with a function, such as the ability to degrade more recalcitrant or previously physically protected soil carbon, is responsible for observed changes in carbon cycling. The increased relative (or absolute) abundance of these functional populations may be independent of changes in overall diversity. In the next sections, we explore the support for each hypothesis, and then evaluate the literature for evidence of diversity or keystone functions in previously published climate warming studies. Diversity 2013, 5 Diversity 2013, 5 competition with other organisms which share some portion of this niche [21]. Organisms may only survive under a narrow set of conditions (specialists), or have a broad niche (generalists). In general, more diverse communities are better at taking advantage of the resource space (i.e., range of conditions present in an environment), and therefore are able to more completely and efficiently convert resources into biomass [13,18]. Environment dictates total resource space, and therefore carrying capacity of the ecosystem, but also the range of niches available to organisms. Furthermore, because organisms differ in their susceptibility to stressors, such as drought and temperature [22–24], changes in the environment affect the relative abundance of organisms in an environment. If lower species richness is indicative of reduced niche overlap, then species loss should have a disproportionately high impact on ecosystem function in species-poor (depauperate) communities. In other words, species-rich assemblages may have another phylotype ready to expand its realized niche into the newly-vacated portion of its fundamental niche, but species-poor ones may not [25]. Functional stability is also expected to be favored by a high proportion of generalists, or in a community with diverse metabolisms represented [15,26]. Furthermore, if diverse communities are more resistant to changes in function, then they may prevent changes in the ecosystem itself, allowing for less resistant organisms to recolonize once the disturbance passes [19]. For example, due to variation in environmental tolerances, an initially diverse arbuscular mycorrhizal fungal community may be necessary to survive an extended drought and maintain a soil structure resistant to erosion [27,28]. Although the community diversity hypothesis is rooted in much theory, it originated in macroecology, which considers communities with apparently lower richness and functional redundancy than soil microbial communities. Therefore, it is unclear how well this hypothesis can predict soil microbial responses to warming. For example, while Hol et al. found that reducing microbial species richness through dilution increased plant biomass and nutritional quality [29], Wertz et al. found no reduction in nitrification or denitrification activity when they diluted soils, and richness did not increase resistance to or recovery from a short period of high temperatures [30]. However, Wertz and colleagues did confirm that functional groups differed in their sensitivity to warming; this knowledge can help us identify steps in the carbon cycle, for example, where ecosystem function is most likely to break down. 2. Functional Implications of Diversity The community diversity hypothesis predicts that diversity-taxonomic diversity, but especially species count (richness)—has a value in and of itself. More diverse communities are not only more productive [18], but they tend to be better able to maintain ecological functions under stress (resistance) and more able to recover function when the stressor is relieved (resilience) [19,20]. These patterns are often explained using niche theory; each organism has a niche defined by the range of environmental conditions it requires to survive, but only realizes a fraction of this potential because of 411 Diversity 2013, 5 Diversity 2013, 5 412 The authors concluded that changes in community composition and loss of keystone species are likely to have a stronger effect on ecosystem function than loss of richness, per se. The authors concluded that changes in community composition and loss of keystone species are likely to have a stronger effect on ecosystem function than loss of richness, per se. Together, these findings imply that sequential loss of taxa in initially rich habitats such as soil should not lead to a significant loss of ecosystem function until one of these keystone species is lost. These patterns open the door to the possibility that a reduction in diversity may lead to a loss of functional stability. Thus, while many soil microbes may appear to be functionally redundant, they differ in their environmental tolerance [35–39], and substrate preferences [40,41], and in some instances may be the sole mediators of an ecological process. Diversity 2013, 5 This is particularly important in the context of the cross-feeding which characterizes many detritivorous pathways [31]. If these syntrophic interactions are highly-specific or dependent on a specific organism, we expect loss of diversity to reduce productivity or some other measure of ecosystem function. As a counter point to the community diversity hypothesis, the keystone species hypothesis predicts that some taxa have a disproportionate effect on ecosystem function. For example, Leptospirillum group III only accounted for 10% of the cells in an acid mine drainage biofilm, but since this organism was the only one with a complete nitrogen fixation pathway, it likely supplied all the nitrogen used by the community [32]. Single phylotypes can be important for ecosystem function in much richer communities too [33]; while members of the Desulfosporosinus only accounted for 0.006% of the microbial population in a peatland soil, they were responsible for the majority of soil sulfate reduction, competing effectively with methanogens for resources and producing the less potent greenhouse gas carbon dioxide instead [34]. These results are consistent with a meta-analysis of studies that manipulated soil microbial diversity, in which Nielsen et al. [31] found that increasing species diversity only improves ecosystem function in species poor (<10 species), but not species rich, soils. Diversity 2013, 5 413 The net effect of warming on soil microbes tends to be increased microbial activity over the short term, which translates to increased soil respiration [12,57,58]. An early lab incubation study of temperate forest soils demonstrated that this increase in activity can be accompanied by shifts in microbial biomass and community composition [59]. However, of the field studies conducted that ran for three years or more [57,58], few that observed increases in microbial activity also examined changes in microbial community composition (Table 2). Despite this, some general patterns are beginning to emerge. For example, there is evidence that not all soil microbial communities respond similarly to warming, with warming more likely to have a negative effect on microbial abundance (density) in cool, dry locations [60]. Furthermore, response to warming is rapid, and perhaps more rapid than shifts in community structure. Therefore it is likely that some of the warming effect is attributable to changes in the active fraction of the biomass, rather than the community’s constituents [59,61,62]. Here, we will discuss how warming has affected microbial community diversity, biomass, and activity in a series of long-term warming experiments in different biomes, before turning to potential drivers of these changes and the consequences for the carbon cycle. Long-term experiments at the Kessler Farm Field Laboratory (KFFL) in the plains of central Oklahoma found increased diversity under warming and drought, suggesting that warming may have somehow ―primed‖ the community to be more resilient and resistant to further disturbance. These tall- grass prairie plots have been continuously warmed 2 °C above ambient since 1999, with half of each plot also being clipped annually [42]. In the second and third full years of treatment, Zhang et al. found that warming treatment increased fungal and decreased bacterial biomass as measured using PLFA analysis, but only in unclipped plots, demonstrating the importance of plant effects in driving the response. Furthermore, the authors found that while there was no effect of warming on total microbial biomass, net N mineralization decreased and there was a significant shift in the substrate utilization profiles, indicating a change in the metabolic capacity of the community. In years five to seven of the KFFL soil warming study, Sheik et al. found the effect of warming on microbial community structure and activity was strongly dependent on whether or not the site was facing drought [43]. 3. Warming Effects on Diversity Climate warming is an abiotic stressor, with the potential to alter the diversity of the soil microbial community, and therefore the range of processes they complete [15]. Of particular interest is the effect of elevated temperatures on soil organic matter decomposition, which a growing number of studies are addressing. A small subset of these incorporate some measure of microbial community diversity, using a biochemical approach such as phospholipid fatty acid (PLFA) analysis, or a molecular approach such as community fingerprinting or sequencing, with some metric of microbial activity or biomass (Table 1). These two approaches can provide different—though complementary—views of microbial communities. Table 1. Geographic and climatic data for sites compared in this paper. Biome Site Coordinates MAT MAP References Tall-grass prairie Kessler Farm Field Laboratory, Washington OK 34.98°N, 97.52°W 16.3 °C 967mm [42–44] Old field grassland National Ecological Research Park, Oak Ridge TN 35.90°N, 84.33°W 14.9 °C 1360 mm [45,46] Mixed hardwood forest Harvard Forest LTER, Petersham MA 42.5°N, 72.18°W 7.6°C 1100mm [6] Temperate mountain forest North Tyrolean Limestone Alps, Austria 47.58°N, 11.64°E 5.7 °C 1480mm [47] Taiga boreal forest Delta Junction, AK 63.92°N, 145.73°W −2.6 °C 1290mm [48–50] Sub-Arctic blanket bog Abisko, Sweden 68.21°N, 18.49°E −0.6 °C 352mm [51,52] Sub-Arctic heath Abisko, Sweden 68.19°N, 18.51°E −0.6 °C 352mm [53] Sub-Antarctic Signy Island 60.72°S, 45.38°W −2 °C 400mm [54–56] Sub-Antarctic Falkland Islands 51°S, 59.05°W 7.9 °C, 575mm [54–56] Antarctic Anchorage Island 67.57°S, 68.13°W −2 °C 500mm [54–56] Table 1. Geographic and climatic data for sites compared in this paper. Diversity 2013, 5 In regular precipitation years, warming increased population size, but that the community was less rich and diverse. Under drought, however, warming decreased population size and increased its diversity, richness, and evenness without any change in community composition. The authors suggested that shifts in relative abundance of extant species rather than recruitment of novel ones drives ecosystem function under altered climate. As a follow up to this study, Zhou et al. used high-throughput technologies to determine the functional significance of these shifts in diversity and potential feedback response of microbial communities to eight years of warming [44]. Pyrosequencing of the 16S ribosomal RNA gene showed a large shift in the community composition, and despite data collection in a non-drought year, higher microbial abundance using GeoChip and PLFA analysis [44]. However, not all populations involved in SOM decay were equally stimulated by warming; while those involved in the degradation of labile compounds such as starch, hemicellulose, cellulose, and chitin were stimulated, those involved in lignin degradation were not, and activity of the ligninase peroxidase even decreased. Soil respiration increased at this site, but its temperature sensitivity decreased, suggesting a weakened positive feedback to the climate system. Since there was a shift in the taxa detected without a change in Diversity 2013, 5 Diversity 2013, 5 414 diversity at this site, this implies immigration or recruitment of microbes from the rare biosphere may be responsible for the shift towards labile substrate use. Table 2. In situ field studies examining the effect of warming on soil microbial communitya. Biome Location Heatingb Duration Key Findingsc Ref. Tall-grass prairie Kessler Farm Field Lab, Washington OK +1.8–2.7 °C (2 °C), IR, continuous 2−3 years MC: Strong overall shift in absence of clipping MB: No overall change in biomass; increased fungid MA: No change in C mineralization [42] 4−6 years MC: Increased diversity but no shift under drought; strong overall shift if above-normal precipitation MB: Decreased population size in drought years; increased if above-normal precipitation. MA: n.d. [43] 8 years MC: Strong overall shift MB: Increased microbial PLFA’s; non-significant increase in bacterial and fungal biomass, no change in F:B ratio MA: Increased C mineralization; increased labile C- degrading genes, unchanged recalcitrant C-degrading genes [44] Old field grassland National Ecological Research Park, Oak Ridge TN +3 °C, OTC, continuous 2−3 years MC: Strong overall shift MB: Increased Firmicutes, decreased Gram- negative bacteria, arbuscular mycorrhizal fungi and saprophytic fungi MA: n.d. [45] 4 years MC: Strong overall shift MB: Increased fungal abundance; decreased bacteria (QPCR) MA: n.d. [46] Mixed hardwood forest Harvard Forest LTER, Petersham MA +5 °C,HC, continuous 12 years MC: Strong overall shift MB: Decreased microbial biomass, decreased fungi, increased Gram-positives and Actinomycetes MA: Decreased biomass-specific respiration [6] Temperate mountain forest North Tyrolean Limestone Alps, Austria +4 °C, HC, snow-free seasons only 4−6 years MC: No overall changes MB: No overall change in biomass; decreased Actinomycetes and Gram-negatives. MA: Increased biomass-specific respiration, stress biomarkers [47] 2. In situ field studies examining the effect of warming on soil microbial communitya. Diversity 2013, 5 Diversity 2013, 5 415 Table 2. Cont. Biome Location Heatingb Duration Key Findingsc Ref. Sub- Arctic Sub-Arctic heath, Abisco, Sweden +1.2−2 °C, OTC, snow-free seasons only 15 years MC: No overall changes MB: decreased microbial biomass C; increased fungal:bacterial ratioe; no change in G+: G-MA: n.d. [53] Antarctic and sub- Antarctic Falkland, Signy,& Anchorage Islands +0.5–2 °C,OTC, continuous 3 years MC: No overall changes MB: Increased fungi, bacteria, and ratio of Alphaproteobacteria to Acidobacteria MA: Increased N-cycling [56] a Studies were included in this table if they looked at microbial community composition and at least one of microbial biomass, and microbial community were examined. Diversity 2013, 5 416 system. This is consistent with the finding of Rinnan et al., where it took more than 15 years for 1.2–2 °C of experimental warming to affect microbial community composition in a subarctic heath at the same site [53] although addition of nitrogen at a rate designed to mimic warming-induced mineralization led to an effect sooner [64]. The authors hypothesized that nitrogen addition increased plant productivity, but there was a time delay between the increased plant productivity and support of greater microbial biomass. In a final example, after three years of continuous 0.5–2 °C warming at three sites in the Antarctic and sub-Antarctic, Yergeau et al. found no effect of warming on the rRNA profile of the community, but higher microbial abundance and Alphaproteobacteria:Acidobacteria ratio. Sequences for a number of processes were reduced under warming, including cellulose, chitin, and lignin degradation, although those for nitrogenase were higher under warming [56]. Together these results imply that the ecological functions were relatively redundant in the initial community and distributed across the phylogenetic tree, indicating both functional and phylogenetic diversity aided with the survival of the community through time. However, Allison and Treseder found that the fungal community in a taiga boreal forest of Alaska was highly-sensitive to just 0.5 °C of seasonal warming; microbial biomass, soil respiration and chitinase activity were reduced by about half late in the growing season [48]. Using the nucleotide analog BrdU, they also observed a shift of the active fungal community away from a Thelephorid fungus and towards Ascomycetes and Zygomycetes. The relatively large change in community composition for a small increase in temperature implies a keystone species may have been lost, changing the functionality of the community and allowing an otherwise apparently small pressure to cascade. Long-term field warming experiments in two temperate forested sites provide contrasting consequences of warming on microbial community composition. Following four years of 4 °C seasonal warming in the North Tyrolean Limestone Alps of Austria, Schindlbacher et al. found an increase in respiration and a non-significant increase in the rRNA:DNA ratio, another measure of activity [47]. There was no change in microbial biomass C or the PLFA profile of the community as a whole, but there was a slight reduction in one PLFA associated with Actinomycetes and Gram-negative bacteria. Diversity 2013, 5 They must also have at least three full growing seasons of data, unless there is subsequent data from the same site to corroborate the early warming effects. b Heating methods include ―OTC,‖ passive open-top chambers; ―HC,‖ resistance heating cables; ―CTC,‖ closed-top chambers; and ―IR,‖ infrared radiators suspended 1.5m off the ground. In cases where there is no feedback regulation of warming treatment, temperature is provided as a range followed by the mean treatment in brackets. c Key findings are for ―MC,‖ microbial community profiles; ―MB,‖ microbial biomass; and ―MA,‖ microbial activity, where ―n.d.‖ indicates no data for this category. d This trend observed only in treatments without clipping. e top 5cm only. Th ff f i i bi l d b hi h l i d [1 63] Taiga boreal forest Delta Junction, AK +0.5 °C,CTC, continuous 0–3 years MC: Change in active (BrdU) fungi MB: >50% decrease in fungi and bacteria MA: Lower chitinase, lower respiration rate in late growing season. [48] Taiga boreal forest Delta Junction, AK +1.2 °C, CTC growing season only 0-3 years MC: No overall changes in fungal community MB: n.d. M.A.:Increased β-glucosidase and N-acetyl- glucosaminidase activity; no change in respiration [49] Sub- Arctic Blanket bog, Abisko, Sweden +0.3−2.8 °C (1°C), OTC, seasonal 9 years MC: No overall changes MB: Decreased microbial biomass under summer warming MA: No change in soil peptidase activity; increased N-flux [52] [56] The effects of warming on microbial processes are expected to be greatest at higher latitudes [1,63], though studies indicate that effects are mixed, and some ecosystems may in fact be more resistant to warming than originally thought. For example, in a blanket bog in northern Sweden, Weedon et al. [52] found that 9 years of seasonal 0.2–2.2 °C warming increased soil respiration, particularly from older, deeper carbon [51], and increased nitrogen cycling, but did not lead to any significant changes in vegetation [52]. However, warming did not change the DGGE profile of the microbial community or peptidase activity, but decreased the microbial abundance detected using qPCR. The relative resistance of the microbial community to elevated temperatures may reveal a tight association to plants in this 4. Mechanisms and Consequences of Warming Effects on Microbial Diversity A number of patterns emerge from the warming experiments above, the first of which is the lack of congruence between phylogenetic and functional diversity in the studies that measured both. The second pattern which emerges is that warming only sometimes affects the efficiency with which microbes convert carbon to biomass (carbon use efficiency), leading to shifts to a community characteristic of higher carbon use efficiency in some instances [6], with no predictable change [45] or a reduction in others [47,56]. The third pattern is that changes in microbial biomass are inconsistent, increasing at some sites [43,44,56] and decreasing at others [6,7,45,48,52] while at some sites there was no change [47] or a delayed response [42,64]. These inconsistencies may be methodological, site- specific, or due to plant communities, which are well known links to below ground communities [66]. The lack of apparent congruence between phylogeny and function may be due to a ―priming‖ effect not observed in all studies, where of one stress (for example, drought or fire) favors growth of species or functions that aid in providing resistance to a secondary stress (experimental warming). The primed species could derive from an initially diverse community (community diversity hypothesis), or a handful of organisms whose keystone role is their ability to weather the change in the environment. The increased diversity under drought and warming at the Great Plains site implies the former [43], while Yergeau’s Antarctic and sub-Antarctic study indicates the latter. In this instance, warming reduced functional gene richness with no effect on microbial abundance or activity [56]. This may indicate that functional redundancy of the community was reduced by warming, and so the community may be more sensitive to further disturbances. Carbon use efficiency is a key determinant of the long-term stability of carbon in soil, because microbial biomass, rather than recalcitrant plant matter, is most resistant to decay [67–69], and because microbial activity contributes significantly to terrestrial CO2 emissions [11,12]. Fierer et al. loosely classified bacteria into two groups based on the kinds of carbon they prefer and the efficiency with which they grow: copiotrophs, which grow rapidly but with low efficiency in high resource environments, and oligotrophs, which grow slowly but efficiently, thriving in low resource environments [70]. While Proteobacteria and Bacteroidetes tended towards copiotrophy, Acidobacteria, Verrucomicrobia, and the Gram-positive phyla Actinobacteria and Firmicutes leaned towards oligotrophy [70]. Diversity 2013, 5 Diversity 2013, 5 417 or functions are more important. Approaches that resolve finer changes in the microbial community, such as the metagenomic study we are completing at the Harvard Forest, are necessary if we are to better understand how microbial community composition affects soil carbon cycling, and how this will change under future climate. Diversity 2013, 5 These large increases in activity without much change in community identity suggest that warming improved conditions for the extant microbes without necessarily favoring one group over another, creating a more productive community. Alternatively, twenty years of warming at in a mixed deciduous stand at the Harvard Forest in Massachusetts led to an initial increase in respiration, followed by a subsequent decrease to control levels, and then an increase to above ambient again (Jerry Melillo, personal communication) [65]. This secondary increase in respiration may be explained under the community diversity hypothesis, which suggests that an overall increase in diversity has resulted in communities more resistant to higher and more extreme temperatures, or by the keystone species hypothesis, which suggests an increase in populations capable of degradation of more recalcitrant or previously physically protected soil carbon. In a PLFA survey of the soil microbial community at this site just before soil respiration increased again, Frey et al. found evidence to support the keystone species hypothesis; while fungal and bacterial biomass decreased overall, there was a relative increase in Gram-positives and Actinomycetes, and a strong shift in PLFA profile despite no change in its diversity[6]. However, PLFA markers don't align well with phylogeny, and metabolic capacities vary non-randomly with phylogeny [17], so the data do not have enough resolution to decisively say whether whole community diversity or keystone species 4. Mechanisms and Consequences of Warming Effects on Microbial Diversity This implies that in environments where Bacteroidetes or β-Proteobacteria increase in relative abundance, carbon use efficiency has declined, favoring respiration, soil carbon loss, and positive feedbacks to climate. However, studies have provided mixed support for this so far (Table 2), with one study finding increased oligotrophs and lower biomass-specific respiration [6], while a second found no obvious change [45], and two more found increased copiotrophs [56] and biomass-specific respiration [47], an indication of low carbon use efficiency. Unfortunately, explicit conclusions regarding the effects of experimental warming on carbon use efficiency are few and far between, and inconsistencies in the methods used [8,71] precludes a more direct generalization of this microbial response. Furthermore, we have found that when we extract data from long-term warming Diversity 2013, 5 418 studies and calculate proxies for carbon use efficiency, the magnitude and direction of the change depends on the metric used. Changes in community structure may mask effects of warming on carbon use efficiency. All microbes inherently use labile substrates at a higher efficiency than complex ones [8,72], copiotrophs and oligotrophs differ in their substrates of choice [70], warming changes substrate quality [6,44], and carbon use efficiency decreases with temperature [72]. At the Harvard Forest, warming induced a significant shift in the microbial community [6], which was paralleled by a reduction in soil respiration and thermal adaptation of microbial carbon use efficiency on phenol [8]. It is unclear whether changes in carbon use efficiency are directly driven by changes in soil carbon chemistry or indirectly through warming- or substrate-mediated shifts in community structure. Unfortunately, in many cases the lack of taxonomic resolution in community data following disturbance prevents determination of whether shifts towards or away from oligotrophy was driven by changes in few keystone or many taxa. It is likely edaphic factors such as secondary soil drying with warming, and changes in soil carbon availability, also play a role in microbial response to warming. In studies which test both warming and precipitation, it is often observed that precipitation treatment has an as strong if not stronger effect on soil microbial communities than warming [45,73,74]. However, this trend is muddied by inconsistencies in methods and associated errors in estimation. Diversity 2013, 5 419 Diversity 2013, 5 warming on a time scale much faster than the dominant vegetation, would lead to depletion of the soil carbon pool. While this was true for one temperate forest [6,7], it was not the case for another [47,71], or in a subarctic heath, despite the apparently slow vegetation response there [53]. Microbes adapt to changes in carbon quality and temperature, through plasticity in their metabolisms or through shifts in community. If conditions favor more efficient microbes, more of the carbon assimilated will be directed to microbial biomass, a relatively stable carbon pool, but more biomass also means greater respiration and greater carbon loss. As soil carbon chemistry becomes more complex [85], or as temperatures increase, carbon use efficiency decreases independent of any change in the community [72]. Understanding which of these processes has dominated—as well as how diversity may affect the resistance of the soil microbial community to further change—is important for understanding how feedbacks to climate are mediated in the soil. 4. Mechanisms and Consequences of Warming Effects on Microbial Diversity For example, PLFA profile can differ depending on the soil type and lipid extraction method used [75–77], ―universal‖ primers for qPCR are never universal [41], and chloroform fumigation extraction efficiency is known to be pH sensitive [78]. However, microbes differ in their sensitivity to moisture stress [23,79], so moisture likely directly affect community composition and function, though the relative contributions of warming to drying are not well understood. Inconsistencies in biomass may also be due to difference in plant effects across studies, many of which conclude that the strength and direction of the effect depends on how plants respond [47,56,74,80]. Soil warming induces nitrogen mineralization, which increases plant productivity [12,44], and plant community composition often also changes with warming [81–84]. Since plants stressed by warming may increase production of structural carbohydrates and secondary compounds that reduce decomposability of senesced litter [85], species shifts may stimulate soil microbes and result in increased N mineralization as a side effect. In fact, while warming at the Great Plains site led to an increase in labile (and microbially available) carbon [44], it led to a reduced capacity to use labile substrates at the Harvard Forest [6], where fine root production [86] and soil carbon quality declined [7]. These differences in microbial activity are unlikely to be purely due to direct effects of increased nitrogen availability; rate of nitrogen cycling [11,44] and soil N availability [42,44,87] have been affected similarly in both sites. While aboveground biomass removal has demonstrated that moderate warming is unlikely to have direct effects on the microbial community [42], direct determination of the relative role of changes in carbon and nitrogen availability on microbial community and activity is complicated by the fact that clipping reduces both C and N availability [88]. Nonetheless, the increase in diversity of genes involved in labile carbon utilization at the grassland site shows that many taxa were involved in the warming response, supporting the community diversity hypothesis. The patterns outlined above have important implications for the carbon cycle and feedbacks to climate. Warming initially increases respiration, which, if heterotrophic soil microbes respond to References 1. Davidson, E.A.; Janssens, I.A. Temperature sensitivity of soil carbon decomposition and feedbacks to climate change. Nature 2006, 440, 165–173. 1. Davidson, E.A.; Janssens, I.A. Temperature sensitivity of soil carbon decomposition and feedbacks to climate change. Nature 2006, 440, 165–173. 2. Schlesinger, W.H.; Andrews, J.A. Soil respiration and the global carbon cycle. Biogeochemistry 2000, 48, 7–20. 2. Schlesinger, W.H.; Andrews, J.A. Soil respiration and the global carbon cycle. Biogeochemistry 2000, 48, 7–20. 3. Conrad, R. 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To meet the goal of an improved understanding of processes driving the microbial response to warming, we need improved understanding of microbes, from the level of the gene and the physiology of individual microbes all the way to microbial community composition and interactions. Long term warming studies, especially at LTER sites such as the Harvard Forest, and the cross-latitudinal NEON sites, are especially suited for this because of the immense amount of biogeochemical and other site data. To understand the role of microbes in ecosystem functioning, there are a wide range of choices to make in terms of measuring diversity, and in the end, relatively few direct measures of the contributions of microbes to ecosystem function. Microbes function on a scale which by definition we cannot see, yet drive the majority of energy and nutrient transformations which shape ecosystem function. New technologies such as NanoSIMS which, when coupled with FISH staining, enable us to visualize how microbes interact with each other and their environment, are changing this. Simpler techniques such as co-culture of novel isolates may also help improve our understanding for less money, in addition to ―ground-truthing‖ some of the functions assigned to phylotypes in the masses of metagenomic data we are now able to collect. Understanding the ecological function of microbial communities in their natural environment is essential, but microbial processes are often placed in a ―black box‖ because the microbes present could not be determined or quantified. Rapid advances in genomic sequencing technology are transforming microbiology and in particular ecological research, and yet there is still value in making more traditional measures of microbial biomass and diversity in order to understand the portion of carbon or biomass that populations occupy, which is also a measure of functional potential. Though this is not always the case, genomic, genetic or informatics approach should be undertaken along with functional or absolute measures of diversity. Overall, we have much to learn about microbial diversity and its importance in the context of carbon cycling, and we have better tools than ever to do so. 420 Diversity 2013, 5 Conflict of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. Diversity 2013, 5 421 Diversity 2013, 5 14. Ptacnik, R.; Solimini, A.G.; Andersen, T.; Tamminen, T.; Brettum, P.; Lepistö, L.; Willén, E.; Rekolainen, S. 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Zhou, Y.; Tang, J.; Melillo, J.M.; Butler, S.; Mohan, J.E. Root standing crop and chemistry after six years of soil warming in a temperate forest. Tree Physiol. 2011, 31, 707–717. 87. Bradford, M.A.; Watts, B.W.; Davies, C.A. Thermal adaptation of heterotrophic soil respiration in laboratory microcosms. Global Change Biol. 2010, 16, 1576–1588. 88. Belay-Tedla, A.; Zhou, X.; Su, B.; Wan, S.; Luo, Y. Labile, recalcitrant, and microbial carbon and nitrogen pools of a tallgrass prairie soil in the US Great Plains subjected to experimental warming and clipping. Soil Biol. Biochem. 2009, 41, 110–116. © 2013 by the authors; licensee MDPI, Basel Switzerland. Diversity 2013, 5 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2013 by the authors; licensee MDPI, Basel Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2013 by the authors; licensee MDPI, Basel Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W4391252625	https://www.ijfmr.com/papers/2024/1/12493.pdf	English	null	Diabetes and Oral Health: A Review	International Journal For Multidisciplinary Research	2,024	cc-by-sa	2,090	Abstract A healthy mouth is important for everyone, but high blood sugar can make it harder to keep mouth healthy. Diabetes mellitus is a major source of morbidity and mortality and a global epidemic that should be taken very seriously. Diabetes Mellitus is a chronic, non-communicable metabolic disease characterized by abnormalities in insulin production, action, or both. The prevalence of diabetes mellitus, a prevalent systemic disease, is rising in the modern world. Diabetes is a prevalent illness that has an effect on dental care due to its concurrent oral symptoms. This review aims to provide an overview of diabetes prevalence, symptoms, diagnosis, and treatment options, along with dental care considerations for those with the condition. The general population and people who have diabetes have demonstrated an unacceptable level of awareness regarding the bidirectional association between diabetes mellitus and general oral health, while physicians and dental practitioners have a satisfactory level of understanding. Keywords: Oral Health, Dental caries, hyperglycemia, diabetes mellitus, salivary dysfunction, periodontal disease, infection, awareness, and a multidisciplinary approach Diabetes and Oral Health: A Review Dr.Daxesh Shasi kant Modi1, Dr.Sheshkaran Singh2, Dr.Suraj Ojha3, Dr.Kumari Pramila4 Dr.Daxesh Shasi kant Modi1, Dr.Sheshkaran Singh2, Dr.Sura Dr.Kumari Pramila4 1Consultant Eye Surgeon –Drishti Eye & Dental Hospital Barmer,Rajasthan. 2Consultant Dental Surgeon –Drishti Eye & Dental Hospital Barmer,Rajasthan. 3Consultant Ayush Physician K.K.Medicentre Barmer,Rajasthan. 4MPH Scholar AIIMS Jodhpur,Rajasthan. Diabetes Symptoms Diabetes Symptoms • Urinate (pee) a lot, often at night • Are very thirsty • Lose weight without trying • Are very hungry • Have blurry vision • Have numb or tingling hands or feet • Feel very tired • Have very dry skin • Have sores that heal slowly • Have more infections than usual • Have more infections than usual Prevalence of the most common oral diseases Periodontal diseases-A majority of any population in the world suffer from periodontal disease (gum disease). The reversible form, gingivitis, manifests itself as bleeding and swelling of only the soft tissues around the teeth and affects 50%–90% of the adult population worldwide Other disease like Caries, Tooth loss, Periapical lesions, Peri-implant diseases, Early tooth eruption, Dry mouth, Candidiasis, Delayed wound healing, Iron precipitation in dental tissue, Oral cancer etc. PREVALENCE AND INCIDENCE OF DIABETES- PREVALENCE AND INCIDENCE OF DIABETES- The prevalence of diabetes has risen by 30 to 40% over the last 20 years, and as the population ages, so too will the burden of diabetes and its complications. At any age, obesity is a significant risk factor for the development of diabetes, and throughout the past 20 years, there has been a noticeable rise in the proportion of overweight kids and teenagers. In conclusion, diabetes is a medical condition that is on the rise and that can impact individuals of all ages. It also has associated morbidity and death. Introduction Diabetes is a well-known chronic systemic illness characterized by elevated blood glucose levels mostly brought on by low insulin levels or improper insulin action. Type 1 diabetes mellitus, or insulin- dependent diabetes(IDDM), and type 2 diabetes mellitus, or Non-insulin-dependent diabetes, are the two primary categories into which diabetes is typically classified (NIDDM) And Gestational diabetes (diabetes while pregnant) Adolescents and younger people are more likely to develop type 1 diabetes because of total insulin insufficiency brought on by the pancreatic β-cells' degeneration. 90-95% of diabetics have type 2 diabetes, which is mostly caused by insulin resistance in adults. Symptoms of diabetes for both types 1 and 2 are common with hyperglycemia, polyphagia, polydipsia, and polyuria. Complications of diabetes mellitus have been seen in the long-term comprise of neuropathy, nephropathy, retinopathy, genitourinary, cardiovascular, and sexual dysfunction. Cardiovascular manifestations mainly include hypertension, atherosclerosis, peripheral vascular disease, and cerebrovascular disease Research has shown that the oral cavity is one of the body's systems that can be impacted by diabetes. Common symptoms include dental caries, an inhibition of salivary flow, diseases of the oral mucosa such as glossodynia, lichen planus, candidiasis, xerostomia, burning mouth Volume 6, Issue 1, January-February 2024 IJFMR240112493 1 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com syndrome, recurrent aphthous stomatitis, and most significantly, delayed wound healing, tongue abnormalities, halitosis gingivitis and periodontitis. E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com syndrome, recurrent aphthous stomatitis, and most significantly, delayed wound healing, tongue abnormalities, halitosis gingivitis and periodontitis. syndrome, recurrent aphthous stomatitis, and most significantly, delayed wound healing, tongue abnormalities, halitosis gingivitis and periodontitis. Diagnosis Clinicians can evaluate the blood glucose control of their patients using a variety of diagnostic methods. The fasting plasma glucose (FPG) test gauges blood glucose levels after a minimum of eight hours without consuming any calories. FPG levels of 5.6 mmol/L or less are regarded as normal.The hemoglobin A1C (HbA1c) test yields data on the three months' average blood glucose levels. Clinicians utilize this test, which is expressed as a percentage, to evaluate DM control and management. A normal HbA1C level in a healthy, non-diabetic patient is 5.7% or less. Diabetes systemic complications: Diabetic neuropathy-Diabetic neuropathy is a type of nerve damage that can occur if you have diabetes. High blood sugar (glucose) can injure nerves throughout the body. Diabetic neuropathy most often damages nerves in the legs and feet. Diabetic retinopathy-Diabetic retinopathy is a diabetes complication that affects eyes. It's caused by damage to the blood vessels of the light-sensitive tissue at the back of the eye (retina). Diabetic nephropathy-Diabetic nephropathy is a serious complication of type 1 diabetes and type 2 diabetes. It's also called diabetic kidney disease. diabetic nephropathy slowly damages the kidneys' filtering system. Early treatment may prevent this condition or slow it and lower the chance of complications.Diabetic kidney disease can lead to kidney failure. Oral manifestations in patients with diabetes mellitus-Dry mouth (xerostomia), tooth decay (including root caries), periapical lesions, gingivitis, periodontal disease, oral candidiasis, burning mouth (especially glossodynia), altered taste, geographic tongue, coated and fissured tongue, oral lichen planus Volume 6, Issue 1, January-February 2024 IJFMR240112493 2 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com (OLP), recurrent aphthous stomatitis, increased susceptibility to infections, and impaired wound healing are among the oral manifestations and complications associated with diabetes mellitus. (OLP), recurrent aphthous stomatitis, increased susceptibility to infections, and impaired wound healing are among the oral manifestations and complications associated with diabetes mellitus. (OLP), recurrent aphthous stomatitis, increased susceptibility to infections, and impaired wound healing are among the oral manifestations and complications associated with diabetes mellitus. Dental Management Considerations- Dental Management Considerations- Dentists need to be aware of crucial dental management issues before beginning treatment for a diabetic patient. By doing this, the dental surgeon can lessen the possibility of an oral diabetes consequence of the condition and the risk of an intraoperative diabetic emergency. The first step in treating a diabetic patient effectively is for the dentist to perform a system assessment and obtain a complete medical history. The patient's recent blood glucose levels, at-home monitoring habits, frequency of HbA1C tests and their results, and frequency of hypo- or hyperglycemic episodes should all be gathered by dentists. Additionally, the dentist should go over the current DM management plan, which includes all medication dosages and schedules as well as any dietary or activity modifications. • Consult with patient’s physician to assess diabetes control. • Consult with patient’s physician to assess diabetes control. • Update medical history and medications and review systems at each appointment. • Confirm that patient has eaten and taken medications before initiating treatment. • Anticipate and be prepared to manage hypoglycemia. • Prevent, treat and eliminate infections promptly. • Do not use or recommend aspirin-containing compounds. • Ensure excellent oral hygiene and provide profound preventive care. Reinforce regular diet and medication regimen before and after dental appointments. • Reinforce regular diet and medication regimen before and after dental appointments T k l t di if ti t i hi h i k i li h i • Take glucometer reading if patient is high risk, on insulin or having surgery. rnational Journal for Multidisciplinary Research (IJFMR) Missing or moving teeth can make it difficult to consume a healthy diet, which can result in malnutrition and a lower quality of life due to oral health issues. Additionally, replacing lost teeth by prosthodontics with fixed bridges or dentures may enhance eating habits, food preferences, self-esteem, and overall quality of life. Healthcare practitioners have a new weapon at their disposal for managing and preventing diabetes of any kind awareness of the critical role that oral health plays in the disease. Diabeties mellitus (DM) screening in dentistry and oral health education in medicine, when combined with appropriate reciprocal referrals and possible co-management of patients by both medical and dental professionals, should have a good chance of improving mutual patients' quality of life as well as their overall health. Conclusion According to recent estimates, 318 million individuals globally are thought to have diabetes. Any dentist practicing in india would surely see a lot of patients with DM throughout the course of their career. Given the wide range of oral symptoms of diabetes mellitus and the potential for an intraoperative diabetic emergency, it is critical for dental professionals to understand the disease's effects on dental treatment. By having a comprehensive awareness of diabetes mellitus (DM) and its dental management implications, the dental healthcare team can collaborate efficiently to deliver superior oral health care to patients with diabetes Dental health and diabetes are negatively correlated in a vicious cycle.DM has an adverse effect on the majority of oral illnesses and ailments.The administration of non-surgical periodontal therapy and the extraction of teeth affected by periodontitis or pulpal necrosis resulting from significant dental caries decrease blood levels of inflammatory markers and glycated hemoglobin. Volume 6, Issue 1, January-February 2024 IJFMR240112493 3 14. Jain A, Chawla M, Kumar A, et al. Management of periodontal disease in patients with diabetes- good clinical practice guidelines: a joint statement by Indian society of periodontology and research society for the study of diabetes in India. J Indian Soc Periodontol. 2020;24(6):498–524. doi:10.4103/jisp.jisp_688_20 Badiah B. A preliminary survey on awareness of periodontal risk and oral health practices among diabetic patients in hospital Kuala Lumpur. Malaysian Dent J. 2012;34(1):1–7. REFERENCES 1. American Diabetes Association. (2010). Diagnosis and classification of diabetes mellitus. Diabetes Care. 33(Suppl 1):S62–69.doi:10.2337/dc10-S062 2. Al Qahtani NA, Joseph B, Deepthi A, Vijayakumari BK. Prevalence of chronic periodontitis and its risk determinants among female patients in the Aseer region of KSA. J Taibah Univ Med Sci 2017;12:241–8. https://doi.org/ 10.1016/j.jtumed.2016.11.012 3. Adams PF, Hendershot GE, Marano MA. Current estimates from the National Health Interview Survey, 1996. Hyattsville, Md.: National Center for Health Statistics, Vital Health Statistics; 1999:10(200). 4. Diabetes Canada Clinical Practice Guidelines Expert Committee, Punthakee Z, Goldenberg R, Katz P. Definition, classification and diagnosis of diabetes, prediabetes and metabolic syndrome. Can J Diabetes. 2018;42(Suppl. 1):S10-5. 5. Gosmanov AR, Gosmanova EO, Dillard-Cannon E. Management of adult diabetic ketoacidosis. Diabetes Metab Syndr Obes. 2014;7:255-64. 6. Al-Maskari AY, Al-Maskari MY, Al-Sudairy S. Oral manifestations and complications of diabetes mellitus: a review. Sultan Qaboos Univ Med J. 2011;11(2):179-86. 7. McKenna SJ. Dental management of patients with diabetes. Dent Clin North Am. 2006;50(4):591- 606. 8. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet 2005;366:180920. https://doi.org/10.1016/ S0140-6736(05)67728-8 9. Khan T. Oral manifestations and complications of diabetes mellitus: a review. Int J Med Health Res. 2018;4:50–52. doi:10.22271/ ijmhr 10. 10 . Al-Maskari AY, Al-Maskari MY, Al-Sudairy S. Oral manifestations and complications of diabetes mellitus: a review. SultanQaboos Univ Med J. 2011;11(2):179–186. 11. Mohanty S, Mohanty N, Rath S. Analysis of oral health complications in diabetic patients – a diagnostic perspective. J Oral Res. 2018;7(8):278–281. doi:10.17126/joralres.2018.072 12. Poudel P, Griffiths R, Wong VW, et al. Perceptions and practices of diabetes educators in providing oral health care: a Qualitative Study. Diabetes Educ. 2018;44(5):454–464. doi:10.1177/ 0145721718796055 Volume 6, Issue 1, January-February 2024 IJFMR240112493 4 International Journal for Multidisciplinary Research (IJFMR) 13. Davis TME, Bruce DG, Curtis BH, Barraclough H, Davis WA.The relationship between intensification of blood glucose-lowering therapies, health status and quality of life in type 2 diabetes: the Fremantle Diabetes Study Phase II. Diabetes Res Clin Pract. 2018;142:294–302. doi:10.1016/j. diabres.2018.05.047 14. Jain A, Chawla M, Kumar A, et al. Management of periodontal disease in patients with diabetes- good clinical practice guidelines: a joint statement by Indian society of periodontology and research society for the study of diabetes in India. J Indian Soc Periodontol. 2020;24(6):498–524. doi:10.4103/jisp.jisp_688_20 Badiah B. A preliminary survey on awareness of periodontal risk and oral health practices among diabetic patients in hospital Kuala Lumpur. Malaysian Dent J. 2012;34(1):1–7. Volume 6, Issue 1, January-February 2024 IJFMR240112493 5
W4300934116.txt	https://zenodo.org/records/5399781/files/source.pdf	fr		Un nouveau genre de Pseudachorutinae (Collembola) des sables littoraux de la Province du Natal (République Sud africaine)	Zenodo (CERN European Organization for Nuclear Research)	2,002	public-domain	1,394	Un nouveau genre de Pseudachorutinae (Collembola) des sables littoraux de la Province du Natal (République Sud africaine) Jean-Auguste BARRA Université Louis Pasteur, Laboratoire de Zoologie, 12, rue de l’Université, F-67000 Strasbourg (France) barra@neurochem.u-strasbg.fr Barra J.-A. 2002. — Un nouveau genre de Pseudachorutinae (Collembola) des sables littoraux de la Province du Natal (République Sud Africaine). Zoosystema 24 (1) : 177-180. MOTS CLÉS Collembola, Pseudachorutinae, Najtafrica n. gen., nouveau genre, Province du Natal. RÉSUMÉ Un nouveau genre, Najtafrica n. gen., pour l’espèce Stachorutes riebi Barra, 1994 est créé et une redescription de cette espèce est donnée. Elle est caractérisée par un aspect général de type pseudachorutinien sans paratergite saillant et par un grain tégumentaire fort. L’organe postantennaire est moruliforme ; 2 + 2 cornéules. L’article antennaire IV porte sept sensilles ; la chétotaxie est réduite ; les tibiotarses I, II, III ont respectivement 11, 11, 10 soies ; la furca est courte avec trois soies par dens. Cette espèce a été trouvée dans les sables humides littoraux. KEY WORDS Collembola, Pseudachorutinae, Najtafrica n. gen., new genus, Natal Province. ABSTRACT A new Pseudachorutinae genus (Collembola) in the littoral sand of Natal Province (South African Republic). A new genus, Najtafrica n. gen., is created for the species Stachorutes riebi Barra, 1994 and a redescription of this species is given. It is characterised by a pseudachorutinian aspect without leaping paratergits and a strong tegumental grain. The postantennal organ is moruliform; 2 + 2 eyes. The antennal article IV sustains seven blunt setae. Chaetotaxy is reduced. Tibiotarsi I, II, III with 11, 11, 10 respectively. Furca short with three setae per dens. The species was found in humid littoral sand. ZOOSYSTEMA • 2002 • 24 (1) © Publications Scientifiques du Muséum national d’Histoire naturelle, Paris. www.mnhn.fr/publication/ 177 Barra J.-A. INTRODUCTION Parmi les espèces décrites (Barra 1994) des sables littoraux de la plage de Santa Lucia, l’une d’elles a été classée dans le genre Stachorutes, essentiellement sur une interprétation incorrecte de l’organe postantennaire. Une étude plus approfondie des spécimens et la confrontation avec les genres proches de Stachorutes n’a pas permis d’intégrer cette espèce dans un genre connu. Les caractères différentiels suffisamment divergents ont conduit à créer le genre nouveau : Najtafrica n. gen. SYSTÉMATIQUE Famille NEANURIDAE Börner, 1901 Sous-famille PSEUDACHOTINAE Massoud, 1967 Genre Najtafrica n. gen. (Fig. 1) ESPÈCE-TYPE. — Stachorutes riebi Barra, 1994: 184, 185, figs 8-15. L OCALITÉ - TYPE . — Afrique du Sud, Province du Natal, plage de Santa Lucia, à environ 100 km au sud de Sodwana Bay, dans des sables marins dunaires sous la végétation pionnière, 6.II.1992, leg. J.-P. Rieb. MATÉRIEL EXAMINÉ. — Holotype femelle et cinq paratypes. Les échantillons-types déposés au Musée royal de l’Afrique Centrale de Tervuren sous Stachorutes riebi (holotype et trois paratypes) ont été transférés au Muséum national d’Histoire naturelle de Paris. ÉTYMOLOGIE. — Nom dédié à Judith Najt qui a initié et largement contribué au reclassement de cette espèce. DIAGNOSE. — Aspect général de type « pseudachorutinien » sans paratergite saillant. Couleur blanche. Grain tégumentaire fort. Article antennaire IV avec des sensilles bien différenciées, organite subapical et microsensille dorso-externe présents, vésicule apicale non rétractile. Deux cornéules de chaque côté de la tête. Organe postantennaire moruliforme. Maxilles à lames fines emboitées, mandibules dentées. Furca courte. Absence de soie médiane sur le sternite abdominal III. REDESCRIPTION De 0,4 à 0,5 mm de longueur, blanc en alcool à l’exception des taches oculaires. En plus des caractères déjà décrits (Barra 1994), on peut mettre l’accent sur les points suivants : l’article 178 antennaire IV possède bien sept sensilles cylindriques, un organe subapical, une microsensille dorso-externe et une vésicule apicale trilobée (Fig. 1A). Organe sensoriel de l’article antennaire III composé de cinq sensilles. Organe postantennaire moruliforme (OPA) avec 16-18 vésicules simples (Fig. 1B). 2 + 2 cornéules pigmentées de même taille que les grains tégumentaires. Absence de la soie a0 sur la tête. Cône buccal court. Chétotaxie labrale avec un nombre restreint de soies, formule : 2/2,2,2. Grain tégumentaire fort légèrement plus grand que les embases des soies. Chétotaxie dorsale réduite et courte, les soies sensorielles qui se trouvent normalement en position 3,3/4,4,4,4,2, de th. II à abd. V ne sont pas différenciées, tout au plus sont-elles un peu plus grandes que les soies voisines (Fig. 1C). Le sternite abdominal III comporte 4 + 4 soies ventro-externes (VE) et 2 + 2 soies ventro-latérales (VL) (Fig. 1D). Chétotaxie des tibiotarses I, II, III réduite avec 11, 11, 10 soies sans ergot capité, griffe épaisse sans dent et sans empodium (Fig. 1E, F). Furca courte ; le mucron, mal différencié de la dens, est triangulaire, légèrement crochu. La dens porte trois soies. DISCUSSION Najtafrica n. gen. présente de nombreux caractères inégalement partagés par cinq autres genres : Neotropiella Handschin, 1942, Grananurida Yosii, 1954, Stachorutes Dallai, 1973, Intermediurida Najt, Thibaud & Weiner, 1990, et Venezuelida Diaz & Najt, 1994. Parmi les six genres présentés dans le tableau, trois sont monospécifiques : Intermediurida, Venezuelida et Najtafrica. Tous ces genres se caractérisent par la réduction du nombre de cornéules (6 + 6 à 0 + 0) et par un OPA moruliforme à l’exception de Stachorutes. Le genre Stachorutes a été redéfini par Thibaud & Palacios-Vargas (2000), les caractères donnés par les auteurs ont été pris en compte dans le tableau comparatif. Najtafrica n. gen. présente plus d’affinités avec Grananurida qu’avec les autres genres sur les points suivants : formule labrale réduite, soies sternales de l’abd. III, cône ZOOSYSTEMA • 2002 • 24 (1) Najtafrica, un nouveau genre de Collembole d’Afrique du Sud C B A E D E, F F FIG. 1. — Najtafrica riebi (Barra, 1994) n. comb. ; A, article antennaire III et IV, face dorsale ; B, cornéules et organe postantennaire gauche ; C, chétotaxie dorsale ; D, chétotaxie abdominale ventrale ; E, tibiotarse et griffe I ; F, tibiotarse et griffe III. Échelles : A, 15 µm ; B, E, F, 10 µm ; C, D, 50 µm. buccal court et nombre de dents sur les mandibules. La paurochétose des tibiotarses sépare nettement Najtafrica n. gen. des autres genres, ZOOSYSTEMA • 2002 • 24 (1) toutefois, la variabilité de la chétotaxie des tibiotarses dans les espèces du genre Stachorutes rapproche celle de Najtafrica riebi (Barra, 1994) 179 Barra J.-A. TABLEAU 1. — Principaux caractères différentiels. Caractères Venezuelida Diaz & Najt, 1994 Intermediurida Najt et al. 1990 Cornéules 4+4 0+0 5 + 5, 6 + 6 0+0 0 + 0, 1 + 1, 2 + 2 ou 5 + 5 2+2 nbre sensilles Ant. IV 6 6 5-7 5 5-6 7 microsensille Ant. IV absente absente présente présente présente présente vésicule apicale Ant. IV trilobée non exsertile trilobée non exsertile trilobée exsertile trilobée exsertile simple/trilob. non exsertile trilobée non exsertile Labre : formule 4/2352 4/2352 2/3422 2/3222 2/4352 2/222 Rétinacle absent présent présent absent présent présent Abd. III soies sternales 3 + 3 VE 0 + 0 VL 3 + 3 VE 2 + 2 VL 0 + 0 VE 3 + 3 VL 4 + 4 VE 3 + 03 VL 8 + 8 VE (ou moins) 2 + 2 VL 4 + 4 VE 2 + 2 VL Cône buccal allongé allongé allongé court court/allongé court Mandibules nbre dents 6 5 4 à 11 2-3 2-3 3 (2) OPA moruliforme moruliforme moruliforme moruliforme 1 rangée moruliforme Furca mamelon normale normale mamelon normale/réduite courte Dens : nbre de soies Neotropiella Grananurida Stachorutes Handschin, Yoshii, Dallai, 1942 1954 1973 Najtafrica n. gen. 5 6 4à6 4 3à6 3 Mucron absent présent normal présent normal absent présent/réduit absent présent réduit Tibiotarses I-II-III nbre soies 19-19-18 19-19-18 18-18-17 18-18-17 19-19-18 à 12-11-11 11-11-10 néotropicale néotropicale holotropicale paléarctique paléarctique Afrique du Sud Distribution géographique n. comb. de celle de Stachorutes escobarae (Palacios-Vargas, 1990). Les caractères différentiels sont présentés dans le Tableau 1. Remerciements Je tiens à remercier Judith Najt pour ses conseils et son aide dans la réalisation des figures et du tableau qui sont présentés dans ce travail. RÉFÉRENCES B A R R A J.-A. 1994. — Nouveaux Collemboles Poduromorphes de la Province du Natal (Rép. Sud Africaine) (Insecta: Collembola). Journal of African Zoology 108: 181-189. THIBAUD J.-M. & PALACIOS-VARGAS J. G. 2000. — Remarks on Stachorutes (Collembola: Pseudachorutidae) with a new Mexican species. Folia Entomologica Mexicana 109: 107-112. Soumis le 6 juin 2001 ; accepté le 24 septembre 2001. 180 ZOOSYSTEMA • 2002 • 24 (1)
https://openalex.org/W2413544111	https://www.nature.com/articles/srep16529.pdf	English	null	Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress	Scientific reports	2,015	cc-by	6,620	Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress Weiguo Chen1, Yulia Epshtein1, Xiuquin Ni2, Randal O. Dull3, Anne E. Cress4, Joe G.N. Garcia4 & Jeffrey R. Jacobson1 received: 23 June 2015 accepted: 15 October 2015 Published: 17 November 2015 Simvastatin, an HMG-CoA reductase inhibitor, has lung vascular-protective effects that are associated with decreased agonist-induced integrin β4 (ITGB4) tyrosine phosphorylation. Accordingly, we hypothesized that endothelial cell (EC) protection by simvastatin is dependent on these effects and sought to further characterize the functional role of ITGB4 as a mediator of EC protection in the setting of excessive mechanical stretch at levels relevant to ventilator-induced lung injury (VILI). Initially, early ITGB4 tyrosine phosphorylation was confirmed in human pulmonary artery EC subjected to excessive cyclic stretch (18% CS). EC overexpression of mutant ITGB4 with specific tyrosines mutated to phenylalanine (Y1440, Y1526 Y1640, or Y1422) resulted in significantly attenuated CS-induced cytokine expression (IL6, IL-8, MCP-1, and RANTES). In addition, EC overexpression of ITGB4 constructs with specific structural deletions also resulted in significantly attenuated CS-induced inflammatory cytokine expression compared to overexpression of wildtype ITGB4. Finally, mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were found to have attenuated lung injury after VILI-challenge (VT = 40 ml/kg, 4 h). Our results provide mechanistic insights into the anti-inflammatory properties of statins and may ultimately lead to novel strategies targeted at ITGB4 signaling to treat VILI. Acute lung injury (ALI) is a challenging clinical problem characterized by increased lung vascular per- meability and inflammation and associated with significant morbidity and mortality1. Clinically, ALI can be precipitated or exacerbated by excessive lung stretch associated with mechanical ventilation, a condi- tion known as ventilator-induced lung injury (VILI). However, aside from the use of low tidal volume mechanical ventilation which was first reported over a decade ago2, there are no therapies currently in practice that are able to definitively improve outcomes in patients with ALI/VILI. We previously reported the protective effects of simvastatin, an HMG coA-reductase inhibitor, in a murine model of LPS-induced ALI3. The mechanisms underlying the protective effects of simvastatin in this context are complex and involve differential effects on endothelial cell (EC) Rho GTPases4,5, actin cytoskeletal rearrangement4, and NADPH oxidase inhibition5 as well as differential EC gene expression including the upregulation of the tight junctional protein, claudin-56. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 23 June 2015 accepted: 15 October 2015 Published: 17 November 2015 Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress Weiguo Chen1, Yulia Epshtein1, Xiuquin Ni2, Randal O. Dull3, Anne E. Cress4, Joe G.N. Garcia4 & Jeffrey R. Jacobson1 While these effects all contribute to murine ALI protection by statins, we identified integrin β 4 (ITGB4) as the most upregulated gene in EC treated with simvastatin4 and confirmed that ITGB4 is a critical mediator of EC inflammatory responses relevant to ALI, evi- denced by the abrogation of murine ALI protection by simvastatin in response to the use of an ITGB4 blocking antibody7. Subsequently, we have begun to characterize the role of ITGB4 in normal lung EC signaling and function8,9 and have identified ITGB4-mediated vascular inflammatory responses. In this study we sought to further define functional role of ITGB4 in lung EC inflammatory responses mediated by excessive mechanical stress consistent with levels able to precipitate VILI. 1Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, Chicago, IL. 2Department of Anatomy, Harbin Medical University at Daqing, China. 3Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL. 4Department of Medicine, University of Arizona, Tucson, AZ. Correspondence and requests for materials should be addressed to J.R.J. (email: jrjacob@uic.edu) Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 1 1 www.nature.com/scientificreports/ tificreports/ Figure 1. Mechanical stress of ITGB4 phosphorylation in human lung EC. (A) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). (B) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, p < 0.05 compared to control). Figure 1. Mechanical stress of ITGB4 phosphorylation in human lung EC. (A) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). (B) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, p < 0.05 compared to control). The integrins, expressed on a variety of cell types, are transmembrane heterodimers consisting of α and β subunits that mediate both inside-out and outside-in signaling. While the role of ITGB4 in ALI is not well characterized, a growing literature has implicated other β integrins as effectors of lung vas- cular permeability and EC barrier function10–12. Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress Weiguo Chen1, Yulia Epshtein1, Xiuquin Ni2, Randal O. Dull3, Anne E. Cress4, Joe G.N. Garcia4 & Jeffrey R. Jacobson1 For example, ITGB5 has been identified as a mediator of ALI in separate models of rat ischemia-reperfusion lung injury and murine ALI10. Moreover, ITGB5 inhibition in vitro was found to attenuate agonist-induced EC actin stress fiber formation and monolayer permeability. Additionally, in separate reports, ITGB212, ITGB313 and ITGB611 have also been implicated as mediators of lung vascular permeability associated with ALI, although the precise mechanisms of integrin-mediated effects in these contexts remain to be fully characterized.i gf y While 8 β subunits have been identified, the laminin-5 receptor ITGB4, which forms a heterodimer only with integrin α 6, is uniquely characterized by its long cytoplasmic tail of over 1000 amino acids. This tail is comprised of a proximal Calx Na-Ca exchanger domain followed by two pairs of fibronectin type III repeats separated by a tyrosine activation motif (TAM)14. The extracellular portion of the β 4 subunit sequence is found to have 35% homology with other integrin β subunits but is the most different within this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known β sequences or to other protein sequences. The exceptionally long cytoplasmic domain of ITGB4 suggests potentially distinct protein interactions and downstream signaling relative to the other β integrins. In this study we utilized various ITGB4 constructs with specific mutations or deletions within the cytoplasmic tail to characterize its role in the elaboration of inflammatory responses of mechanically stressed lung EC in vitro. In separate experiments, we subjected mice expressing a mutant ITGB4 lacking a cytoplasmic sig- naling domain (terminating at amino acid 1355) to VILI and assessed lung vascular leak and inflamma- tion. Collectively, these studies further define the functional role of EC ITGB4 as a mediator of ALI/VILI and suggest selective targeting of ITGB4 signaling may represent a novel therapeutic strategy clinically. Results Interestingly, two of the three assays that did not demonstrate a significant effect involved constructs in which Y1422 and Y1440 were both mutated suggesting that this combined mutation has effects on EC inflammatory signaling induced by mechanical stress that effectively coun- teracts the predominant anti-inflammatory effects associated with the mutation of either one of these tyrosines by itself. There was no appreciable effect of overexpression of either wildtype ITGB4 or any of the mutants on cytokine levels detectable in the media of static cells. Role ITGB4 cytoplasmic domain in EC inflammatory responses to cyclic stretch. We con- ducted identical experiments using a separate series of ITGB4 constructs in which specific cytoplasmic domains were deleted (Fig. 3A). Western blotting again confirmed overexpression of constructs with bands noted at variable molecular weights corresponding to the expression of both endogenous ITGB4 and the individual transfected constructs (Fig. 3B). Compared to cells overexpressing wildtype ITGB4, transfection of EC with any of these ITGB4 mutants resulted in a significant attenuation of the expres- sion of inflammatory cytokines IL-6, IL-8, MCP-1 and RANTES in the media after CS (18%, 6 h), with only one exception noted (Fig. 3C–F). This single outlier was observed with CS-induced MCP-1 expres- sion in cells transfected ITGB4 completely lacking a cytoplasmic domain (A). Conversely, we observed a non-significant trend that transfection with ITGB4 lacking the proximal region of the cytoplasmic domain (C) was associated with the most prominent and consistent effects on the reduced elaboration of inflammatory cytokines after mechanical stress. Consistent with the previous series of experiments, there was again no appreciable effect of overexpression of either wildtype ITGB4 or any of the mutants on cytokine levels detectable in the media of static cells. Role of ITGB4 cytoplasmic domain in the elaboration of murine VILI. To investigate the signif- icance of our results in an in vivo VILI model we initially confirmed the protective effects of simvastatin in this context consistent with other reports18. Mice were treated with simvastatin (20 mg/kg, IP injec- tion) or vehicle 16 h prior to being subjected to high tidal volume mechanical ventilation (VT = 40 ml/ kg, 4 h) to induce VILI. Compared to controls, animals that received simvastatin were found to have significantly reduced bronchoalveolar lavage (BAL) fluid protein levels and total cell counts consistent with an attenuated VILI-associated inflammatory response (Fig. 4). Results ITGB4 phosphorylation in response to mechanical stress. In initial experiments, human pulmo- nary artery EC were grown to confluence on Bioflex plates and then subjected to 18% cyclic stretch (CS) for up to 4 h prior to collection of cell lysates for immunoprecipitation of ITGB4 followed by Western blotting for p-tyrosine (Fig. 1). These studies confirmed increased tyrosine phosphorylation of ITGB4 in response to CS evident within 30 min with peak phosphorylation at 2 h. These data are consistent with our previous report of increased EC ITGB4 phosphorylation in response to lipopolysaccharide (LPS)7, as both LPS and excessive mechanical stress induce EC barrier disruption in vitro and inflammatory lung injury in vivo. Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 2 www.nature.com/scientificreports/ Role ITGB4 tyrosine phosphorylation in EC inflammatory responses to cyclic stretch. As a model of VILI in vitro we utilized a Flexcell Strain Unit (FX-3000; FlexCell International) and subjected human pulmonary artery EC grown on Bioflex plates to 18% cyclic stretch (CS) as we have previously described15. This level of stretch is consistent with pathologic lung stretch that occurs clinically in the setting of VILI while 5% CS corresponds to physiologic changes associated with normal respiration in the healthy lung15. To investigate the role of ITGB4 tyrosine phosphorylation in the expression of inflam- matory cytokines induced by the mechanical stress of lung EC we first utilized a series of six ITGB4 constructs in which one of four tyrosines (Y1440, Y1526, Y1640, or Y1422) were mutated to phenyala- nine (F), either individually or in combination (Fig. 2A). Initially, we confirmed effective overexpression by Western blotting of whole cell lysates (Fig. 2B). These blots demonstrated two distinct bands upon probing for ITGB4 which we speculate may be due to either variable posttranslational modifications such as glycosolation16 or proteolysis of ITGB417. Subsequently, in all but three cases, transfection of EC with an ITGB4 mutant resulted in a significant attenuation of the expression of inflammatory cytokines IL-6, IL-8, MCP-1 or RANTES in the media after 18% CS (6 h) compared to cells overexpressing wild- type ITGB4 (Fig. 2C–F). Results l Next, we subjected genetically engineered mice expressing ITGB4 lacking a cytoplasmic signaling domain to high tidal volume mechanical ventilation and assessed indices of lung vascular permeability and inflammation. Compared to wildtype control animals, mutant ITGB4 mice were found to have sig- nificantly attenuated inflammatory response to VILI-challenge (VT = 40 ml/kg, 4 h) as assessed by BAL fluid protein and total cell counts (Fig. 5A,B). Notably, while simvastatin was protective in our VILI model, there was no appreciable effect of simvastatin pretreatment (20 mg/kg, IP, 16 h prior to mechanical ventilation) in ITGB4 mutant mice subjected to VILI. These results suggest that protection by simvastatin in our murine VILI model is strongly influenced by its effects on ITGB4.il g yl yf In the same experiments lung histology demonstrated infiltration of inflammatory cell and edema in wildtype mice in response to VILI that was markedly reduced in mutant ITGB4 mice after VILI (Fig. 5C). Finally, BAL fluid collected for measurement of inflammatory cytokines, IL-6 and KC (a murine IL-8 homolog), confirmed significantly lower levels of both cytokines in the BAL fluid of ITGB4 mutant mice subjected to VILI compared to VILI-challenged controls (Fig. 6). Similarly, there was no effect of simvastatin pretreatment on IL-6 and KC levels in ITGB4 mutant mice after VILI. Discussion In this study we confirmed the functional importance of ITGB4 in the elaboration of lung EC inflamma- tory responses to mechanical stress both in vitro and in vivo. Specifically, we observed increased tyrosine phosphorylation of ITGB4 in human pulmonary artery EC subjected to excessive CS while the overex- pression of full length ITGB4 was associated with increased CS-induced EC expression of inflammatory cytokines. In contrast, overexpression of any one of several distinct ITGB4 constructs harboring muta- tions in the cytoplasmic tail, defined broadly by either mutations of tyrosine phosphorylation sites or deletion of structural domains, resulted in decreased mechanical stress-induced inflammatory cytokines Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 3 cientificreports/ Figure 2. ITGB4 tyrosine phosphorylation mediates CS-induced EC inflammatory responses. Human pulmonary artery EC were transfected with vector (control), wildtype ITGB4 or specific constructs in which individual tyrosine residues (Y) were mutated to phenylalanine (F, panel A) prior to being subjected to excessive mechanical stretch (18% CS, 6 h). (B) Whole cell lysates were used for Western blotting and were probed for ITGB4 to confirm overexpression. (C–F) Inflammatory cytokines IL-6, IL-8, MCP-1 and RANTES were measured in the media after CS as shown (n = 3/condition, p < 0.05 compared to static wildtype controls, p < 0.05 compared to wildtype cells subjected to CS). (TM = transmembrane domain, PSI = plexin-semaphorin-integrin domain, VWA = von Willebrand factor A domain, TM = transmembrane domain, FN = fibronectin type III domain, TAM = tyrosine activation motif). www.nature.com/scientificreports/ Figure 2. ITGB4 tyrosine phosphorylation mediates CS-induced EC inflammatory responses. Human Figure 2. ITGB4 tyrosine phosphorylation mediates CS-induced EC inflammatory responses. Human pulmonary artery EC were transfected with vector (control), wildtype ITGB4 or specific constructs in which individual tyrosine residues (Y) were mutated to phenylalanine (F, panel A) prior to being subjected to excessive mechanical stretch (18% CS, 6 h). (B) Whole cell lysates were used for Western blotting and were probed for ITGB4 to confirm overexpression. (C–F) Inflammatory cytokines IL-6, IL-8, MCP-1 and RANTES were measured in the media after CS as shown (n = 3/condition, p < 0.05 compared to static wildtype controls, *p < 0.05 compared to wildtype cells subjected to CS). (TM = transmembrane domain, PSI = plexin-semaphorin-integrin domain, VWA = von Willebrand factor A domain, TM = transmembrane domain, FN = fibronectin type III domain, TAM = tyrosine activation motif). Figure 2. Discussion ITGB4 tyrosine phosphorylation mediates CS-induced EC inflammatory responses. Human pulmonary artery EC were transfected with vector (control), wildtype ITGB4 or specific constructs in which individual tyrosine residues (Y) were mutated to phenylalanine (F, panel A) prior to being subjected to excessive mechanical stretch (18% CS, 6 h). (B) Whole cell lysates were used for Western blotting and were probed for ITGB4 to confirm overexpression. (C–F) Inflammatory cytokines IL-6, IL-8, MCP-1 and RANTES were measured in the media after CS as shown (n = 3/condition, p < 0.05 compared to static wildtype controls, *p < 0.05 compared to wildtype cells subjected to CS). (TM = transmembrane domain, PSI = plexin-semaphorin-integrin domain, VWA = von Willebrand factor A domain, TM = transmembrane domain, FN = fibronectin type III domain, TAM = tyrosine activation motif). released by EC. Moreover, mice expressing ITGB4 lacking a cytoplasmic signaling domain altogether were protected from VILI compared to VILI-challenged wildtype animals. These results are consistent with our hypothesis that phosphorylated ITGB4 is pro-inflammatory and, conversely, a relative decrease in phosphorylated ITGB4 is anti-inflammatory. These findings further support emerging evidence for the critical role of ITGB4 in the elaboration of ALI/VILI. Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 4 www.nature.com/scientificreports/ Figure 3. ITGB4 cytoplasmic domains mediate cyclic CS-induced EC inflammatory responses. Human pulmonary artery EC were transfected with wildtype ITGB4 or specific deletion constructs (A) prior to being subjected to excessive mechanical stretch (18% CS, 6 h). (B) Whole cell lysates were used for Western blotting and were probed for ITGB4 to confirm overexpression. (C–F) IL-6, IL-8, MCP-1 and RANTES were measured in the media after CS as shown (n = 3/condition, p < 0.05 compared to static wildtype controls, *p < 0.05 compared to wildtype cells subjected to CS). (TM = transmembrane domain, PSI = plexin- semaphorin-integrin domain, VWA = von Willebrand factor A domain, TM = transmembrane domain, FN = fibronectin type III domain, TAM = tyrosine activation motif). Figure 3. ITGB4 cytoplasmic domains mediate cyclic CS-induced EC inflammatory responses. Human pulmonary artery EC were transfected with wildtype ITGB4 or specific deletion constructs (A) prior to being subjected to excessive mechanical stretch (18% CS, 6 h). (B) Whole cell lysates were used for Western blotting and were probed for ITGB4 to confirm overexpression. Discussion (C–F) IL-6, IL-8, MCP-1 and RANTES were measured in the media after CS as shown (n = 3/condition, p < 0.05 compared to static wildtype controls, *p < 0.05 compared to wildtype cells subjected to CS). (TM = transmembrane domain, PSI = plexin- semaphorin-integrin domain, VWA = von Willebrand factor A domain, TM = transmembrane domain, FN = fibronectin type III domain, TAM = tyrosine activation motif). Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 5 www.nature.com/scientificreports/ Figure 4. Simvastatin attenuates vascular permeability and inflammation in mice subjected to VILI. Wildtype mice were treated with simvastatin (20 mg/kg, IP injection) or vehicle 16 h prior to being subjected to VILI challenge (VT = 40 ml/kg, 4 h). Spontaneously breathing mice were used as controls. BAL fluid was then collected and analyzed for (A) protein content and (B) total cell counts (p < 0.05, n = 3 animals/ condition). Figure 4. Simvastatin attenuates vascular permeability and inflammation in mice subjected to VILI. Wildtype mice were treated with simvastatin (20 mg/kg, IP injection) or vehicle 16 h prior to being subjected to VILI challenge (VT = 40 ml/kg, 4 h). Spontaneously breathing mice were used as controls. BAL fluid was then collected and analyzed for (A) protein content and (B) total cell counts (p < 0.05, n = 3 animals/ condition). The functional role of ITGB4 tyrosine phosphorylation has previously been studied in a variety of other contexts. For example, the binding of the adaptor protein, Shc, to ITGB4 is dependent on phos- phorylation of Y1140 and Y1526 and is associated with both downstream MAPK signaling and inhibition of hemidesmosome formation in epithelial cells19. More relevant to the current study, we previously reported that inhibition of ITGB4 tyrosine phosphorylation is associated with an attenuation of EC barrier augmentation by hepatocyte growth factor20, an agonist that is also protective in murine ALI21. We have also previously identified ITGB4 as a robust mediator of murine ALI/VILI protection by statins associated with potent anti-inflammatory effects. Specifically, simvastatin induces increased expression of ITGB4 in lung EC while the protective effects of simvastatin in murine ALI can be mitigated by pretreatment with an ITGB4 blocking antibody7. The dramatic upregulation of ITGB4 by simvastatin is associated with decreased agonist-induced tyrosine phosphorylation of ITGB4 which in turn attenuates MAPK signaling and downstream expression of inflammatory mediators, including IL-6 and IL-87. Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 Discussion Thus, our previous findings suggest that increased expression of ITGB4 harboring unphosphorylated tyrosines within its cytoplasmic tail effectively serves as a “brake” on lung EC inflammatory responses. Our current Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 6 www.nature.com/scientificreports/ Figure 5. Decreased lung vascular permeability and inflammati VILI. Wildtype mice and mice expressing a mutant ITGB4 lackin pretreated with simvastatin (20 mg/kg, IP injection, 24 h) or vehicl 4 h). Spontaneously breathing mice were used as controls. BAL flu protein content and (B) total cell counts (p < 0.05, n = 5 animals lungs were collected from spontaneously breathing and VILI-chall animals and used for histology. Representative images are shown ( Figure 5. Decreased lung vascular permeability and inflammation in ITGB4 mutant mice subjected to VILI. Wildtype mice and mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were pretreated with simvastatin (20 mg/kg, IP injection, 24 h) or vehicle prior to VILI challenge (VT = 40 ml/kg, 4 h). Spontaneously breathing mice were used as controls. BAL fluid was then collected and analyzed for (A) protein content and (B) total cell counts (p < 0.05, n = 5 animals/condition). (C) In separate experiments, lungs were collected from spontaneously breathing and VILI-challenged wildtype and ITGB4 mutant animals and used for histology. Representative images are shown (10× magnification, insets 40× ). Figure 5. Decreased lung vascular permeability and inflammation in ITGB4 mutant mice subjected to VILI. Wildtype mice and mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were pretreated with simvastatin (20 mg/kg, IP injection, 24 h) or vehicle prior to VILI challenge (VT = 40 ml/kg, 4 h). Spontaneously breathing mice were used as controls. BAL fluid was then collected and analyzed for (A) protein content and (B) total cell counts (p < 0.05, n = 5 animals/condition). (C) In separate experiments, lungs were collected from spontaneously breathing and VILI-challenged wildtype and ITGB4 mutant animals and used for histology. Representative images are shown (10× magnification, insets 40× ). data extend our prior observations and confirm a critical role for ITGB4 in EC barrier regulation with complex effects dependent on the specific context and support the idea that increased tyrosine phospho- rylation is requisite for the pro-inflammatory effects of ITGB4. Discussion yl yf While we did not investigate the events responsible for the attenuation of ITGB4 tyrosine phospho- rylation by simvastatin one potential mechanism is the inhibition of specific Src family kinases (SFKs), including Fyn and/or Yes, that are known to mediate these events22. The downstream consequences of these effects may include the attenuation of Shc and Shp-2 recruitment resulting in decreased Ras and Erk signaling19,23. Consistent with this, we previously reported decreased agonist-induced Shp-2 Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 7 www.nature.com/scientificreports/ Figure 6. Decreased lung inflammatory cytokines in ITGB4 mutant mice subjected to VILI. Wildtype mice and mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were pretreated with simvastatin (20 mg/kg, IP injection, 24 h) or vehicle prior to VILI challenge (VT = 40 ml/kg, 4 h). BAL fluid was then collected and inflammatory cytokine levels measured including (A) IL-6 and (B) KC. (p < 0.05, n = 5 animals/condition). Figure 6. Decreased lung inflammatory cytokines in ITGB4 mutant mice subjected to VILI. Wildtype mice and mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were pretreated with simvastatin (20 mg/kg, IP injection, 24 h) or vehicle prior to VILI challenge (VT = 40 ml/kg, 4 h). BAL fluid was then collected and inflammatory cytokine levels measured including (A) IL-6 and (B) KC. (p < 0.05, n = 5 animals/condition). phosphorylation and Erk activation in EC expressing decreased ITGB4 (via silencing RNA)8. In addition, although our studies focused on the role of ITGB4 tyrosine phosphorylation, three of the constructs studied (constructs A, B, and D; Fig. 3A) were also characterized by the deletion of serines (S1356, S1360, and S1364) that are known to drive ITGB4 signaling upon phosphorylation via epidermal growth factor (EGF) and/or protein kinase C-α (PKCα )24.l p Several other questions remain to be explored regarding EC inflammatory signaling by ITGB4. Among these are the mechanisms by which overexpression of ITGB4 lacking a proximal cytoplasmic domain, but with otherwise preserved tyrosine phosphorylation sites (construct C, Fig. 3A), is associ- ated with reduced inflammatory responses as we observed in mechanically-stressed EC. Notably, as has been previously reported, this construct is associated with differential effects on ITGB4 phosphoryla- tion compared to wildtype ITGB4 which may account for this observation19. Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 Materials and Methods Cell culture. HPAEC cell lines were purchased from Lonza (Walkersville, MD) and were cultured in EGM-2 supplemented with 2% FBS, hydrocortisone, hFGF, VEGF, ascorbic acid, hEGF, GA-1000, hep- arin, and R3-IGF-1. Cells were incubated in 75 cm2 flasks and cultured at 37 °C in 5% CO2 and 95% air. All cells were used at passages 4–8. Materials and reagents. ITGB4 antibody (SC-9090) specific for the extracellular domain of ITGB4 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A separate antibody specificially rec- ognizing the ITGB4 cytosolic domain (LS-C14098) was purchased from Lifespan BioSciences (Seattle, WA). Human and mouse cytokines measurements were performed using commercially available kits as per the manufacturer’s instructions (Bio-Rad, Hercules, CA). All other antibodies and reagents were pur- chased from Santa Cruz Biotechnology (Santa Cruz, CA). ITGB4 mutated and deletion constructs were a generous gift from Dr. Filipo Giancotti (Memorial Sloan Kettering Cancer Center, New York, NY)19. Transgenically engineered mice expressing ITGB4 lacking the C-terminal signaling signaling segment of the ITGB4 tail were also provided by Dr. Giancotti’s lab. These animals express an ITGB4 that terminates at amino acid 1355, upstream of the four tyrosine phosphorylation sites32. Immunoprecipitation and Western blotting. For immunoprecipitation, cell lysates prepared from EC were incubated in immunoprecipitation buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 1% Nonidet P-40, 0.4 mM Na3VO4, 40 mM NaF, 50 μ M okadaic acid, 0.2 mM phenylmethylsulfo- nyl fluoride, and Calbiochem protease inhibitor mixture III at 1:250 dilution). The immunoprecipitation was carried with an anti-ITGB4 antibody.f y For Western blotting, samples were prepared in RIPA buffer containing proteinase and phosphatase inhibitors as per standard protocols. After sonication and centrifugation, the supernatant was collected, Laemmli sample buffer added, and sampels were then boiled and analyzed by SDS–PAGE. After trans- fer to a nitrocellulose membrane (Bio-Rad, Inc., Hercules, CA), Western blotting was performed using appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies prior to visualization via chemiluminescence (Amersham Biosciences, Piscataway, NJ). Blot density was deter- mined by Alpha Imager software (Alpha Innotech, San Leandro, CA). Transfection of EC prior to mechanical stretch. Cells were plated (60–80% confluent) and trans- fected with a vector plasmid (PRK5) or specific ITGB4 plasmids using jetPRIME® transfection reagent (Polyplus Transfection, New York, NY). After incubating for 24 h, transfection efficiency of plasmids was verified by Western blotting. www.nature.com/scientificreports/ cytoplasmic tail. Separately, we did not investigate a potential role for integrin α 6 (ITGA6), the only α integrin subunit with which ITGB4 associates. We speculate that ITGA6 may preferentially heterodimer- ize with overexpressed mutated ITGB4 in our experiments with subsequent consequences on EC inflam- matory responses, an idea supported by evidence of a functional role for signaling by ITGA6 in other experimental models25,26. We recognize that ITGB4-mediated signaling is complex and the identification of relevant molecular targets remains to be fully investigated. g y g One potential target of interest is syndecan-1, a cell-surface proteoglycan that binds extraceullar matrix components but also binds the ITGB4 cytoplasmic domain27, is expressed by EC, and has been implicated as a mediator of ALI28. Moreover, we have observed markedly decreased syndecan-1 expres- sion in simvastatin-treated EC (unpublished data) which we speculate may account for the attenuation of ITGB4 tyrosine phosphorylation despite significant increases in ITGB4 protein levels induced by sim- vastatin7,29. Further studies aimed at characterizing the role of syndecan-1 in EC inflammatory responses mediated by ITGB4 may further inform our current findings and, in turn, lead to additional insights into the elaboration and propagation of ALI clinically. p p g y Finally, it should be noted that although our interest in ITGB4 is derived from our prior research focused on the potential role of statins in ALI, two randomized, controlled clinical trials of ALI patients were recently published which in fact reported no benefit with the administration of statins30,31. However, there were potential limitations to both studies, not the least of which was the administration of statins to patients relatively late in their clinical course. It may also be the case that while statins may have effects beneficial in ALI patients, effective therapeutic strategies may ultimately require more targeted approaches aimed at augmenting the specific statin effects most responsible for their benefits. If this is the case, our data support signaling mediated by ITGB4 as just such a target and warrants further investigation. Discussion Additionally, our results indicate anti-inflammatory effects associated with the overexpression of ITGB4 lacking a cytoplasmic tail altogether (construct A) that were less robust than that observed with each of the constructs with only partial deletions of the cytoplasmic tail (B–D). While further study is needed to fully understand the mechanisms underlying this observation, it is possible that the ITGB4 cytoplasmic tail harbors both pro- and anti-inflammatory regions and the constructs with only partial deletions may actually have a net anti-inflammatory effect that is more prominent than that associated with complete deletion of the Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 8 www.nature.com/scientificreports/ References 1. Rubenfeld, G. D. et al. Incidence and outcomes of acute lung injury. N. Engl. J. Med. 353, 1685–1693, (2005). , g j y g J , , ( ) 2. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network. N. Engl. J. Med. 342, 1301–1308 (2000).ll 2. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network. N. Engl. J. Med. 342, 1301–1308 (2000).ll yh p y y g 3. Jacobson, J. R. et al. Simvastatin attenuates vascular leak and inflammation in murine inflammatory lung injury. Am. J. Physiol Lung Cell. Mol. Physiol. 288, L1026–1032 (2005). g y 4. Jacobson, J. R. et al. Cytoskeletal activation and altered gene expression in endothelial barrier regulation by simvastatin. Am. J. Resp. Cell. Mol. Biol. 30, 662–670, (2004). 5. Chen, W., Pendyala, S., Natarajan, V., Garcia, J. G. & Jacobson, J. R. Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L575–583, (2008). 6. Chen, W. et al. Role of claudin-5 in the attenuation of murine acute lung injury by simvastatin. Am. J. Resp. Cell. Mol. Biol. 50 328–336, (2014). 7. Chen, W. et al. Critical role for integrin-beta4 in the attenuation of murine acute lung injury by simvastatin. Am. J. Physiol. Lung. Cell. 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Absence of integrin alphavbeta3 enhances vascular leak in mice by inhibiting endothelial cortical actin formation. Am. J. Resp. Crit. Materials and Methods Of note, EC express relatively low levels of endogenous ITGB4 which was present in all cells in addition to the specific ITGB4 constructs transfected. Mechanical stretch of EC. EC were plated onto six-well silicone elastomer Bioflex plates coated with type I collagen (FlexCell International, Hillsborough, NC) and grown to 75–80% prior to trans- fection as described. Mechanical stretch was performed via the Flexcell Strain Unit (FX-3000; FlexCell International) placed in a 5% CO2 incubator at 37 °C and 95% humidity. The device uses a controlled vac- uum to induce CS with 18% elongation at a frequency of 30 cycles per minute (0.5 Hz) for 6 h. The media Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 9 www.nature.com/scientificreports/ was then collected and briefly centrifuged to measure cytokine levels with Bio-Rad Bio-Plex ELISA kits (Hercules, CA) according to the manufacturer’s instructions. was then collected and briefly centrifuged to measure cytokine levels with Bio-Rad Bio-Plex ELISA kits (Hercules, CA) according to the manufacturer’s instructions. Murine VILI model. All experiments and animal care procedures were approved by the University of Illinois at Chicago Animal Care and Use Committee and were performed in accordance with their guidelines and regulations. The ITGB4 mutant mice and wildtype C57/Bl6 mice, 8–12 weeks-old, were administered high tidal volume mechanical ventilation (VT = 40 ml/kg, 65 breaths/min) for 4 h to induce VILI as we have previously described33. Mice were then sacrificed and bronchoalveolar lavage (BAL) was performed3. 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CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin. J. Biol. Chem. 282, 30643–30657, (2007). 22. Giancotti, F. G. Targeting integrin beta4 for cancer and anti-angiogenic therapy. Trends Pharmacol. Sci. 28, 506–511, (2007 23. Bertotti, A., Comoglio, P. M. & Trusolino, L. Beta4 integrin activates a Shp2-Src signaling pathway that sustains HGF-induced anchorage-independent growth. J. Cell. Biol. 175, 993–1003, (2006).i g g 24. Rabinovitz, I., Tsomo, L. & Mercurio, A. M. Protein kinase C-alpha phosphorylation of specific serines in the connecting seg f th b t 4 i t i l t th d i f t II h id M l C ll Bi l 24 4351 4360 (2004) 24. Rabinovitz, I., Tsomo, L. & Mercurio, A. M. Protein kinase C-alpha phosphorylation of specific serines in the connecting segment of the beta 4 integrin regulates the dynamics of type II hemidesmosomes. Mol. Cell. Biol. 24, 4351–4360 (2004).f i of the beta 4 integrin regulates the dynamics of type II hemidesmosomes. Mol. Cell. Biol. 24, 4351–4360 (2004). www.nature.com/scientificreports/ 2. Nikolopoulos, S. N., Blaikie, P., Yoshioka, T., Guo, W. & Giancotti, F. G. Integrin beta4 signaling promotes tumor angiogenesis Cancer Cell 6, 471–483, (2004). 32. Nikolopoulos, S. N., Blaikie, P., Yoshioka, T., Guo, W. & Giancotti, F. G. Integrin beta4 signaling promotes tumor angiogenesis. Cancer Cell 6, 471–483, (2004). 33. Finigan, J. H. et al. Activated protein C protects against ventilator-induced pulmonary capillary leak. Am. J. Physiol. Lung Cell. Mol Physiol 296 L1002 1011 (2009) 3. Finigan, J. H. et al. Activated protein C protects against ventilator-induced pulmonary capillary leak. Am. J. Physiol. Lung Cell Mol. Physiol. 296, L1002–1011, (2009). Author Contributions W.C., Y.E. and X.N. conducted the experiments described in the manuscript. J.J. oversaw all experimental designs. W.C. and J.J. prepared the manuscript and figures. A.C. and J.G. both contributed to experimental design and data interpretation. R.D. was also involved in data interpretation and provided helpful discussions. All authors reviewed the manuscript prior to submission. References 5 Jiang R & Grabel L B Function and differential regulation of the alpha 6 integrin isoforms during parietal endoderm i of the beta 4 integrin regulates the dynamics of type II hemidesmosomes. Mol. Cell. Biol. 24, 4351–4360 (2004). 25. Jiang, R. & Grabel, L. B. Function and differential regulation of the alpha 6 integrin isoforms during parietal endo diff ti ti E C ll R 217 195 204 (1995) 5. Jiang, R. & Grabel, L. B. Function and differential regulation of the alpha 6 integrin isoforms during parietal endoderm differentiation. Exp. Cell. Res. 217, 195–204, (1995).ht f 26. King, T. E. et al. The role of alpha 6 integrin in prostate cancer migration and bone pain in a novel xenograft model. PLOS One 3, e3535, (2008). 27. Wang, H., Jin, H., Beauvais, D. M. & Rapraeger, A. C. Cytoplasmic domain interactions of syndecan-1 and syndecan-4 with alpha6beta4 integrin mediate human epidermal growth factor receptor (HER1 and HER2)-dependent motility and survival. J. Biol. Chem. 289, 30318–30332, (2014). 8. Li, Q., Park, P. W., Wilson, C. L. & Parks, W. C. Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 111, 635–646 (2002). fl 29. Wang, H., Leavitt, L., Ramaswamy, R. & Rapraeger, A. C. Interaction of syndecan and alpha6beta4 integrin cytoplasmic dom regulation of ErbB2-mediated integrin activation. J. Biol. Chem. 285, 13569–13579, (2010). g g 30. National Heart, L. et al. Rosuvastatin for sepsis-associated acute respiratory distress syndrome. N. Engl. J. Med. 370, 2191–2200, (2014). 31. McAuley, D. F. et al. Simvastatin in the acute respiratory distress syndrome. N. Engl. J. Med. 371, 1695–1703, (2014). Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 10 www.nature.com/scientificreports/ Acknowledgementsh g This work acknowledges support from NIH HL096887 (JJ). Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 Additional Informationi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Chen, W. et al. Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress. Sci. Rep. 5, 16529; doi: 10.1038/srep16529 (2015). How to cite this article: Chen, W. et al. Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress. Sci. Rep. 5, 16529; doi: 10.1038/srep16529 (2015). How to cite this article: Chen, W. et al. Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress. Sci. Rep. 5, 16529; doi: 10.1038/srep16529 (2015). 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 Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 5:16529 \| DOI: 10.1038/srep16529 11
https://openalex.org/W2506498622	http://jatp.ift.or.id/index.php/jatp/article/download/180/148	Indonesian	null	SUBSTITUSI GULA SUKROSA DENGAN GULA FRUKTOSA PADA PROSES PEMBUATAN ROTI TERHADAP SIFAT SENSORI ROTI	Jurnal aplikasi teknologi pangan	2,016	cc-by	3,727	Catatan Penelitian Nilai pH, Total Padatan Terlarut, dan Sifat Sensoris Yoghurt dengan Penambahan Ekstrak Bit (Beta vulgaris L.) The Value of pH, Total Dissolved Solids, and Sensory Properties of Yoghurt with Addition Beet Extract (Beta vulgaris L.) Nur Ismawati, Nurwantoro, Yoyok Budi Pramono Program Studi Teknologi Pangan, Fakultas Peternakan dan Pertanian, Universitas Diponegoro, Semarang Korespondensi dengan penulis (nurismawati19@yahoo.com) Artikel ini dikirim pada tanggal 4 Juni 2016 dan dinyatakan diterima tanggal 01 Juli 2016. Artikel ini juga dipublikasi secara online melalui www.jatp.ift.or.id. Hak cipta dilindungi undang-undang. Dilarang diperbanyak untuk tujuan komersial. Diproduksi oleh Indonesian Food Technologists® ©2016 ( g ) Nur Ismawati, Nurwantoro, Yoyok Budi Pramono Program Studi Teknologi Pangan, Fakultas Peternakan dan Pertanian, Universitas Diponegoro, Semarang Korespondensi dengan penulis (nurismawati19@yahoo.com) Artikel ini dikirim pada tanggal 4 Juni 2016 dan dinyatakan diterima tanggal 01 Juli 2016. Artikel ini juga dipubl www.jatp.ift.or.id. Hak cipta dilindungi undang-undang. Dilarang diperbanyak untuk tujuan komersial. Diproduksi oleh Indonesian Food Technologists® ©2016 Abstract The purpose of this research was to determined the pH value, total dissolved solids, and sensory properties of yoghurt with addition of beet extract. Material used in this research were liquid skimmed milk, beet extract, starter cultures of lactic acid bacteria (BAL) that were Lactobbacillus bulgaricus, Streptococcus thermophillus, and Lactobacillus acidophilus. The research showed that addition of the beet extract given significant (P<0,05) the value of pH, total dissolved solid, and sensory properties of yoghurt. The result obtained showed that the pH value ranged between 3.79 – 3.92; total dissolved solids 7.70 – 8.13 °Brix; and an addition in the taste and sweetness become increasingly concentrated. The additional of the best beet extract happens on 4%. Keywords: pH, total dissolved solids, sensory properties, yoghurt, extract beet Abstrak Penelitian ini bertujuan untuk mengetahui nilai pH, total padatan terlarut, dan sifat sensoris yoghurt dengan penambahan ekstrak bit. Materi yang digunakan adalah susu skim cair, ekstrak bit, kultur bakteri asam laktat (BAL) yaitu Lactobbacillus bulgaricus, Streptococcus thermophillus, dan Lactobacillus acidophilus. Rancangan percobaan yang digunakan adalah Rancangan Acak Lengkap (RAL) dengan perlakuan penambahan ekstrak bit 0%, 1%, 2%, 3%, dan 4%. Masing-masing perlakuan diulang sebanyak 4 kali. Hasil penelitian menunjukkan penambahan ekstrak bit memberikan perbedaan nyata (P<0,05) terhadap nilai pH, total padatan terlarut, dan sifat sensoris yoghurt. Nilai pH menghasilkan angka 3,79 – 3,92; total padatan terlarut 7,70 – 8,13 °Brix; dan semakin tinggi penambahan ekstrak bit yang digunakan menambah agak bau aroma tanah dan rasa lebih agak manis. Penambahan konsentrasi ekstrak bit terbaik yaitu 4%. Kata kunci : nilai pH, total padatan terlarut, sifat sensoris, yoghurt, ekstrak bit 89 89 Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 ©Indonesian Food Technologists http://dx.doi.org/10.17728/jatp.181 Catatan Penelitian Nilai pH, Total Padatan Terlarut, dan Sifat Sensoris Yoghurt dengan Penambahan Ekstrak Bit (Beta vulgaris L.) The Value of pH, Total Dissolved Solids, and Sensory Properties of Yoghurt with Addition Beet Extract (Beta vulgaris L.) Nur Ismawati, Nurwantoro, Yoyok Budi Pramono Program Studi Teknologi Pangan, Fakultas Peternakan dan Pertanian, Universitas Diponegoro, Semarang Korespondensi dengan penulis (nurismawati19@yahoo.com) Artikel ini dikirim pada tanggal 4 Juni 2016 dan dinyatakan diterima tanggal 01 Juli 2016. Artikel ini juga dipublikasi secara online melalui www.jatp.ift.or.id. Hak cipta dilindungi undang-undang. Dilarang diperbanyak untuk tujuan komersial. Diproduksi oleh Indonesian Food Technologists® ©2016 Pendahuluan diperbaiki atau diubah dengan penambahan bit (Beta vulgaris L.) pada proses pembuatan yoghurt. Yoghurt merupakan salah satu produk fermentasi yang dihasilkan dengan menambahkan starter yoghurt aktif yang mengandung kultur campuran Lactobacillus bulgaricus (atau kadang-kadang acidophilus) dan Streptococcus thermophillus yang menghasilkan asam laktat selama proses fermentasi (Nwaoha et al., 2012). Asam laktat yang terbentuk akan menurunkan pH seka0ligus sebagai pengawet karena dalam kondisi asam bakteri patogen tidak dapat tumbuh. Asam laktat yang terbentuk berasal dari hasil pemecahan laktosa dalam susu. Ekstrak umbi bit mengandung gula yang diduga dapat menstimulasi pertumbuhan dan meningkatkan bakteri asam laktat (BAL) dalam menghasilkan asam laktat, sehingga mempengaruhi terhadap pH. Salah satu kelebihan yoghurt karena sifat fungsionalitas terhadap kesehatan diantaranya yaitu mengatur kadar kolesterol dalam darah, memperlancar saluran pencernaan, membantu dalam meningkatkan pertumbuhan, antikanker, membantu penderita lactose intolerance, dan antidiare (Astawan, 2008). Sehingga dengan kelebihan yoghurt dan bit dapat mempengaruhi sifat fisiko kimia dan sensoris yoghurt (pH, padatan terlarut, aroma tanah dan tingkat kemanisan) dapat vulgaris L.) pada proses pembuatan yoghurt. Bit kaya akan senyawa bioaktif yang bermanfaat seperti karotenoid, saponin, glisin betain, polifenol dan flavonoid (Vali et al., 2007). Selain itu mengandung mikronutrisi (Mg, Na, K, Ca dan Vitamin C) yang tinggi, serat pangan serta zat besi, tetapi bit memiliki kandungan protein dan lemak yang rendah. Yoghurt kaya akan protein, lemak, kalsium, potassium, vitamin B (B1, B2, B6, asam nicotinic dan pantothenic) akan tetapi kekurangan zat besi, vitamin C, karotenoid dan serat pangan (Gahruie et al., 2015). Dengan demikian, kombinasi dari ekstrak bit dan yoghurt akan meningkatkan pangan gizi dan fungsional karakteristik yoghurt. Bit memiliki sukrosa yang dapat ditambahkan pada yoghurt untuk memberikan rasa manis yoghurt. Kandungan serat yang terdapat pada bit dapat digunakan sebagai fortifikasi pada yoghurt yang dihasilkan dan diharapkan dengan penambahan bit dapat menambah serat terlarut. Yoghurt dengan tanpa penambahan rasa didominasi asam karena asam laktat yang dihasilkan oleh fermentasi. Maka dari itu, untuk penerimaan yang lebih baik, buah-buahan, bahan perasa atau bahan pemanis ditambahkan ke yoghurt Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 90 Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 ©Indonesian Food Technologists http://dx.doi.org/10.17728/jatp. Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 ©Indonesian Food Technologists http://dx doi Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 ©Indonesian Food Technologists http://dx.doi.org/10.17728/jatp.181 refraktometer (SNI, 2004) dan sifat sensoris (aroma tanah dan tingkat kemanisan) menggunakan panelis (Setyaningsih et al., 2010). untuk meningkatkan keseimbangan rasa dan menutupi sebagian rasa asetaldehid pada yoghurt. Pendahuluan Penelitian ini bertujuan untuk mengetahui perbedaan perlakuan penambahan konsentrasi ekstrak bit terhadap nilai pH, total padatan terlarut, dan sifat sensoris (aroma tanah dan tingkat kemanisan) yoghurt dengan penambahan ekstrak bit. Manfaat penelitian ini yaitu untuk dapat melakukan penilaian terhadap penggunaan ekstrak bit dalam pembuatan yoghurt terhadap nilai pH, total padatan terlarut, dan sifat sensoris yoghurt, serta untuk memberikan diversifikasi dari produk yoghurt produk pangan yang mempunyai nilai fungsional dan meningkatkan pangan gizi dari karakteristik yoghurt dengan menambahkan ekstrak bit. Pengukuran pH (derajat asam) dengan pH meter “HANNA instruments” yang dikalibrasi dengan larutan buffer pH 4 dan 7 sebelum digunakan. Pengukuran sampel dilakukan dengan sebanyak 20 ml diambil, kemudian elektroda dibilas dengan aquades. Elektroda dikeringkan dengan tisu kemudian dicelupkan dalam sampel. Elektroda dibiarkan tercelup beberapa saat. Nilai yang terbaca adalah nilai pH yang telah stabil (AOAC, 1995). Pengujian Total Padatan Terlarut Materi dan Metode Materi e guj a ota adata e a ut Pengukuran total padatan terlarut menggunakan refractometer menurut SNI 01-3546-2004. Total kandungan padatan terlarut dari ekstrak bit dan yoghurt dengan penambahan ekstrak bit ditentukan dengan menggunakan refraktometer genggam digital (Model: PAL-1, Atago co, Ltd, Tokyo, Jepang) pada 25°C dan dilakukan kalibrasi menggunakan aquades, sebanyak 1-2 sampel dimasukkan pada prisma refraktometer dan jumlah kandungan padatan terlarut dinyatakan sebagai °Brix. Bahan yang digunakan dalam penelitian ini adalah bit, air, susu skim cair, starter campuran L. bulgaricus, L. acidophilus, dan S. thermophillus, alkohol, aquadest, larutan buffer, dan susu UHT. Alat yang digunakan adalah juicer, plastik PE, botol kaca, panci, kompor, gelas beker, inkubator, freezer, cawan petri, pipet tetes, tabung reaksi, pH meter “HANNA instruments”, refraktometer, viskometer, tisu, refrigerator, cup kecil, erlenmeyer, timbangan analitik, karet, corong, mikrotube, mikropipet kuning, mikropipet biru, plastik wrap, alumunium foil, gelas ukur, dan bunsen. Metode Penelitian dilakukan pada bulan September – Oktober 2015. Perlakuan yang diterapkan terdiri dari tanpa penambahan ekstrak bit (T0), penambahan ekstrak bit 1% dari 500 ml susu skim (T1), penambahan ekstrak bit 2% dari 500 ml susu skim (T2), penambahan ekstrak bit 3% dari 500 ml susu skim (T3), dan penambahan ekstrak bit 4% dari 500 ml susu skim (T4). masing-masing perlakuan diulang sebanyak 4 kali. Penelitian terdiri dari beberapa langkah yaitu pembuatan ekstrak bit, pembuatan starter kerja yoghurt, pembuatan yoghurt dengan penambahan ekstrak bit dan pengujian parameter yang meliputi nilai pH, total padatan terlarut dan sifat sensoris (aroma tanah dan tingkat kemanisan). Umbi bit diambil ekstraknya dengan menggunakan juicer untuk didapatkan hasil ekstrak bit. Pembuatan starter kerja digunakan dengan mencampurkan bibit bubuk yoghurt dengan aquades hingga diperoleh starter kerja yoghurt (Harjiyanti et al., 2013). Mekanisme pembuatan yoghurt dengan penambahan ekstrak bit yaitu susu skim cair masing-masing sebanyak 500 ml yang telah ditambahkan dengan ekstrak bit sebanyak 0%, 1%, 2%, 3%, dan 4% (sesuai perlakuan) dan di pasteurisasi pada 73 °C selama 15 detik, lalu suhunya diturunkan sampai 43 °C. Susu hasil pasteurisasi kemudian diinokulasi dengan starter BAL sebanyak 5%, selanjutnya diinkubasi selama 4 jam pada suhu 43 °C (Legowo et al., 2009 dengan modifikasi). Parameter pengujian yoghurt yaitu pengujian nilai pH dengan pH meter(AOAC, 1995), total padatan terlarut dengan Pengujian Sensoris Pengujian sensoris dilakukan oleh 25 orang panelis agak terlatih. Panelis dipilih secara acak yang diminta untuk menilai tingkat kemanisan, aroma tanah, dan citarasa asam yoghurt dengan penambahan ekstrak bit. Metode yang digunakan adalah dengan mempersiapkan sampel terlebih dahulu dengan ditempatkan pada cup kecil 50 ml. Tabel uji disediakan berupa form kuisioner uji tingkat aroma tanah yoghurt dengan rentang nilai 1 sampai 5 dengan nilai 1 yaitu sangat tidak bau tanah, nilai 2 yaitu tidak bau tanah, nilai 3 yaitu agak bau tanah, nilai 4 yaitu bau tanah, dan nilai 5 yaitu sangat bau tanah. Uji tingkat kemanisan yoghurt dengan rentang nilai 1 sampai 5 dengan nilai 1 yaitu sangat tidak manis, nilai 2 yaitu tidak manis, nilai 3 yaitu agak manis, nilai 4 yaitu manis, dan nilai 5 yaitu sangat manis (Setyaningsih et al., 2010). Hasil dan Pembahasan Nilai pH Berdasarkan Tabel 1, nilai pH yoghurt dengan penambahan ekstrak bit menunjukkan adanya perbedaan nyata penambahan ekstrak bit terhadap pH yoghurt. Rata-rata pH akhir yoghurt berkisar antara 3,79 - 3.92 dengan waktu fermentasi selama 6 jam. Kisaran pH tersebut merupakan akibat dari Streptoccus yang menurunkan pH menjadi 5,0 – 5,5 yang kemudian dilanjutkan dengan aktivitas Lactobacillus yang menurunkan pH menjadi 3,8 – 4,4 (Rahman et al., 1992). ) Dalam penelitian ini, rata-rata nilai pH yoghurt yang ditambahkan dengan ekstrak bit 1% dan 2% terlihat lebih rendah dari kontrol (Tabel 1). Dukungan dari umbi bit yang memiliki karbohidrat berupa gula sukrosa sebagai sumber energi bagi berlangsungnya bakteri untuk proses fermentasi. Hal ini sesuai dengan Icier et al. (2015) bahwa tersedianya karbohidrat sebagai sumber energi menyebabkan peningkatan metabolisme aktivitas oleh bakteri L. acidophilus yang memberikan kontribusi terhadap penurunan pH. Sedangkan pH yoghurt dengan penambahan ekstrak bit 3% dan 4% terlihat berada diatas kontrol dan hasil pH yoghurt dengan penambahan ekstrak bit 1%, 2%, 3%, 4%, dan 5% terlihat mengalami peningkatan nilai pH. Hal tersebut terjadi diduga karena efek penghambatan oleh gula-gula sederhana (sukrosa) yang terkandung dalam umbi bit karena habitat asli dari BAL yaitu memecah laktosa dan hanya memanfaatkan laktosa sebagai sumber karbon dan sumber energi. Tabel 1. Nilai Rata-rata Nilai pH dan Total Padatan Terlarut Yoghurt dengan Perlakuan Penambahan Ekstrak Bit Perlakuan Nilai pH Total Padatan Terlarut T0 3,89 ± 0,37b 7,70 ± 0,20a T1 3,79 ± 0,59a 7,90 ± 0,09ab T2 3,88 ± 0,77b 8,01 ± 0,63bc T3 3,92 ± 0,52b 8,13 ± 0,16c T4 3,92 ± 0,71b 8,10 ± 0,14bc Keterangan: Superskrip huruf kecil yang berbeda pada baris rata-rata menunjukkan perbedaan nyata (P<0,05) Tabel 1. Nilai Rata-rata Nilai pH dan Total Padatan Terlarut Yoghurt dengan Perlakuan Penambahan Ekstrak Bit Keterangan: Superskrip huruf kecil yang berbeda pada baris rata-rata menunjukkan perbedaan nyata (P<0,05) Tabel 2. Rata-rata Sifat Sensori Yoghurt dengan Perlakuan Penambahan Ekstrak Bit Tabel 2. Rata-rata Sifat Sensori Yoghurt dengan Perlakuan Penambahan Ekstrak Bit Perlakuan Aoma Tanah Tingkat Kemanisan T0 1,24 ± 0,44a 2,00 ± 0,64a T1 1,96 ± 0,54 b 2,04 ± 0,61a T2 2,64 ± 0,50c 2,20 ± 0,64a T3 3,12 ± 0,53d 2,96 ± 0,74b T4 3,16 ± 0,47de 2,92 ± 1,00bc Keterangan: Superskrip huruf kecil yang berbeda pada baris rata-rata menunjukkan perbedaan nyata (P<0,05) g g Menurut Patrick et al. Analisis Data Data yang diperoleh dari pengujian nilai pH dan total padatan terlarut yoghurt diolah dengan Analisis of Varians (ANOVA) pada taraf 5% karena sebaran datanya normal untuk mengetahui perbedaan perlakuan konsentrasi ekstrak bit terhadap nilai pH dan total padatan terlarut. Apabila terdapat perbedaan nyata maka dilanjutkan dengan uji Wilayah Ganda Duncan (Duncan’s Multi Range Test). Uji Duncan ini digunakan untuk mengetahui perlakuan konsentrasi yang menunjukkan perbedaan secara nyata. Pengujian sifat sensoris (aroma tanah dan tingkat kemanisan) diuji dengan menggunakan uji Kruskal-Wallis, dan apabila terdapat perbedaan nyata maka dilanjutkan ke uji Mann-Whitney untuk mengetahui perlakuan konsentrasi ekstrak bit yang menunjukkan perbedaan secara nyata. Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 ©Indonesian Food Technologists http://dx.doi.org/10.17728/jatp.1 Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 ©Indonesian Food Technologists http://dx.do 91 Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 ©Indonesian Food Technologists http://dx.doi.org/10.17728/jatp.181 Perolehan data diolah dengan bantuan komputer program SPSS 16.0 for windows. Berdasarkan hasil penelitian yang telah dilakukan semakin tinggi perlakuan penambahan ekstrak bit maka total padatan terlarutnya juga semakin tinggi. Peningkatan ekstrak bit menyebabkan jumlah total padatan terlarut (TPT) yang dihasilkan semakin meningkat pula. Selama berlangsungnya proses fermentasi laktosa dan sukrosa akan dirombak oleh kultur starter. Menurut Sintasari et al. (2014) sisa laktosa, sukrosa dan asam-asam organik lain tersebut yang terhitung sebagai total padatan terlarut. Sehingga semakin banyak penambahan dengan ekstrak bit akan meningkatkan total padatan terlarutnya. Asam laktat termasuk asam organik yang merupakan salah satu jenis total padatan terlarut selain gula, pigmen, dan vitamin. Total Padatan Terlarut Tabel 1. menunjukkan bahwa perlakuan penambahan ekstrak bit memilki perbedaan nyata (P<0,05) terhadap total padatan terlarut. Total padatan terlarut yang diperoleh yaitu antara 7,70 – 8,10 °Brix. Proses fermentasi bakteri asam laktat menghasilkan metabolit berupa asam laktat. Menurut Fardiaz (2003) metabolit tersebut akan terseksresikan keluar sel dan akan terakumulasi dalam cairan fermentasi. Sisa hasil total gula, asam laktat, dan asam organik yang terbentuk terhitung sebagai total padatan terlarut. Komponen padatan terlarut terdiri dari total gula. pigmen, asam-asam organik, dan protein. Hasil dan Pembahasan Nilai pH (2004) BAL memanfaatkan laktosa dan menghasilkan asam laktat sebagai zat metabolit karena laktosa merupakan sumber utama karbon dan sumber energi bagi BAL terutama oleh spesies bakteri S. thermophilus. Ditambahkan oleh Kiros et al. (2016) menyatakan bahwa meningkatnya nilai pH yang ditambahkan oleh ekstrak bit adalah karena aktivitas yang rendah oleh bakteri asam laktat selama masa inkubasi dibandingkan dengan kontrol yang memiliki kandungan solid non fat (SNF) atau padatan bukan lemak dan memiliki kadar laktosa dalam susu yang lebih tinggi. Keterangan: Superskrip huruf kecil yang berbeda pada baris rata-rata menunjukkan perbedaan nyata (P<0,05) Total padatan terlarut dalam ekstrak bit adalah sebesar 6 °Brix yang menunjukkan bahwa isi padatan terlarut adalah gula dan asam organik. Menurut Manjunatha dan Raju (2015) kepadatan dan aktivitas air dalam ekstrak bit masing-masing adalah sekitar 1024,4 kg/m3 dan 0,988. Total padatan meningkat seiring dengan meningkatnya presentase ekstrak bit. Hal ini dikarenakan bit memiliki sejumlah padatan terlarut sehingga setelah ditambahakan ke yoghurt, padatan terlarut dalam yoghurt meningkat. Hal tersebut sesuai dengan pendapat Osundahusi et al. (2007) yang menyatakan bahwa kandungan gula tinggi yang tinggi dari buah-buahan atau lainnya yang ditambahkan ke yoghurt telah berkontribusi terhadap komponen padatan terlarut yang lebih tinggi. Aroma Tanah Berdasarkan rata-rata hasil penelitian aroma tanah yoghurt dengan penambahan ekstrak bit, seiring dengan level pemberian ekstrak bit menambah tingkat aroma tanah pada yoghurt. Aroma atau bau suatu produk pangan menentukan kenikmatan pangan tersebut. Penilaian aroma suatu produk pangan tidak Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 Jurnal Aplikasi Teknologi Pangan 5 (3) 2016 ©Indonesian Food Technologists http://dx.doi.org/10.17728/jatp.181 92 terlepas dari fungsi indera pembau. Hal sesuai menurut Winarno (2004) yang menyatakan bahwa bau yang diterima oleh hidung dan otak lebih banyak merupakan campuran empat bau yaitu asam, tengik, harum, dan hangus. Aroma dan rasa yang dihasilkan dari umbi bit disebabkan oleh senyawa volatil, senyawa tersebut dipancarkan karena jaringan terluka, terpotong, dipanaskan atau dihancurkan. Berdasarkan Tabel 2 terlihat bahwa yoghurt dengan penambahan ekstrak bit 1% sampai 4% masih menghasilkan yoghurt dengan kriteria tidak manis sampai agak manis. Yoghurt yang dihasilkan belum mendapati kriteria manis ataupun sangat manis. Hal tersebut berkaitan dengan kandungan gula sukrosa yang terdapat dalam bit memilki nilai rasa manis yang berbeda dengan glukosa dan fruktosa. Hal ini sesuai dengan Belitz et al. (2009) yang menyatakan bahwa glukosa memiliki nilai rasa manis 69% dari sukrosa, sedangkan fruktosa memiliki nilai rasa manis 114% dari sukrosa. Hal ini ditambahkan oleh Sintasari et al. (2014) bahwa sukrosa yang pada fungsi utama sebagai pemberi rasa manis yang juga dapat memberikan nutrisi pada bakteri asam laktat secara optimal agar bakteri tersebut mampu menghasilkan rasa yang pas dan tidak terlalu masam / khas akibat pembentukan asam laktat dan asam-asam organik lain sebagai hasil dari metabolitnya sehingga menimbulkan kombinasi yang pas bagi panelis saat menguji coba produk yoghurt dengan penambahan ekstrak bit ini. Perlakuan yoghurt T0 (0%) atau kontrol yang dihasilkan adalah sangat tidak bau. Hal ini dikarenakan tidak ada penambahan ekstrak bit sedikitpun pada yoghurt sehingga aroma tanah yang dihasilkan tidak ada. Penambahan ekstrak bit 1% memiliki nilai rata-rata terendah yaitu 1,96 yaitu dengan kriteria sangat tidak bau sampai tidak bau. Sedangkan untuk rata-rata nilai aroma tanah paling tinggi adalah 4% yaitu 3,16 dengan kriteria agak bau tanah. Aroma tanah yang terkandung pada bit disebabkan oleh suatu senyawa yang terkandung, sehingga semakin banyak penambahan ekstrak bit yang ditambahkan maka semakin tinggi pula tingkat aroma bau tanah yoghurt tersebut. Sesuai dengan Lu et al. (2003) yang menyatakan bahwa aroma tanah pada bit disebabkan oleh suatu senyawa yang terdapat di bit yaitu geosmin dan beberapa senyawa pirazin saat menambahkan ekstrak bit. Kesimpulan Berdasarkan hasil penelitian yang telah dilakukan dapat disimpulkan bahwa semakin besar penambahan ekstrak bit maka semakin menaikkan nilai pH dan total padatan terlarut. Yoghurt dengan penambahan ekstrak bit cenderung agak bau tanah (kriteria sangat tidak bau hingga agak bau), dengan tingkat kemanisan cenderung agam manis (kriteria tidak manis hingga agak manis). Aroma Tanah Geosmin merupakan senyawa metabolit sekunder yang bersifat aromatik. Menurut Lu et al. (2003) bahwa senyawa organik tersebut dihasilkan oleh mikroba dalam tanah, geosmin mengeluarkan bau seperti aroma tanah atau seperti bau yang dihasilkan ketika hujan turun dan mengenai permukaan bumi. Hidung manusia sangat sensitif terhadap geosmin. Beberapa orang tidak menyukai aroma tanah tersebut sedangkan yang lainnya menyukai. Daftar Pustaka Andarwulan, N. dan F. Faradilla. 2012, Pewarna Alami Untuk Pangan. Seafast Center. Institut Pertanian Bogor. Bogor. Tingkat Kemanisan g Berdasarkan hasil penelitian yang dilakukan tersebut didapatkan hasil bahwa kontrol mempunyai nilai 2,00 yang berarti memiliki kriteria tidak manis. Begitu pula dengan tingkat kemanisan yoghurt dengan penambahan ekstrak bit 1% dan 2% yang mempunyai nilai masing-masing sebesar 2,04 dan 2,20 dengan kriteria tidak manis. Sedangkan hasil yang berbeda ditunjukkan oleh yoghurt dengan penambahan ekstrak bit 3% dan 4% yaitu dengan nilai 2,96 dan 2,92 dengan kriteria tidak manis sampai agak manis. Hal tersebut mengindikasikan bahwa penambahan ekstrak bit dapat menambah rasa manis pada yoghurt. Kandungan gula dalam bit diduga bertanggung jawab terhadap hal tersebut yang mempengaruhi penilaian panelis terhadap tingkat kemanisan. Hal ini sesuai dengan Andarwulan dan Faradilla (2012) bahwa tanaman bit dibudidayakan terutama untuk produksi gula karena umbi bit mengandung gula sukrosa dalam kadar yang tinggi, sehingga digunakan sebagai pemanis. Sesuai dengan hasil penelitian kadar gula dalam bit mengandung sekitar 6%. Menurut Drake et al. (2001) menyatakan bahwa rasa gula yang manis memiliki efek yang besar dengan penilaian konsumen terhadap rasa yoghurt. AOAC. 1995. Official Methods of Analisys Chemist. Vol. 1A. AOAC Inc., Washington. Astawan, M. 2008. Sehat dengan Hidangan Hewani. Penebar Swadaya. SNI 01-3546. 2004. TSS Gravimetri. Standar Nasional Indonesia. Jakarta. Belitz, H. D., W. Grosch., dan P. Schieberle. 2009. Food chemistry. Berlin, Germany: Springer Verlag. Drake, M. 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https://openalex.org/W4286669030	https://bmcvetres.biomedcentral.com/counter/pdf/10.1186/s12917-022-03381-7	English	null	Pathogenic and genomic characterisation of a rabbit sourced Pasteurella multocida serogroup F isolate s4	BMC veterinary research	2,022	cc-by	8,909	© 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. Wang et al. BMC Veterinary Research (2022) 18:288 https://doi.org/10.1186/s12917-022-03381-7 Open Access Abstract Background: Pasteurella multocida is one of the most significant pathogens for a number of animals. In rabbits, the infection is generally associated with the P. multocida serogroups A and D, and the knowledge about the serogroup F is limited. In the present study, a P. multocida serogroup F isolate designated s4 was recovered from the lungs of rab- bits died of respiratory disease in Fujian, in the southeast of China. The pathogenicity and genomic features of the s4 were then determined. Results: The serotype and sequence type of s4 were F:L3 and ST12, respectively. The s4 was pathogenic for rabbits, but it was a low virulent strain comparing to the previously reported highly pathogenic P. multocida serogroup F strains J-4103, C21724H3km7, P-4218 and HN07. The whole genome of the s4 was then sequenced to understand the genomic basis for pathogenicity. Particularly, a large-sized fragment of approximate 275 kb in length was truncated from the chromosome to form a plasmid. Moreover, the in-frame deletion of natC and N-terminal redundance of gatF would resulted in the production of a mutant L3 outer core structure that was distinct from those of the other P. multocida strains belonging to the lipopolysaccharide genotype L3. We deduced that these features detected in the genome of s4 might impair the pathogenicity of the bacterium. Conclusions: This study evaluated the pathogenicity and determined the genomic features of the rabbit sourced P. multocida serogroup F isolate s4, the observations and findings would helpful for the understanding of the patho- genicity variability and genetic diversity of P. multocida. Keywords: Pasteurella multocida serogroup F, Rabbit, Pathogenicity, Genomic feature in rabbit [1, 2]. P. multocida strains can be classified into five capsular serogroups or capsular genotypes (A, B, D, E and F) and further classified into sixteen lipopolysac- charide (LPS) serotypes (1–16) or eight LPS genotypes (L1-L8) [3, 4]. P. multocida strains can also be typed by using the multi-locus sequence typing (MLST) scheme based on the seven housekeeping genes [5, 6]. Rabbit pasteurellosis is generally associated with the strains of capsular serogroups A and D, LPS genotypes L3 and L6, and MLST sequence types ST10, ST11 and ST12 [7, 8]. However, some reports showed that strain of serogroup F that predominately circulated in poultry had also been Pathogenic and genomic characterisation of a rabbit sourced Pasteurella multocida serogroup F isolate s4 Jinxiang Wang, Shikun Sun, Yanfeng Chen, Dongjin Chen, Lei Sang and Xiping Xie Background Pasteurella multocida is recognized as one of the important pathogens for a wide range of animals. The infection of P. multocida is often associated with eco- nomically important diseases, such as fowl cholera in poultry, haemorrhagic septicaemia in bovine, progres- sive atrophic rhinitis in swine, and respiratory disease *Correspondence: wjx841227@126.com; xxp702@163.com Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, No. 100 Pudang Road, Xindian Town, Jin’an District, Fuzhou, Fujian 350013, People’s Republic of China © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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. . Open Access T d i di i Virulence genes detectionh The virulence genes ptfA, tadD, hgbB, ompA, omph and oma87 were positive for the 144 isolates, whereas the virulence genes pfhA, toxA, fur, tbpA, nanB and pmHAS were negative for the all isolates. The sequence of each of the virulence genes (ptfA, tadD, hgbB, ompA, omph and oma87) of the 144 isolates were identical, and the sequences of these six virulence genes shared highest identity (ranged from 99.90 to 100%) with the corre- sponding virulence genes of P. multocida. Taken together, the serotypes, sequence types and the virulence gene profiles of the 144 isolates were identi- cal, suggesting that all the isolates were derived from the same progenitor (Fig. 1). In this study, one of the isolates named s4 was selected as the representative for the following animal experiments and whole genome sequencing. In present study, the s4 was defined by capsular typ- ing, LPS genotyping, MLST, screened virulence factors, and evaluated the pathogenicity in rabbits. Moreover, the whole genome sequence of s4 was determined, and the whole genome comparison between the s4 and the other P. multocida serogroup F strains was performed. The aim of the present study was to evaluate the pathogenicity and determine the genetic features of the rabbit sourced P. multocida serogroup F isolate s4. Animal experiments To evaluate the virulence of P. multocida serogroup F isolate s4 in rabbits, groups of animals were subcutane- ously or intranasally challenged with 6.0 × 104 colony forming units (CFU) of the s4. All the eight rabbits in the subcutaneous inoculation group survived the challenge, and all of the rabbits showed no clinical signs of disease. The only gross lesion of local subcutaneous abscess at the inoculation site was observed in the all rabbits from the subcutaneous inoculation group (Fig. 2A), and the tissue samples including tracheas, lungs, livers, hearts, spleens, kidneys and blood of the all rabbits from the subcutane- ous inoculation group were negative for P. multocida. One rabbit in the intranasal inoculation group developed distinct clinical signs of cough and nasal discharge on 6 days post challenge (Fig. 2B), and the rabbit was caught . Open Access T d i di ib Wang et al. BMC Veterinary Research (2022) 18:288 Page 2 of 12 Page 2 of 12 detected in rabbits, and the bacterium was highly patho- genic for rabbits [9–12]. 1 mm in diameter. Three colonies from each lung sam- ple were randomly picked up. The sequences of the 16S rRNA genes of the 144 isolates recovered from the 48 lung samples were identical and shared the highest iden- tity (range from 99.90 to 100%) with that of P. multocida. The PCR assays for capsular and LPS typing of the iso- lates yielded segments of approximate 850 bp and 470 bp in length, and the sequences of the two segments were 100% identical with the fcbD and gatF of P. multocida, respectively. The results indicated that the 144 isolates were P. multocida serotype F:L3. Rabbit farming is popular in Fujian, in the southeast of China [8, 13]. The number of rabbits farmed and the amount of rabbit meat yielded in Fujian were about 10.67 million and 16.2 thousand tons by the end of 2019 (Fujian Statistical Yearbook, 2020), respectively. Our previous work showed that P. multocida was widespread in rabbits in Fujian, but only strains of serogroups A and D were detected [8]. In July 2020, a respiratory infectious disease broke out on a local rabbit farm in Fujian, and around 500 rabbits died of the disease during a 30-day period. Clini- cal signs of cough and nasal discharge were observed in the diseased rabbits. Gross pathological lesions including hemorrhagic pneumonia and pulmonary consolidation were showed in the dead rabbits. Four PCR assays were used to screen the potential causative agents including Rabbit hemorrhagic disease virus (RHDV) [14], P. mul- tocida [3], Bordetella bronchiseptica (B. bronchiseptica) [15] and Staphylococcus aureus (S. aureus) [16] in the 48 lung samples collected from the dead rabbits. The results showed that the RHDV, B. bronchiseptica and S. aureus were negative, but the P. multocida was positive for the all 48 lung samples and a P. multocida serogroup F iso- late named s4 was recovered from the all samples. To our knowledge, the information on the isolation and/or iden- tification of P. multocida serogroup F strain in the rabbits in Fujian is limited, and especially, there was no report concerning the pathogenicity of rabbit sourced P. multo- cida serogroup F strain isolated from Fujian.i MLSTh The allelic numbers of the 7 housekeeping genes (adk, aroA, deoD, gdhA, g6pd, mdh and pgi) of the 144 isolates were identical and assigned as 8–8–5-3-5-7-5, and the sequence types of the 144 isolates were then defined as ST12. P. multocida isolation and identificationh aureus (arcC, aroE, glpF, gmk, pta, tpi and yqiL) were concatenated, and then the neighbour-joining tree was constructed using MEGA 5.0 (1000 of bootstrap replications). The senve MLST genes of the strains were obtained under the accession numbers: P. multocida strains Pm70 (AE004439), HN07 (CP007040), CIRMBP-0884 (CP020345), CIRMBP-0873 (CP020347), HN06 (CP003313) and CQ2 (CP033599); B. bronchiseptica strains F709 (CP020818) and RB50 (BX470250); S. aereus strains ST59 (CP076823) and V8 (CP079715) Fig. 1 Neighbour-joining tree indicating the position of P. multocida isolate s4 based on the seven MLST genes. The seven MLST genes of P. multocida (adk, aroA, deoD, gdhA, g6pd, mdh and pgi), B. bronchiseptica (adk, fumC, glyA, tyrB, icd, pepA and pgm) and S. aureus (arcC, aroE, glpF, gmk, pta, tpi and yqiL) were concatenated, and then the neighbour-joining tree was constructed using MEGA 5.0 (1000 of bootstrap replications). The senve MLST genes of the strains were obtained under the accession numbers: P. multocida strains Pm70 (AE004439), HN07 (CP007040), CIRMBP-0884 (CP020345), CIRMBP-0873 (CP020347), HN06 (CP003313) and CQ2 (CP033599); B. bronchiseptica strains F709 (CP020818) and RB50 (BX470250); S. aereus strains ST59 (CP076823) and V8 (CP079715) Fig. 1 Neighbour-joining tree indicating the position of P. multocida isolate s4 based on the seven MLST genes. The seven MLST genes of P. multocida (adk, aroA, deoD, gdhA, g6pd, mdh and pgi), B. bronchiseptica (adk, fumC, glyA, tyrB, icd, pepA and pgm) and S. aureus (arcC, aroE, glpF, gmk, pta, tpi and yqiL) were concatenated, and then the neighbour-joining tree was constructed using MEGA 5.0 (1000 of bootstrap replications). The senve MLST genes of the strains were obtained under the accession numbers: P. multocida strains Pm70 (AE004439), HN07 (CP007040), CIRMBP-0884 (CP020345), CIRMBP-0873 (CP020347), HN06 (CP003313) and CQ2 (CP033599); B. bronchiseptica strains F709 (CP020818) and RB50 (BX470250); S. aereus strains ST59 (CP076823) and V8 (CP079715) Fig. 2 Clinical signs and gross lesions of the rabbits challenged with s4. P. multocida isolation and identificationh The presence of the potential pathogens including RHDV, P. multocida, B. bronchiseptica and S. aureus in the 48 lung samples collected from the natural infected dead rabbits were determined by using PCR assays. The results showed that RHDV, B. bronchiseptica and S. aureus were negative for the all 48 lung samples, whereas P. multocida was positive for the all 48 lung samples. All the lung sam- ples were then subjected to P. multocida isolation. All the 48 lung samples produced identical bacterial colonies on brain heart infusion (BHI) blood agar plate. The colonies were grey, round, smooth and less than Wang et al. BMC Veterinary Research (2022) 18:288 Page 3 of 12 Fig. 1 Neighbour-joining tree indicating the position of P. multocida isolate s4 based on the seven MLST genes. The seven MLST genes of P. multocida (adk, aroA, deoD, gdhA, g6pd, mdh and pgi), B. bronchiseptica (adk, fumC, glyA, tyrB, icd, pepA and pgm) and S. aureus (arcC, aroE, glpF, gmk, pta, tpi and yqiL) were concatenated, and then the neighbour-joining tree was constructed using MEGA 5.0 (1000 of bootstrap replications). The senve MLST genes of the strains were obtained under the accession numbers: P. multocida strains Pm70 (AE004439), HN07 (CP007040), CIRMBP-0884 (CP020345), CIRMBP-0873 (CP020347), HN06 (CP003313) and CQ2 (CP033599); B. bronchiseptica strains F709 (CP020818) and RB50 (BX470250); S. aereus strains ST59 (CP076823) and V8 (CP079715) Fig. 2 Clinical signs and gross lesions of the rabbits challenged with s4. A Local subcutaneous abscess of the rabbits subcutaneously infected with 6.0 × 104 CFU of the s4; B Nasal discharge of the rabbit intranasally infected with 6.0 × 104 CFU of the s4; C Matted forepaws of the rabbits intranasally infected with 6.0 × 104 CFU of the s4; D Hemorrhagic pneumonia of the rabbit intranasally infected with 6.0 × 104 CFU of the s4; E Pulmonary consolidation and weak hemorrhagic pneumonia of the rabbits intranasally infected with 6.0 × 104 CFU of the s4; F Weak hemorrhagic pneumonia of the rabbits intranasally infected with 6.0 × 104 CFU of the s4 Fig. 1 Neighbour-joining tree indicating the position of P. multocida isolate s4 based on the seven MLST genes. The seven MLST genes of P. multocida (adk, aroA, deoD, gdhA, g6pd, mdh and pgi), B. bronchiseptica (adk, fumC, glyA, tyrB, icd, pepA and pgm) and S. P. multocida isolation and identificationh The s4 Wang et al. BMC Veterinary Research (2022) 18:288 Page 4 of 12 Fig. 3 Histological lesions in the lungs of the rabbits intranasally infected with 6.0 × 104 CFU of the s4 (hematoxylin-eosin staining, × 400). A Inflammatory exudates appeared in the bronchiole and alveoli as well as degeneration of the alveolar epithelial cells in the one rabbit with hemorrhagic pneumonia; B Inflammatory exudates in the bronchiole and alveoli as well as proliferation of alveolar epithelial cells in the two rabbits with pulmonary consolidation and weak hemorrhagic pneumonia; C Proliferation of alveolar epithelial cells in the five rabbits with weak hemorrhagic pneumonia; D The lung tissues from the rabbits of negative control group Fig. 3 Histological lesions in the lungs of the rabbits intranasally infected with 6.0 × 104 CFU of the s4 (hematoxylin-eosin staining, × 400). A Inflammatory exudates appeared in the bronchiole and alveoli as well as degeneration of the alveolar epithelial cells in the one rabbit with hemorrhagic pneumonia; B Inflammatory exudates in the bronchiole and alveoli as well as proliferation of alveolar epithelial cells in the two rabbits with pulmonary consolidation and weak hemorrhagic pneumonia; C Proliferation of alveolar epithelial cells in the five rabbits with weak hemorrhagic pneumonia; D The lung tissues from the rabbits of negative control group and 2 intact prophages were determined (Fig. 4A). The plasmid of s4 was 274,750 bp in length with the G + C content of 39.62%, in which 302 protein coding sequences, 7 tRNA genes, 2 GIs and one intact prophage were determined (Fig. 4B). Interestingly, BLAST analysis showed that the plasmid of s4 was highly homologous (up to 85% identity) with the other P. multocida genomes at nucleotide level (Fig. 5). By blasting against the Com- prehensive Antibiotic Resistance Database (CARD), two elfamycin resistance genes and one cephalosporin resist- ance gene were predicted, and all of the three drug resist- ance genes were located in the chromosome of s4. shown in lungs of the two rabbits with pulmonary con- solidation and weak hemorrhagic pneumonia (Fig. 3B), only proliferation of alveolar epithelial cells was shown in lungs of the five rabbits with weak hemorrhagic pneu- monia (Fig. 3C). Interestingly, all the eight rabbits in the subcutaneous inoculation group and five rabbits in the intranasal inoculation group were serological positiv- ity for P. multocida IgG on 15 days post challenge. P. multocida isolation and identificationh Dur- ing the 15-day experimental period, all the rabbits in the control group remained P. multocida free, and showed no clinical signs and gross lesions of disease. P. multocida isolation and identificationh A Local subcutaneous abscess of the rabbits subcutaneously infected with 6.0 × 104 CFU of the s4; B Nasal discharge of the rabbit intranasally infected with 6.0 × 104 CFU of the s4; C Matted forepaws of the rabbits intranasally infected with 6.0 × 104 CFU of the s4; D Hemorrhagic pneumonia of the rabbit intranasally infected with 6.0 × 104 CFU of the s4; E Pulmonary consolidation and weak hemorrhagic pneumonia of the rabbits intranasally infected with 6.0 × 104 CFU of the s4; F Weak hemorrhagic pneumonia of the rabbits intranasally infected with 6.0 × 104 CFU of the s4 Fig. 2 Clinical signs and gross lesions of the rabbits challenged with s4. A Local subcutaneous abscess of the rabbits subcutaneously infected with 6.0 × 104 CFU of the s4; B Nasal discharge of the rabbit intranasally infected with 6.0 × 104 CFU of the s4; C Matted forepaws of the rabbits intranasally infected with 6.0 × 104 CFU of the s4; D Hemorrhagic pneumonia of the rabbit intranasally infected with 6.0 × 104 CFU of the s4; E Pulmonary consolidation and weak hemorrhagic pneumonia of the rabbits intranasally infected with 6.0 × 104 CFU of the s4; F Weak hemorrhagic pneumonia of the rabbits intranasally infected with 6.0 × 104 CFU of the s4 was re-isolated from the following tissue samples col- lected from rabbits in the intranasal inoculation group: tracheas (n = 8/8), lungs (n = 8/8), livers (n = 8/8), hearts (n = 0/8), spleens (n = 0/8), kidneys (n = 0/8) and blood (n = 0/8). Histologically, inflammatory exudates appeared in the bronchiole and alveoli as well as degener- ation of the alveolar epithelial cells were observed in the lung of the rabbit with hemorrhagic pneumonia (Fig. 3A), inflammatory exudates in the bronchiole and alveoli as well as proliferation of alveolar epithelial cells were up in the endpoint on 15 days post challenge because of inability to access feed and water. However, the only clini- cal sign of matted forepaws was showed in the remain- ing 7 rabbits in the intranasal inoculation group (Fig. 2C), and the seven rabbits survived the challenge. Gross lesions including hemorrhagic pneumonia (one rabbit) (Fig. 2D), pulmonary consolidation and weak hemor- rhagic pneumonia (two rabbits) (Fig. 2E), and weak hem- orrhagic pneumonia (five rabbits) (Fig. 2F) were observed in the rabbits in the intranasal inoculation group. Genome sequencing and comparative analysish Phylogenetic analysis showed that the s4 was closely related to the well- characterized avian sourced P. multo- cida serogroup F strain Pm70 (Fig. 1) [17]. Therefore, the genome of s4 was compared with that of Pm70. By com- paring with the genome of Pm70, gene rearrangement, gain and loss were observed in that of s4. Especially, three large regions (R) of specific sequences were found in the genome of s4 (Fig. 6). Two regions (R1 and R2) located in the chromosome and were approximately 31.9 kb and 37.0 kb in length, respectively. The third region (R3) located in the plasmid and was approximately 42.3 kb in length. Interestingly, all of the three regions located The complete genome of the s4 was approximately 2.06 Mbp in length, which is 201,495 bp smaller than that of avian sourced P. multocida serogroup F strain Pm70 [17]. Interestingly, the complete genome of s4 consisted of a chromosome and a plasmid. The complete genome of the s4 has been deposited to the NCBI GenBank data- base under the accession numbers of CP084165 (chro- mosome) and CP084164 (plasmid). The chromosome of s4 was 1,781,242 bp in length with the G + C content of 40.42%, in which 1619 protein coding sequences, 45 tRNA genes, 19 rRNA genes, 3 Genomic islands (GIs) Wang et al. BMC Veterinary Research (2022) 18:288 Page 5 of 12 Fig. 4 Circular maps of the s4 genome generated by using DNAPlotter. A The chromosome of s4; B The plasmid of s4. From the outside to the inside, circle 1 (black): DNA base position; circle 2 (cyan-blue): protein-coding regions in forward strand; circle 3 (cyan-blue): protein-coding regions in reverse strand; circle 4 (red): tRNA genes; circle 5 (blue): rRNA genes; circle 6 (green): genomic islands; circle 7 (purple): prophages; the two innermost circles represent the G + C content and GC skew, respectively Fig. 4 Circular maps of the s4 genome generated by using DNAPlotter. A The chromosome of s4; B The plasmid of s4. Genome sequencing and comparative analysish From the outside to the inside, circle 1 (black): DNA base position; circle 2 (cyan-blue): protein-coding regions in forward strand; circle 3 (cyan-blue): protein-coding regions in reverse strand; circle 4 (red): tRNA genes; circle 5 (blue): rRNA genes; circle 6 (green): genomic islands; circle 7 (purple): prophages; the two innermost circles represent the G + C content and GC skew, respectively (in positions 559–741) was observed in the natC of s4, which resulted in a deletion of 61 amino acids (in posi- tions 183–243) of the glycosyltransferase NatC of s4 by comparison with that of Pm70 (Fig. 8). Additionally, the gatF of s4 possessed a 210-bp nucleotide sequence redundance at the N-terminal by comparison with that of Pm70 (Fig. 8). in the intact phage regions that were determined in s4 genome by using PHAST [18]. Additionally, one unique gene (LCY73_01710) encoding the TonB-dependent receptor was identified in the genome of s4 but absent in that of Pm70. The entire cap locus of s4 were compared with those of Pm70 (capsular type F), HN07 (capsular type F), CIRMBP-0873 (capsular type F), CIRMBP-0884 (capsu- lar type F), CQ2 (capsular type A) and HN06 (capsular type D). The result showed that the entire cap locus of s4 was highly matched with those of capsular type F strains (Pm70, HN07, CIRMBP-0873 and CIRMBP-0884), and the entire cap locus of s4 was closely matched with that of capsular type A strain (CQ2) but was distinct from that of capsular type D strain (HN06) (Fig. 7). Discussion From the outside to the inside, circle 1 (black): positions of capsule locus and LPS outer core locus; circle 2 (gray): plasmid of s4; circle 3 (gray): chromosome of s4; circle 4 (yellow): plasmid of CIRMBP-0884; circle 5 (yellow): chromosome of CIRMBP-0884; circle 6 (green): CQ2; circle 7 (purple) HN06; circle 8 (orange): CIRMBP-0873; circle 9 (blue): HN07; circle 10 (red): Pm70; circles 11 and 12 represent the G + C content and GC skew, respectively; the innermost circle represents DNA base position Fig. 5 Comparation between the genome of s4 and those of CIRMBP-0884, HN06, CQ2, CIRMBP-0873, HN07 and Pm70 at nucleotide level. From the outside to the inside, circle 1 (black): positions of capsule locus and LPS outer core locus; circle 2 (gray): plasmid of s4; circle 3 (gray): chromosome of s4; circle 4 (yellow): plasmid of CIRMBP-0884; circle 5 (yellow): chromosome of CIRMBP-0884; circle 6 (green): CQ2; circle 7 (purple): HN06; circle 8 (orange): CIRMBP-0873; circle 9 (blue): HN07; circle 10 (red): Pm70; circles 11 and 12 represent the G + C content and GC skew, respectively; the innermost circle represents DNA base position the pathogenicity of a rabbit sourced P. multocida sero- group F isolate s4 was evaluated and the whole genome of the isolate was sequenced, which would helpful for the understanding of the pathogenitity of rabbit sourced P. multocida serogroup F. rabbit without prior adaption [23, 24]. The results sug- gested that the variation in virulence among P. multo- cida serogroup F strains, and the s4 was a low virulent strain by comparison with the J-4103, C21724H3km7, P-4218 and HN07. Additionally, it should be alert to the potential transmission of P. multocida serogroup F from chicken, turkey and pig into rabbit, which might cause high mortality and subsequent huge economic loss to rabbit farming. Unexpectedly, the inoculation of s4 did not cause death and result in severe gross lesions in most of the chal- lenged rabbits. The results were in contradict with the previous study, in which the inoculation of rabbit sourced P. multocida serogroup F isolate J-4103 caused high mor- tality and resulted in severe pathological lesions in most of the challenged rabbits (acute septicemic syndrome and extensive hemorrhage in subcutis in subcutaneously challenged rabbits, and fibrinopurulent pleuropneumo- nia and extensive hemorrhagic pneumonia in intrana- sally challenged rabbits) [12]. Moreover, P. Discussion P. multocida serogroup F was first isolated from turkeys in the USA in 1987 [19], and since then it has been mainly associated with the infections in avian hosts [20–22]. P. multocida is one of the important pathogens results in morbidity and mortality in rabbits, whereas only the serogroups A and D had been previously recognized as the causative agents of rabbit pasteurellosis [7, 12]. It was not until 2008 that the pathogenicity of P. multocida serogroup F for rabbit was documented [12]. Since then, the presence of P. multocida serogroup F has been iden- tified in the important rabbit farming areas worldwide [9–11]. However, the knowledge about the pathogenicity and genetic characteristics of the rabbit sourced P. mul- tocida serogroup F is still limited. In the present study, The LPS outer core biosynthetic genes of s4 were compared with that of Pm70. All of the LPS outer core biosynthetic genes of Pm70 were located in the chro- mosome, whereas these genes were located in the plas- mid of s4. With the exception of natC and gatF, the LPS outer core biosynthetic genes of s4 including gatG, natB, gctC and hptE were highly matched with those of Pm70. Interestingly, an in-frame deletion of 183-bp nucleotide Wang et al. BMC Veterinary Research (2022) 18:288 Page 6 of 12 Fig. 5 Comparation between the genome of s4 and those of CIRMBP-0884, HN06, CQ2, CIRMBP-0873, HN07 and Pm70 at nucleotide level. From the outside to the inside, circle 1 (black): positions of capsule locus and LPS outer core locus; circle 2 (gray): plasmid of s4; circle 3 (gray): chromosome of s4; circle 4 (yellow): plasmid of CIRMBP-0884; circle 5 (yellow): chromosome of CIRMBP-0884; circle 6 (green): CQ2; circle 7 (purple): HN06; circle 8 (orange): CIRMBP-0873; circle 9 (blue): HN07; circle 10 (red): Pm70; circles 11 and 12 represent the G + C content and GC skew, respectively; the innermost circle represents DNA base position Fig. 5 Comparation between the genome of s4 and those of CIRMBP-0884, HN06, CQ2, CIRMBP-0873, HN07 and Pm70 at nucleotide level. Discussion multocida serogroup F isolates from chicken (C21724H3km7), tur- key (P-4218) and pig (HN07) were also highly virulent to P. multocida possesses a number of virulence genes, which contributes to the fitness and pathogenicity of the pathogen [1]. The s4 carried a panel of virulence genes including ptfA, tadD, hgbB, ompA, omph and oma87. The ptfA, tadD, ompA, omph and oma87 encode the proteins that are associated with P. multocida virulence because of their roles in the attachment and colonization of host [25, 26]. The hgbB encodes an outer membrane protein, which is involved in iron acquisition [25]. Interestingly, the P. Wang et al. BMC Veterinary Research (2022) 18:288 Page 7 of 12 Fig. 6 Colinear analyses between the genomes of s4 and Pm70. s4-Chr and s4-P represent the chromosome and plasmid of s4, respectively. Rectangle of the same colour indicates the similar local colinear block of the s4 and Pm70. R1, R2 and R3 indicate the three large regions of specific sequences in the genome of s4 but not in that of Pm70 Fig. 6 Colinear analyses between the genomes of s4 and Pm70. s4-Chr and s4-P represent the chromosome and plasmid of s4, respectively. Rectangle of the same colour indicates the similar local colinear block of the s4 and Pm70. R1, R2 and R3 indicate the three large regions of specific sequences in the genome of s4 but not in that of Pm70 Additionally, the plasmids of the rabbit sourced P. mul- tocida serogroup F strains s4 and CIRMBP-0884 were highly identical (up to 85% indentity) to the other P. multocida genomes at nucleotide level. Therefore, it was deduced that the sequences of the plasmids of s4 and CIRMBP-0884 might derive from their own genomes. Interestingly, only a small number of P. multocida strains possess plasmids according to the P. multocida genomes deposited in the NCBI genome database. The plasmids of P. multocida strains often carry antibiotic resistance genes and mobilization genes [28], and plasmids of some P. multocida strains carry the toxA gene encoding the dermonecrotic toxin [28]. Unexpectedly, the antibiotic resistance genes, mobilization genes and toxin gene were not determined in the plasmid of s4. multocida serogroup D isolates carrying the hgbB were significantly associated with the clinical presentation of respiratory disease in rabbits [9]. Additionally, com- parative genomic analyses between s4 and Pm70 identi- fied a unique gene in s4 but absent in Pm70. Discussion The unique gene (LCY73_01710) encoded TonB-dependent receptor that is essential for transport of organic iron chelators (siderophores) into the periplasm to establish commen- sal and pathogenic relationships with the hosts [27]. In Pm70, more than 2.5% of the genome was devoted to genes encoding proteins involved in iron uptake and acquisition, suggesting that iron uptake and acquisition is important for P. multocida survival and pathogenesis [17]. Taken together, the presence of these genes might contribute to the pathogenicity of the s4. LPS is an important virulence determinant of P. mul- tocida [29]. The LPS of the s4 belongs to the genotype of L3. It was showed that P. multocida strains belonging to the LPS genotype L3 might be highly virulent because these strains were the most common causative agents of fowl cholera [4, 29]. The L3 outer core structure is similar to the self-antigen of host, which facilitates the bacteria evading the host innate immune system [29]. The in- frame deletion of natC and the N-terminal redundance of gatF would result in the production of a mutant L3 outer core structure, which might contribute to the low pathogenicity of s4. Interestingly, the genetic diversi- ties of natC and gatF from P. multocida serogroup F:L3 The complete genome of s4 was sequenced and then the comparative genomic analyses were performed to better understand the genetic basis for the pathogenic- ity of s4. The genome of s4 shared a high level of DNA identity to that of Pm70. However, the genome of s4 was smaller (approximate 200 kb) than that of Pm70, which might result in the loss of a number of functional genes and subsequently impaired the pathogenicity of the s4. Interestingly, the s4 possessed a plasmid of approxi- mate 275 kb in length, and the sequence of the plasmid shared a high level of DNA identity (up to 90% identity) to the plasmid (approximate 325 kb in length) of rabbit sourced P. multocida serogroup F strain CIRMBP-0884. Wang et al. BMC Veterinary Research (2022) 18:288 Page 8 of 12 Fig. 7 Comparative analyses of the entire cap locus between the s4 and other P. multocida strains. The entire cap locus of the s4 were compared with those of Pm70 (capsular type F), HN07 (capsular type F), CIRMBP-0873 (capsular type F), CIRMBP-0884 (capsular type F), CQ2 (capsular type A) and HN06 (capsular type D). Discussion The color code represents the BLASTn identity Fig. 7 Comparative analyses of the entire cap locus between the s4 and other P. multocida strains. The entire cap locus of the s4 were compared with those of Pm70 (capsular type F), HN07 (capsular type F), CIRMBP-0873 (capsular type F), CIRMBP-0884 (capsular type F), CQ2 (capsular type A) and HN06 (capsular type D). The color code represents the BLASTn identity Fig. 7 Comparative analyses of the entire cap locus between the s4 and other P. multocida strains. The entire cap locus of the s4 were compared with those of Pm70 (capsular type F), HN07 (capsular type F), CIRMBP-0873 (capsular type F), CIRMBP-0884 (capsular type F), CQ2 (capsular type A) and HN06 (capsular type D). The color code represents the BLASTn identity strains of CIRMBP-0873, CIRMBP-0884 and HN07 were also detected (Fig. 8) [30]. Taken together, the effects of the polymorphism in the LPS outer core genes natC and gatF on the pathogenicity of P. multocida would be worth eliciting in the further work. features would provide a new genome for the P. multo- cida genome database. Particularly, the identification of natural truncation of large-sized sequence from genome to form a plasmid, and the in-frame deletion and N-ter- minal redundance of the LPS outer core assembly genes would helpful for understanding the genetic diversity of P. multocida. Conclusion In the present study, the pathogenic and genetic char- acteristics of a rabbit sourced P. multocida serogroup F isolate s4 were determined. This study revealed that the s4 was low pathogenicity for rabbits by comparison with the previously reported highly virulent P. multo- cida serogroup F isolates J-4103, C21724H3km7, P-4218 and HN07, which was helpful for the awareness of the pathogenicity variation among P. multocida serogroup F isolates. Besides, the complete genome of the s4 was sequenced. The obtaining of its genomic sequence and P. multocida isolation and identification One hundred microliter of the lung suspension was spread on the BHI agar plate containing 5% defibrinated sheep blood and incubated at 37 °C for 24 to 48 h. Three isolates from each plate were randomly picked up and each isolate was inoculated in 5 mL of BHI containing Sample collection and detection of potential pathogen A total of 48 lung samples were collected from the natu- rally infected dead rabbits. Each lung sample was placed in a sterile tube, kept on ice and delivered to our lab for examination within 24 h. Each lung sample was homog- enized to make a 50% suspension in sterile normal saline. For the detection of RHDV, the suspension was cen- trifuged at 1500×g for 15 min at 4 °C, and two hundred Wang et al. BMC Veterinary Research (2022) 18:288 Page 9 of 12 Fig. 8 Comparative analyses of the LPS outer core locus of s4. The genes required for the assembly of the LPS outer core of s4 were compared with those of CIRMBP-0873 (LPS genotype L3), CIRMBP-0884 (LPS genotype L3) and Pm70 (LPS genotype L3). The color code represents the BLASTn identity Fig. 8 Comparative analyses of the LPS outer core locus of s4. The genes required for the assembly of the LPS outer core of s4 were compared with those of CIRMBP-0873 (LPS genotype L3), CIRMBP-0884 (LPS genotype L3) and Pm70 (LPS genotype L3). The color code represents the BLASTn identity nalyses of the LPS outer core locus of s4. The genes required for the assembly of the LPS outer core of s4 were compared 0873 (LPS genotype L3), CIRMBP-0884 (LPS genotype L3) and Pm70 (LPS genotype L3). The color code represents the BLASTn Fig. 8 Comparative analyses of the LPS outer core locus of s4. The genes required for the assembly of the LPS outer core of s4 were compared with those of CIRMBP-0873 (LPS genotype L3), CIRMBP-0884 (LPS genotype L3) and Pm70 (LPS genotype L3). The color code represents the BLASTn identity 2% bovine serum. Then, the isolates were shaken at the conditions of 180 rpm and 37 °C for 24 h. To confirm the identities of the isolates, the 16S rRNA genes of the iso- lates were amplified and sequenced [31], and the capsu- lar types and LPS genotypes of the isolates were defined using the PCR assays described by Townsend et al. [3] and Harper et al. [4], respectively. microliter of the supernatant was collected and used for viral RNA extraction using the EasyPure Viral DNA/RNA kit (TransGen Biotech, Beijing, China). The extracted viral RNA was reverse-transcribed to complementary DNA (cDNA) using the EasyScript Reverse Transciptase (TransGen Biotech, Beijing, China) by using the Oligo- dT18 as the primer. Sample collection and detection of potential pathogen The synthetic cDNA was used as the genomic template to screen the presence of the RHDV in the lung samples by using the PCR assay described by Schwensow et al. [14]. For the detection of P. multocida, B. bronchiseptica and S. aureus, one hundred micro- liter of lung suspension was centrifuged at 5000×g for 10 min at room temperature, and the pellet was used for bacterial DNA extraction using the EasyPure Bacteria Genomic DNA kit (TransGen Biotech, Beijing, China). The extracted DNA was used as the genomic template to screen the presence of the P. multocida, B. bronchiseptica and S. aureus in the lung samples by using the PCR assays described by Townsend et al. [3], Wang et al. [15] and Brakstad et al. [16], respectively. MLSTh The MLST analyses of the isolates were conducted using the Multi-host MLST scheme described in the PubMLST (https://pubmlst.org/pmultocida/). The genomic DNA of the isolates were prepared using the EasyPure Bacteria Genomic DNA kit (TransGen Biotech, Beijing, China), and the seven housekeeping genes including adk, aroA, deoD, gdhA, g6pd, mdh and pgi of each isolate were amplified from the genomic DNA of the isolates. The expected PCR products were purified and subjected to sequence. The allelic profile of each isolate was defined by comparing the sequences of the 7 housekeeping genes of each isolate to the corresponding known sequences in the PubMLST database. The sequence type of each iso- late was then defined by submitting the allelic profile to the PubMLST database. Genome sequencing and comparative analysis The whole genome of the isolate was sequenced using PacBio system (Pacific Biosciences, USA) at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shang- hai, China). The PacBio system generated 244,686 total reads and 1,833,208,095 total bases, with the average reads length of 7492.08 bases and the aver- age genome coverage of 891-fold. The raw reads were analyzed on the Majorbio Cloud Platform (www.major bio.com), and then the complete genome of the iso- late was de novo assembled. The complete genome of the isolate was annotated using the NCBI Prokary- otic Genome Annotation Pipeline. Genomic island and prophage were determined using IslandViewer 4 [32] and PHAST [18], respectively. Drug resistance genes were determined by comparing the sequence of the isolate against the comprehensive antibiotic resistance database (CARD) (https://card.mcmas ter.ca/). The circular genome map of the isolate was plotted using DNAPlotter (version 17.0.1) [33]. The complete genomes of the P. multocida strains CQ2 (capsular serogroup A), HN06 (capsular serogroup D), Pm70 (capsular serogroup F), HN07 (capsular serogroup F), CIRMBP-0873 (capsular serogroup F) and CIRMBP-0884 (capsular serogroup F) were freely obtained from NCBI database under the acces- sion numbers of CP033599, CP003313, AE004439, CP007040, CP020347 and CP020345, respectively. The whole genome comparison between the isolate and these strains was performed by using progressive- Mauve alignment procedure on Mauve (version 2.4.0) [34], BLAST (version 2.4.0) [35], BRIG (version 0.95- dist) [36] and Easyfig (version 2.2.5) [37]. Animal experiments Twenty-four 30-day-old rabbits obtained from a local rabbit farm were randomly divided into three groups (subcutaneous inoculation group, intranasal inocula- tion group and negative control group) of eight rabbits each (four males and four females). Each group was placed in a separate room, and two rabbits (one male and one female) from the same group were kept in a cage. Before infection, nasal, conjunctival and rectal swabs as well as whole blood were collected for bacte- riological detection to ensure P. multocida-free status of the rabbits [12]. The sera of the rabbits were also tested for the presence of P. multocida IgG as described by Jaglic et al. [12]. Rabbits in groups of subcutaneous inoculation group and intranasal inoculation group were subcutaneously and intranasally inoculated with 6.0 × 104 CFU of the isolate suspended in 100 μL of sterile normal saline, respectively. Rabbits in the negative control group were intranasally inoculated with 100 μL of sterile nor- mal saline. The challenge does and routs were selected as described by Jaglic et al. [12, 23]. The clinical signs including cough, nasal discharge and dyspnea were monitored twice daily for 15 days for the all rabbits. At the end of the experiment, the gross lesions of the all rabbits were examined and tissue samples includ- ing tracheas, lungs, livers, hearts, spleens, kidneys and whole blood of all rabbits were collected for bacterio- logical examination, and the sera was used for sero- logical examination as described by Jaglic et al. [12]. Lung samples from the rabbits of intranasal inoculation group were histologically examined using hematoxylin- eosin staining. Virulence genes detectionh The genomic DNA of the isolates were prepared using the EasyPure Bacteria Genomic DNA kit (TransGen Wang et al. BMC Veterinary Research (2022) 18:288 Wang et al. BMC Veterinary Research (2022) 18:288 Page 10 of 12 also sacrificed under ketamine narcosis to minimize sufferings. Biotech, Beijing, China) and were screened for the pres- ence of twelve virulence genes including adhesion related proteins (ptfA, tadD and pfhA), dermonecrotoxin (toxA), iron binding proteins (fur, tbpA and hgbB), sialidases (nanB), hyaluronidase (pmHAS) and outer membrane proteins (ompA, ompH and oma87). Primers used for ptfA, tadD, pfhA, toxA, fur, nanB, pmHAS, ompA, ompH and oma87 were described by Tang et al. [26]. Primers used for tbpA and hgbB were described by Ewers et al. [25]. The expected PCR products were purified and sequenced, and the identities of these products were con- firmed by comparing the sequences against the NCBI GenBank database. Abbreviations l d P. multocida: Pasteurella multocida; RHDV: Rabbit hemorrhagic disease virus; B. bronchiseptica: Bordetella bronchiseptica; S. aureus: Staphylococcus aureus; PCR: Polymerase chain reaction; LPS: Lipopolysaccharide; BHI: Brain heart infusion; bp: base pair; CARD: Comprehensive Antibiotic Resistance Database; MLST: Multi-locus sequence typing; CFU: Colony forming units; GIs: Genomic islands. References 24. Peng Z. Isolation and whole genome resequencing of Pasteurella multocida originated from pigs. PhD thesis, College of Animal Veterinary Medicine, Huazhong Agricultural University; 2018. 1. Harper M, Boyce JD, Adler B. Pasteurella multocida pathogenesis: 125 years after Pasteur. FEMS Microbiol Lett. 2006;265:1–10. 1. Harper M, Boyce JD, Adler B. Pasteurella multocida pathogenesis: 125 years after Pasteur. FEMS Microbiol Lett. 2006;265:1–10. 2. Deeb DJ, DiGiacomo RF, Bernard BL, Silbernagel SM. Pasteurella multo- cida and Bordetella bronchiseptica infections in rabbits. J Clin Microbiol. 1990;28:70–5. 2. Deeb DJ, DiGiacomo RF, Bernard BL, Silbernagel SM. Pasteurella multo- cida and Bordetella bronchiseptica infections in rabbits. J Clin Microbiol. 1990;28:70–5. 25. Ewers C, Lübke-Becke A, Bethe A, Kiebling S, Filter M, Wieler LH. Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Vet Microbiol. 2006;114:304–17. 3. Townsend KM, Boyce JD, Chung JY, Frost AJ, Adler B. Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system. J Clin Microbiol. 2001;39:2377. 26. Tang X, Zhao Z, Hu J, Wu B, Cai X, He Q, et al. Isolation, antimicrobial resist- ance, and virulence genes of Pasteurella multocida strains from swine in China. J Clin Microbiol. 2009;47:951–8. 4. Harper M, John M, Turni C, Edmunds M, Michael FS, Adler B, et al. Develop- ment of a rapid multiplex PCR assay to genotype Pasteurella multocida strains by use of the lipopolysaccharide outer core biosynthesis locus. J Clin Microbiol. 2015;53:477–85. 27. Ferguson AD, Deisenhofer J. TonB-dependent receptors-structural perspec- tives. Biochim Biophys Acta. 2002;1562:318–32. 28. Peng Z, Wang X, Zhou R, Chen H, Wilson BA, Wu B. Pasteurella multocida: genotypes and genomics. Microbiol Mol Biol Rev. 2019;83(4):e00014–9. 5. Hotchkiss EJ, Hodgson JC, Lainson FA, Zadoks RN. Multilocus sequence typing of a global collection of Pasteurella multocida isolates from cattle and other host species demonstrates niche association. BMC Microbiol. 2011;11:115. 29. Harper M, Boyce JD. The myriad properties of Pasteurella multocida lipopoly- saccharide. Toxins (Basel). 2017;9:254. 30. Peng Z, Liang W, Wang Y, Liu W, Zhang H, Yu T, et al. Experimental patho- genicity and complete genome characterization of a pig origin Pasteurella multocida serogroup F isolate HN07. Vet Microbiol. 2017;198:23–33. 6. García-Alvarez A, Vela AI, San Martín E, Chaves F, Fernández-Garayzábal JF, Lucas D, et al. Characterization of Pasteurella multocida associated with ovine pneumonia using multi-locus sequence typing (MLST) and 31. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. Acknowledgements N t li bl virulence-associated gene profile analysis and comparison with porcine isolates. Vet Microbiol. 2017;204:180–7. virulence-associated gene profile analysis and comparison with porcine isolates. Vet Microbiol. 2017;204:180–7. virulence-associated gene profile analysis and comparison with porcine isolates. Vet Microbiol. 2017;204:180–7. 7. Dabo SM, Confer AW, Montelongo M, Lu YS. Characterization of rabbit Pasteurella multocida isolates by use of whole-cell, outer-membrane, and polymerase chain reaction typing. Lab Anim Sci. 1999;49:551–9. Consent for publication Not applicable. 20. Rhoades KR, Rimler RB. Capsular groups of Pasteurella multocida isolated from avian hosts. Avian Dis. 1987;31:895–8. 21. Wilson MA, Duncan RM, Nordholm GE, Berlowski BM. Pasteurella multocida isolated from wild birds of North America: a serotype and DNA fingerprint study of isolates from 1978 to 1993. Avian Dis. 1995;39:587–93. Funding 9. García-Alvarez A, Chaves F, Fernández A, Sanz C, Borobia M, Cid D. An ST11 clone of Pasteurella multocida, widely spread among farmed rabbits in the Iberian Peninsula, demonstrates respiratory niche association. Infect Genet Evol. 2015;34:81–7. This work was supported by the Fujian Public Welfare Project (2020R10260015), Construction of Science and Technology Innovation Team of Fujian Academy of Agricultural Sciences (CXTD2021007–2), 5511 Collaborative Innovation Project of Fujian Academy of Agricultural Sciences (XTCXGC2021008) and National Rabbit Industry Technology System of People’s Republic of China (CARS-43-G-5). The funders had no role in the study design, data collection and analysis, interpretation of results, writing of the report, decision to submit the paper for publication. 10. Massacci FR, Magistrali FC, Cucco L, Curcio L, Bano L, Mangili P, et al. Charac- terization of Pasteurella multocida involved in rabbit infections. Vet Microbiol. 2018;213:66–72. 11. Zhu W, Fan Z, Qiu R, Chen L, Wei H, Hu B, et al. Characterization of Pasteurella multocida isolates from rabbits in China. Vet Microbiol. 2020;244:108649. 11. Zhu W, Fan Z, Qiu R, Chen L, Wei H, Hu B, et al. Characterization of Pasteurella multocida isolates from rabbits in China. Vet Microbiol. 2020;244:108649. 12. Jaglic Z, Jeklova E, Leva L, Kummer V, Kucerova Z, Faldyna M, et al. Experi- mental study of pathogenicity of Pasteurella multocida serogroup F in rabbits. Vet Microbiol. 2008;126:168–77. 12. Jaglic Z, Jeklova E, Leva L, Kummer V, Kucerova Z, Faldyna M, et al. Experi- mental study of pathogenicity of Pasteurella multocida serogroup F in rabbits. Vet Microbiol. 2008;126:168–77. Received: 19 January 2022 Accepted: 12 July 2022 Received: 19 January 2022 Accepted: 12 July 2022 23. Jaglic Z, Jeklova E, Christensen H, Leva L, Register K, Kummer V, et al. Host response in rabbits to infection with Pasteurella multocida serogroup F strains originating from fowl cholera. Can J Vet Res. 2011;75:200–8. Availability of data and materials The complete genome sequences of s4 were deposited in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers of CP084165 (chromosome) and CP084164 (plasmid). The BioSample accession number is SAMN21591049, and BioProject accession number is PRJNA765870. Other raw datasets that support this study are available from the correspond- ing authors upon request, Wang J and Xie X. 13. Wang J, Sun S, Chen Y, Chen D, Sang L, Xie X. Characterisation of Bordetella brochiseptica isolated from rabbits in Fujian, China. Epidemiol Infect. 2020;148:e237. 13. Wang J, Sun S, Chen Y, Chen D, Sang L, Xie X. Characterisation of Bordetella brochiseptica isolated from rabbits in Fujian, China. Epidemiol Infect. 2020;148:e237. 14. Schwensow NI, Cooke B, Kovaliski J, Sinclair R, Peacock D, Fickel J, et al. Rab- bit haemorrhagic disease: virus persistence and adaptation in Australia. Evol Appl. 2014;7(9):1056–67. 15. Wang X, Yun S, Wang F, Fan Z. Development and application of PCR assay for detection of Bordetella bronchiseptica in rabbits. Chinese Compar Med. 2008;18(7):40–5. 15. Wang X, Yun S, Wang F, Fan Z. Development and application of PCR assay for detection of Bordetella bronchiseptica in rabbits. Chinese Compar Med. 2008;18(7):40–5. Authors’ contributions Wang J and Xie X conceived the study. Wang J, Sun S, Chen Y, Chen D and Sang L performed the experiments and analysed the data. Wang J drafted the manuscript. All authors read and approved the final manuscript. 8. Wang J, Sang L, Sun S, Chen Y, Chen D, Xie X. Characterization of Pasteurella multocida isolated from dead rabbits with respiratory disease in Fujian, China. BMC Vet Res. 2019;15:438. Supplementary Information Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12917-022-03381-7. Additional file 1: Table 1. The GIs in the genome of s4. Additional file 2: Table 2. The prophages in the genome of s4. Additional file 3. The sequence of the unique gene (LCY73_01710) identified in the s4 but not in the Pm70. The online version contains supplementary material available at https://doi. org/10.1186/s12917-022-03381-7. Additional file 1: Table 1. The GIs in the genome of s4. Additional file 2: Table 2. The prophages in the genome of s4. Additional file 3. The sequence of the unique gene (LCY73_01710) identified in the s4 but not in the Pm70. Prior to challenge, all the rabbits were anaesthetized with intravenous injection of ketamine (40 mg/kg). Rabbits that were caught up in the endpoint (dysp- noea, weight loss of 15% or inability to access feed or water), and rabbits that survived until the 15 days were Page 11 of 12 Page 11 of 12 Wang et al. BMC Veterinary Research (2022) 18:288 Wang et al. BMC Veterinary Research (2022) 18:288 Competing interests The authors declare that they have no competing interests. 22. Jonas M, Morishita TY, Angrick EJ, Jahja J. Characterization of nine Pasteurella multocida isolates from avian cholera outbreaks in Indomesia. Avian Dis. 2001;45:34–42. Ethics approval and consent to participate 16. Brakstad OG, Aasbakk K, Maeland JA. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol. 1992;30(7):1654–60. 16. Brakstad OG, Aasbakk K, Maeland JA. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol. 1992;30(7):1654–60. The protocols of the present study had been reviewed and approved by the Research Ethics Committee of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences (FAAS), with the approved number of FAAS-AHVM2020–0906. All the animal procedures were carried out in accordance with relevant guidelines and regulations with compliance to ARRIVE guidelines. The owner of the rabbit farm was informed and aware about the purpose of the study and written informed consent was obtained to conduct the present study. 17. May BJ, Zhang Q, Li LL, Paustian ML, Whittam TS, Kapur V. Complete genomic sequence of Pasteurella multocida, Pm70. Pro Natl Acad Sci U S A. 2001;98:3460–5. 18. Zhou Y, Liang Y, Lynch K, Dennis JJ, Wishart DS. PHAST: a fast phage search tool. Nucleic Acids Res. 2011;39(suppl 2):W347–52. 19. Rimler RB, Rhoades KR. Serogroup F, a new capsule serogroup of Pasteurella multocida. J Clin Microbiol. 1987;25:615–8. multocida. J Clin Microbiol. 1987;25:615–8. References 16S ribosomal DNA amplifi- cation for phylogenetic study. J Bacteriol. 1991;173:697–703. Page 12 of 12 Wang et al. BMC Veterinary Research (2022) 18:288 Wang et al. BMC Veterinary Research (2022) 18:288 Wang et al. BMC Veterinary Research (2022) 18:288 32. Bertelli C, Laird MR, Williams KP. IslandViewer 4: expanded predic- tion of genomic islands for larger-scale datasets. Nucleic Acids Res. 2017;45(W1):W30–5. 33. Carver T, Thomson N, Bleasby A, Berriman M, Parkhill J, Notes A. DNAPlot- ter: circular and linear interactive genome visualization. Bioinformatics. 2008;25:119–20. 34. Darling AE, Mau B, Perna NT. progressiveMauve: multiple genome align- ment with gene gain, loss and rearrangement. PLoS One. 2010;5:e11147. 35. Camacho C, Coulouris G, Avagya V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421. 36. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST ring image generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011;12:402. 37. Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27:1009–10. 37. Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27:1009–10. Wang et al. BMC Veterinary Research (2022) 18:288 Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 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https://openalex.org/W3199667776	https://europepmc.org/articles/pmc8525183?pdf=render	English	null	The biology of seed discrimination and its role in shaping the foraging ecology of carabids: A review	Ecology and evolution	2,021	cc-by	19,413	Received: 11 March 2021 \| Revised: 23 June 2021 \| Accepted: 25 June 2021 Received: 11 March 2021 \| Revised: 23 June 2021 \| Accepted: 25 June 2021 Received: 11 March 2021 \| Revised: 23 June 2021 \| Accepted: 25 June 2021 DOI: 10.1002/ece3.7898 Abstract p g Agriculture and Bioresources, University of Saskatchewan, Saskatoon, SK, Canada Correspondence Christian J. Willenborg, Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada. Email: chris.willenborg@usask.ca Funding information Natural Sciences and Engineering Research Council of Canada The biology of seed discrimination and its role in shaping the foraging ecology of carabids: A review Khaldoun A. Ali \| Christian J. Willenborg Plant Sciences Department, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, SK, Canada Plant Sciences Department, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, SK, Canada Abstract Species of carabid (ground) beetles are among the most important postdispersal weed seed predators in temperate arable lands. Field studies have shown that car- abid beetles can remove upwards of 65%–90% of specific weed seeds shed in ar- able fields each year. Such data do not explain how and why carabid predators go after weed seeds, however. It remains to be proven that weed seed predation by carabids is a genuine ecological interaction driven by certain ecological factors or functional traits that determine interaction strength and power predation dynam- ics, bringing about therefore a natural regulation of weed populations. Along these lines, this review ties together the lines of evidence around weed seed predation by carabid predators. Chemoperception rather than vision seems to be the primary sen- sory mechanism guiding seed detection and seed selection decisions in carabid weed seed predators. Selection of weed seeds by carabid seed predators appears directed rather than random. Yet, the nature of the chemical cues mediating detection of dif- ferent seed species and identification of the suitable seed type among them remains unknown. Selection of certain types of weed seeds cannot be predicted based on seed chemistry per se in all cases, however. Rather, seed selection decisions are ruled by sophisticated behavioral mechanisms comprising the assessment of both chemical and physical characteristics of the seed. The ultimate selection of certain weed seed types is determined by how the chemical and physical properties of the seed match with the functional traits of the predator in terms of seed handling ability. Seed den- sity, in addition to chemical and physical seed traits, is also an important factor that is likely to shape seed selection decisions in carabid weed seed predators. Carabid responses to seed density are rather complex as they are influenced not only by seed numbers but also by trait-based suitability ranks of the different seed types available in the environment. 2 \| WEED SEED CONSUMPTION BY CARABID PREDATORS Carabid beetles, generally speaking, function as epigaeic polypha- gous predators in agroecosystems (Lovei & Sunderland, 1996). Adults of carabid species can show diurnal or nocturnal activities depending on their habitat and are voracious predators able to consume close to their body weight of food each day (Kromp, 1999; Tuf et al., 2012). Predatory carabids prey upon a wide array of agricultural pests in- cluding aphids, dipteran eggs and midges, lepidopteran caterpil- lars, springtails, earthworms, and slugs (Clark et al., 1994; Floate et al., 1990; Kromp, 1999; Suenaga & Hamamura, 1998). In addition, numerous species of carabid predators are known to feed on seeds of weed species after seed shed (Carbonne et al., 2020; Kulkarni et al., 2015a; Lundgren, Ellsbury, & Prischmann, 2009). Some species of Harpalus sp. and Amara sp. can even attack weed seeds on the mother plant prior to seed shed (Sasakawa, 2010a). Data from field studies have shown that carabid beetles in some cases are respon- sible for removing between 65%–90% of certain weed seeds from arable fields each year (see Table 1 for a summary of selected studies documenting postdispersal removal rates of weed seeds by carabid beetles and other invertebrate groups). Overall, the literature pro- motes carabid beetles as effective natural agents capable of destroy- ing large numbers of weed seeds in arable fields (Bohan et al., 2011). Such elevated seed mortality pressures imposed by carabid weed seed predators are likely to bring about considerable disruption in abundance, distribution, and demography of weed communities in arable fields (Jeanzen, 1971; Jermy, 1984; Davis et al., 2011). Postdispersal weed seed predation, on the other hand, is car- ried out by a wide range of seed predators that span both verte- brate and invertebrate taxa (Moles et al., 2003a; Moles et al., 2003b; White et al., 2007). By and large, arable fields harbor rich faunae of invertebrates that exhibit postdispersal weed seed-feeding habits (Lundgren, Ellsbury, & Prischmann, 2009). Species of ground (cara- bid) beetles (Carabidae: Coleoptera), crickets (Gryllidae: Orthoptera), ants (Formicidae: Hymenoptera), and slugs (Gastropoda: Mollusca) are usually among the main postdispersal weed seed predators in agri- cultural fields of temperate regions (Lundgren & Harwood, 2012; van der Laat et al., 2015). Slugs remain the least studied group, but evi- dence from field and laboratory studies suggests that contributions of slugs to postdispersal weed seed predation are likely minor (Cardina et al., 1996; Dudenhoffer et al., 2016). 1 \| INTRODUCTION (Torra et al., 2016; Westerman et al., 2012). The impactful weed seed removal activities of harvester ants remain limited to warm and dry regions of the temperate climates, however, as other competing grani- vores such as carabid beetles are not highly active in such regions (Evans & Gleeson, 2016). This leaves carabid beetles and crickets as the two dominant invertebrate postdispersal seed predatory groups in warm and wet regions of temperate climates (Carmona et al., 1999; Lundgren et al., 2013). The role of crickets as postdispersal weed seed predators of annual weeds is well documented, but they are difficult to trap and not widely studied (Lundgren, Ellsbury, & Prischmann, 2009; White et al., 2007). By contrast, carabid species are widely distrib- uted, easy to trap, and show high species richness in arable fields (Gaines & Gratton, 2010; Irmler, 2003). Therefore, carabid weed seed predators will be the main species of focus in this review. 1 Predation is one of the fates many weed seeds succumb to either on the mother plant or after seed dispersal (Crawley, 2000). Predispersal seed predation, generally speaking, is largely carried out by specialist species usually belonging to insect orders of Diptera, Lepidoptera, Hymenoptera, and Coleoptera (Kolb et al., 2007). The magnitude of predispersal seed predation pressure and how effective it might be in bio-regulating weed populations remain difficult to generalize. Some studies, for instance, have shown that predispersal weed seed predation could significantly depress seedling recruitment of some shrubs and perennials (Anderson, 1988; Moles et al., 2003; Moles et al., 2003). Likewise, studies on annual weeds revealed that lar- vae of the specialized micro-lepidopterans Coleophora lineapuvella (Chambers) and Scrobipalpa atriplicella (Fischer von Röslerstamm) could destroy large numbers of the seed heads of Amaranthus retrof- lexus L. and Chenopodium album L., respectively, in corn and soybean fields in Eastern Canada (Nurse et al., 2003). However, the seed- destructive effects of those predispersal predators were sporadic and remained rather low throughout the season and showed high variability across locations and years (DeSousa & Swanton, 2003; Nurse et al., 2003). These findings led the authors to suggest that predispersal weed seed predation alone is unlikely to bring about significant suppression of annual weeds in arable fields. By contrast, the presence of the gall midge Clinodiplosis cilicrus (Kieffer) larvae in the flower heads of Centaurea cyanus L. 1 \| INTRODUCTION in a field study in France was found to be associated with a four-fold reduction in seed num- bers, and about 40% drop in seed viability. This suggests C. cilicrus larvae can potentially depress populations of C. cyanus in the field (Koprdova et al., 2015). However, the presence of gall midge larvae was not associated with visible seed damage but rather reduced ovule fertilization. The effect therefore cannot be attributed to gen- uine seed predation but to fertilization disruption by consuming re- sources necessary for successful fertilization or repelling pollinators (Koprdova et al., 2015). Correspondence Correspondence Christian J. Willenborg, Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada. Email: chris.willenborg@usask.ca Correspondence Christian J. Willenborg, Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada. Email: chris.willenborg@usask.ca Funding information Natural Sciences and Engineering Research Council of Canada Carabid (ground) beetles, foraging, postdispersal seed predation, seed bank, weed biocontrol, weed seeds 13702 \| Ecology and Evolution. 2021;11:13702–13722. www.ecolevol.org This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Ecology and Evolution. 2021;11:13702–13722. ALI and WILLENBORG 13703 2 \| WEED SEED CONSUMPTION BY CARABID PREDATORS Ants, by contrast, engage in a wide range of ecological interactions with seeds of weed species, spanning from mutualism to antagonism (Gammans et al., 2005). Harvester ants, for instance, were found to remove large numbers of weed seeds from fields of dryland cereals in some regions of Europe Larval carabids, on the other hand, are also predaceous but their feeding ecology remains poorly studied. Based on the evidence to date, carabid larvae seem to feature seed-feeding habits similar to adults (Sasakawa, 2010b; Sasakawa et al., 2010). For instance, larvae of Amara sp. and Harpalus sp. were reported to consume large num- bers of weed seeds in laboratory feeding bioassays (Saska, 2015; Saska & Jarosik, 2001). Weed seed consumption by larval cara- bids could reach fairly high levels in some cases, similar in number to levels reported for adults (Klimes & Saska, 2010). Such findings 13704 ALI and WILLENBORG (Continues) TA B LE 1 A selection of studies summarizing field data of seed removal rates of some weed species from arable fields by postdispersal weed seed predators Weed species Average seed removal rates Study duration Seed predatory group Crop References Ambrosia artemisifolia L., Amaranthus retroflexs L., Casia obtusifolia L., Datura stramonium L. 4.2%–4.8% day−1 5 weeks Carabid beetles, crickets, and ants Corn and soybean Brust and House (1988) Alopecurus myosuroides Huds., Bromus sterilis L., Avena fatua L. 1.43%–7.2% day−1 1 month Invertebrates Grassy margins of cereal fields Povey et al. (1993) Chenopodium album L., Echinochloa crusgalli (L.) Beauv. 22%–28% day−1 2 years Invertebrates Corn, soybean, and wheat Cromar et al. (1999) Digitaris sanguindis (L.) Scop. 11% day−1 2 weeks Invertebrates Corn Menalled et al. (2000) Setaria faberi Herm. 58% 2 days−1 3 months Carabid beetles and crickets Wheat with red clover Davis and Liebman (2003) Ambrosia trifida L. 57%–70% year−1 12 months Carabid beetles Corn (no-till) Harrison et al. (2003) Stellaria media L., C. album, A. fatua 38%–74% year−1 2 years Carabid beetles and mice Organic cereal fields Westerman et al. (2003) Abutilon theophrasti Medik. 17% 2 days−1 2 years Carabid beetles, crickets, and prairie deer mice Corn and soybean Westerman et al. (2005) A. theophrasti 32% 2 days−1 2 years Carabid beetles, crickets, and prairie deer mice Corn, soybean, triticale, and alfalfa-alfalfa Westerman et al. (2005) Polygonum aviculare L., Sinapis arvensis L., S. media, C. album 35% week−1 2 weeks Carabid beetles Spring barley Mauchline et al. Torra et al. (2016) It remains enigmatic why carabid beetles choose to consume weed seeds or how seed-feeding preferences evolve given the abundance and diversity of prey carabids have access to in arable fields (Booij et al., 1994; Lovei & Sunderland, 1996). These two big questions have surrounded seed predation ecology for a long time, spurring a lot of speculation without reaching definitive conclusions (see Figure 1). Traditionally, seed-eating carabid beetles were con- sidered opportunistic feeders that fell into two main groups; om- nivores and granivores (Fawki & Toft, 2005; Talarico et al., 2016). This dichotomous grouping suggests that seeds are not primary to diets of omnivorous carabids and would be mostly consumed upon random encounter rather than carabids foraging for seeds specif- ically (Cardina et al., 1996; Lovei & Sunderland, 1996). Studies on seed-feeding habits have revealed that the distinction between om- nivory and granivory in carabid species is ambiguous, however, as the two feeding habits often overlap (Fawki et al., 2003; Fawki & Toft, 2005). For example, some carabid species originally proposed to feed strictly on animal prey were found to also include consider- able amounts of weed seeds in their diets (Carbonne et al., 2020). Similarly, multiple species of carabids within the genera Amara sp. and Harpalus sp. previously were assumed to subsist on diets com- posed mainly of seeds. However, many of these species turned out to include nontrivial amounts of animal prey in their diets as well (Frank et al., 2016; Loughride & Luff, 1983). Strict granivory on the whole is quite rare among carabids, and carabid species that feed on seeds exclusively can be found in only a few genera like Ophonus sp., Ditomus sp., Dixus sp., and Carterus sp. (Talarico et al., 2016). Together, these findings indicate that the vast majority of seed- eating carabid species actually belong in the mixed-feeding omniv- orous group, whereas true granivory remains scarce. Molecular gut content analyses spanning both mixed-feeders and carabid species more specialized toward seed feeding in Europe found high levels of seed DNA from weedy species in the guts of both groups (Frei et al., 2019). The unexpected and significant presence of weed seed DNA in guts of mixed-feeding carabids contradicts the opportunistic seed predation reasoning introduced in the early carabid literature. 2 \| WEED SEED CONSUMPTION BY CARABID PREDATORS (2005) Setaria faberi Herm., A. theophrasti 16%–30% day−1 4 months Invertebrates Cereals and legumes Heggenstaller et al. (2006) Lolium rigidum Gaudin 57% 2 weeks−1 3 months Ants and invertebrates Postharvest cropping field Jacob et al. (2006) A. fatua 42% 2 weeks−1 3 months Ants and invertebrates Postharvest cropping field Jacob et al. (2006) Raphanus raphanistrum L. 45% 2 weeks−1 3 months Ants and invertebrates Postharvest cropping field Jacob et al. (2006) Panicum dichotomiflorum Mchx., C. album 10%–90% day−1 3 months Carabid beetles Corn (organic field) Menalled et al. (2007) Taraxacum officinale Weber 34%–40% year−1 2 years Carabid beetles and isopods Grassland Honek et al. (2009) S. faberi, A. trifida, A. theophrasti 11% day−1 4 weeks Carabid beetles Potato Gaines and Gratton (2010) Amaranthus retroflexus L. 5% day−1 4 months Carabid beetles Potato Gaines and Gratton (2010) Lolium multiflorum Lam. 11.5%–29.8% 18 days−1 18 days Carabid beetles Winter wheat Baraibar et al. (2011a) Avena ludoviciana Durieu 33%–63% 6 weeks−1 6 weeks Invertebrates Barley Noroozi et al., 2012 Hordeum spontaneum (K. Koch) Thell. 27%–33% 6 weeks−1 6 weeks Invertebrates Barley Noroozi et al. (2012) Galium spurium L. 60%–70% 2 days−1 2 years Harvester ants Messor barbarus L. Dryland cereals Westerman et al. (2012) Lolium rigidum Gaud 90%–100% 2 days−1 2 years Harvester ants M. barbarus Dryland cereals Westerman et al. (2012) TA B LE Weed s Ambrosi Casi Alopecu Aven Chenopo Beau Digitaris Setaria f Ambrosi Stellaria Abutilon A. theop Polygon C. al Setaria f Lolium r A. fatua Raphanu Panicum Taraxacu S. faberi Amaran Lolium m Avena lu Hordeum Galium s Lolium r ALI and WILLENBORG 13705 Weed species Average seed removal rates Study duration Seed predatory group Crop References Papaver rhoeas L. 90% 2 days−1 2 years Harvester ants M. barbarus Dryland cereals Westerman et al. (2012) Bromus diandrus Roth 20% 2 days−1 2 years Harvester ants M. barbarus Dryland cereals Westerman et al. (2012) Viola arvensis Mur., Capsella bursa-pastoris (L.) Medik., A. myosuroides 30% week−1 5 weeks Carabid beetles Winter cereals Trichard et al. (2013) C. album 53%–65% 2 days−1 3 months Carabid beetles and crickets Corn and soybean Van der Laat et al. (2015) Amaranthus tuberculatus (Moq.) 80%–85% 2 days−1 3 months Carabid beetles and crickets Corn and soybean Van der Laat et al. (2015) Brassica napus L. 42.3%–69.7% week−1 3 weeks Carabid beetles Canola Kulkarni et al. (2016) S. arvensis 41%–58.9% week−1 3 weeks Carabid beetles Canola Kulkarni et al. (2016) Thlaspi arvense L. 16.2%–28.3% week−1 3 weeks Carabid beetles Canola Kulkarni et al. (2016) Avena sativa L. (as a substitute for weed seeds) 78%–100% day−1 3 weeks Harvester ants M. barbarus Dryland cereals Torra et al. (2016) are intriguing but should be approached with caution as they were observed in laboratory feeding experiments only. As yet, no direct measurements of weed seed removal rates by larval carabids have been carried out in the field. Weed species Average seed removal rates Study duration Seed predatory group Crop References Papaver rhoeas L. 90% 2 days−1 2 years Harvester ants M. barbarus Dryland cereals Westerman et al. (2012) Bromus diandrus Roth 20% 2 days−1 2 years Harvester ants M. barbarus Dryland cereals Westerman et al. (2012) Viola arvensis Mur., Capsella bursa-pastoris (L.) Medik., A. myosuroides 30% week−1 5 weeks Carabid beetles Winter cereals Trichard et al. (2013) C. album 53%–65% 2 days−1 3 months Carabid beetles and crickets Corn and soybean Van der Laat et al. (2015) Amaranthus tuberculatus (Moq.) 80%–85% 2 days−1 3 months Carabid beetles and crickets Corn and soybean Van der Laat et al. (2015) Brassica napus L. 42.3%–69.7% week−1 3 weeks Carabid beetles Canola Kulkarni et al. (2016) S. arvensis 41%–58.9% week−1 3 weeks Carabid beetles Canola Kulkarni et al. (2016) Thlaspi arvense L. 16.2%–28.3% week−1 3 weeks Carabid beetles Canola Kulkarni et al. (2016) Avena sativa L. (as a substitute for weed seeds) 78%–100% day−1 3 weeks Harvester ants M. barbarus Dryland cereals Torra et al. (2016) Torra et al. (2016) Instead, seed foraging behaviors in carabid seed predators are more likely driven by specific biological needs that are likely to influence predation dynamics in certain ways (Carbonne et al., 2020; Davis et al., 2011; Headrick & Goeden, 2001). The type of biological needs that are likely to underlie seed- feeding habits and the choice of suitable seeds in carabids remain Dryland cereals Avena sativa L. (as a substitute fo The type of biological needs that are likely to underlie seed- feeding habits and the choice of suitable seeds in carabids remain unknown. Multiple attempts to uncover the key ecological factors that render seed of certain weed species more likely to incur ele- vated carabid attacks in arable fields have not been very fruitful thus far (Kulkarni et al., 2015a). Large-scale weed seed predation studies show that predation pressures fluctuate considerably in space and time and do not follow general trends or patterns (Hatton et al., 2015; Honek et al., 2003; Hulme, 1998; Jacob et al., 2006; Thlaspi arvense L. 13706 \| ALI and WILLENBORG FI G U R E 1 A hypothetical ecological matrix illustrating the possible ecological factors that are likely to influence weed seed predation dynamics in agroecosystems—question marks indicate a possible but not widely understood role for the proposed factor. Wa, fitness of the carabid predator; Wb, fitness of the weed species; E, various environmental factors that possibly influence predation dynamics 13706 ALI and WILLENBORG FI G U R E 1 A hypothetical ecological matrix illustrating the possible ecological factors that are likely to influence weed seed predation dynamics in agroecosystems—question marks indicate a possible but not widely understood role for the proposed factor. Wa, fitness of the carabid predator; Wb, fitness of the weed species; E, various environmental factors that possibly influence predation dynamics Kulkarni et al., 2017a). Although some correlations between cara- bid activity density and regulation of soil seed banks could be es- tablished in a few large-scale studies (Bohan et al., 2011; Carbonne et al., 2020), such relationships were absent in several small-scale studies (Petit et al., 2014; Saska et al., 2008). Such mixed results, in addition to the correlational nature of the evidence, preclude establishing a general and definitive functional link between ac- tivity density of seed predatory carabids and the regulatory pres- sures they impose on soil seed banks. Torra et al. (2016) Mechanistic studies would be more informative in this regard, with a better ability to eluci- date how and why weed seeds are attacked by carabid predators. Studies carried out within the mechanistic paradigm would help to tease out the key factors that underlie correlational relationships in the seed predation literature. They would also help to bridge the knowledge gaps that still remain in weed seed predation ecology (van Regenmortel, 2004; Dean & Thornton, 2007; Baskett, 2012). Therefore, in addition to studying seed predation ecology on large scales in the field, seed predation should also be studied on small scales and at deep mechanistic levels. This way, numerous con- founding factors could be filtered out, and pseudo-replication issues could be avoided to uncover the functional aspects of weed seed predation interactions and identify the ecological factors that deter- mine interaction strength and power predation dynamics (Davies & Gray, 2015; Hurlbert, 1984). Uncovering the mechanistic aspects of seed predation ecology is likely to improve the design and conduct of weed seed predation studies on larger scales (Denny & Benedetti- Cecchi, 2012). Along these lines, we attempt to review the weed seed predation literature in the following sections to synthesize the current knowledge around postdispersal weed seed predation, but in a mechanistic context. Our aim is to tie together the mechanistic results from isolated studies with ecological processes that will pro- vide an improved synthesis of how the biology and ecology of these mechanisms shape weed seed consumption. The discussion will be mainly reserved for adult carabids, and information about larval ca- rabids will be provided, where relevant. 3.2 \| Seed detection and location Interactions between insects and plants that lead to weed seed pre- dation should be viewed as a special case of plant–insect interac- tions (Jermy, 1984). The main difference here, though, is that the insects attack the reproductive units of the plant rather than its so- matic tissues (McArt et al., 2013). It is well established that herbivo- rous insects rely on various types of visual and/or chemical cues to locate their host plants and assess their quality as sources of food (Bruce, 2015; Bruce et al., 2005). Chemical cues in terms of volatile and nonvolatile compounds have been proven to act as major driv- ers of host selection decisions in plant-feeding insects (Eigenbrode & Esplie, 1995; Baldwin, 2010; Bruce & Pickett, 2011; Heil, 2014). By the same token, carabid weed seed predators are expected to rely on similar cue-based systems for seed location and quality assess- ment (Kielty et al., 1996; Merivee et al., 2007, 2008). It is still uncertain which sensory modality (vision or chemopercep- tion) carabid species rely upon most to guide their food location and selection responses. Despite this uncertainty however, the evidence strongly hints that chemoperception is likely the top candidate for roles related to food searching and food choice in carabid predators (Law & Gallagher, 2015; Oster et al., 2014; Wheater, 1989). It can be proposed therefore that food selection decisions in carabid beetles, be that seed or prey, are generally guided by chemical cues encod- ing information that is interpreted based on innate or learned “tem- plates” or “images” in the brain (Oster et al., 2014; Vet & Dicke, 1992). As such, chemical cues emitted from different types of weed seeds in arable fields are expected to mediate seed recognition and seed selection in carabid weed seed predators (Kulkarni et al., 2017b; Law & Gallagher, 2015; Lundgren et al., 2013). However, given the limited number of sensory studies and the highly diverse feeding habits of carabid species in arable fields, it cannot be ruled out that vision may also play some role in seed selection decisions, leading to the forma- tion of complex search images in some cases (Dukas & Kamil, 2001; Postdispersal weed seed predators have to search for seeds that are imbedded in complex, often cryptic environments (Aarstma et al., 2019; Forister et al., 2012). (Kolb et al., 2007). We suggest that such assumptions in the liter- ature have originated from the old “no-choice” laboratory feeding bioassays, where carabids would accept the majority of food types offered to them (e.g., Shough, 1940). Such findings led carabid ecol- ogists to assume that carabid species were scavengers or opportu- nistic feeders for the most part (Forsythe, 1982; Wheater, 1989). This idea still transcends the modern carabid literature, nonetheless. Recent evidence now points to random encounters as the excep- tion rather than the rule, at least for carabid weed seed preda- tors (Saska et al., 2019; Ward et al., 2014). The emerging evidence demonstrates that carabid beetles are likely to employ active forag- ing behaviors in search of weed seeds (Honek et al., 2007; Lundgren & Harwood, 2012). Carabid species in other words not only locate weed seeds in space but also show an ability to discriminate between seeds of different species and choose the most desired (Kulkarni et al., 2016). Therefore, finding weed seeds in space entails multiple exacting tasks for the foraging carabid beetle, especially with regard to seed detection and location. (Kolb et al., 2007). We suggest that such assumptions in the liter- ature have originated from the old “no-choice” laboratory feeding bioassays, where carabids would accept the majority of food types offered to them (e.g., Shough, 1940). Such findings led carabid ecol- ogists to assume that carabid species were scavengers or opportu- nistic feeders for the most part (Forsythe, 1982; Wheater, 1989). This idea still transcends the modern carabid literature, nonetheless. Recent evidence now points to random encounters as the excep- tion rather than the rule, at least for carabid weed seed preda- tors (Saska et al., 2019; Ward et al., 2014). The emerging evidence demonstrates that carabid beetles are likely to employ active forag- ing behaviors in search of weed seeds (Honek et al., 2007; Lundgren & Harwood, 2012). Carabid species in other words not only locate weed seeds in space but also show an ability to discriminate between seeds of different species and choose the most desired (Kulkarni et al., 2016). Therefore, finding weed seeds in space entails multiple exacting tasks for the foraging carabid beetle, especially with regard to seed detection and location. borne out as carabids have been shown to possess highly developed motor abilities along with a formidable arsenal of sensory receptors to guide their behaviors and food choice responses (Forsythe, 1983a; Merivee et al., 2000, 2008). Behavioral data on the whole indicate that food searching and food acceptance in carabid beetles, be that seed or prey, is an active and directed process guided by accurate sensory information collected from the environment (Harrison & Gallandt, 2012; Kulkarni et al., 2015b; White et al., 2007). We sug- gest that carabid predators should be viewed as active and selective foragers, clearly able to decipher between food types irrespective of random encounters. 3.3 \| Discrimination among different seed types and identifying the suitable seed type The logic of optimal foraging theory assumes that food recognition and acceptance in selective foragers is guided by “search images” hardwired in the brain or learned from the environment (Garay et al., 2018; Krieger & Breer, 1999). Technically speaking, search images could be visual, chemical, or complex (visual and chemical) (Vet & Dicke, 1992). Assuch, carabid beetles as selective foragers are expected to employ search images to guide their food discrimi- nation and acceptance decisions so that the most suitable food types are chosen for consumption. Such active decision-making processes need accurate sensory sampling of the environment, and carabid species are thus expected to rely on their different sensory modalities (i.e., vision, olfaction, and gustation) to obtain the sen- sory information essential for food selection (Ploomi et al., 2003; Ramaswamy, 1988). Technically, gustation could be considered a special case of olfaction because olfactory and gustatory receptors show similar physiological structure and function, and collect chemi- cal information of similar nature (Isono & Morita, 2010; Krieger & Breer, 1999). Henceforth, both smell and taste in carabid beetles will be treated as chemoperception in sections that follow. 3.1 \| Seed and carabid predator co-occurrence in space and time Seed predation requires synchronicity between seed availability and predators’ activities both in space and time. Seeds of annual weeds are usually shed into arable fields at certain times, but can persist in the seed bank for extended periods (Baskin & Baskin, 1998). Hence, the window of seed availability for postdispersal carabid weed seed predators could be considerably wide in time (Meiners, 2015). This would seem to suggest that finding weed seeds in time should not be an exacting task for carabid weed seed predators. By contrast, the spatial overlap between weed seed shed (i.e., the seed shadow) and carabids is more complex. Some of the early carabid literature had proposed that carabid weed seed predators locate weed seeds upon random encounter (Cardina et al., 1996; Lovei & Sunderland, 1996). That is, if carabid beetles came across a weed seed, they would simply consume it. Beyond that, they would not spend considerable time and energy foraging for weed seeds that are less nutritious and more difficult to locate compared to prey ALI and WILLENBORG 13707 Factors relating to habitat properties, chemical or biophysical seed traits, taxonomic identity of the dominant species in the local carabid community, fear, and effects of learning and experience could all influence the processes of seed selection decision-making, or perhaps override it altogether (De Heij & Willenborg, 2020; Ishii & Shimada, 2010). Overlooking such influential ecological and environmental factors is likely one main reason why field data involving seed predation remain ambig- uous (e.g., Hatton et al., 2015; Honek et al., 2003; Petit et al., 2014). Therefore, we suggest that weed seed selection by carabid weed seed predators entails complex and multilayered processes that are unlikely to be explained by chemoperception-based decisions alone. Hence, in sections that follow we will attempt to identify other pos- sible ecological forces likely to shape seed selection decisions in carabid weed seed predators. The discussion will be reserved for an- alyzing functional traits for both weed seeds and carabid weed seed predators, and how their interactions might determine the predation strength under realistic situations. For a review of the possible ways in which the environmental factors influence weed seed predation dynamics, we direct readers to Kulkarni et al. (2015a), Sarabi (2019), or De Heij and Willenborg (2020). Evidence in support of chemically mediated food detection, by contrast, is more abundant and comes from sensory and behavioral data pertaining to carnivorous and omnivorous carabids. Some cara- bid carnivores, for instance, showed positive orientation toward vol- atile chemicals originating from prey habitat (e.g., wheat extracts), as well as volatile chemicals specific to prey itself or to its phero- mones (Kielty et al., 1996; Mundy et al., 2000; Tréfás et al., 2001). Moreover, electroantennographic detection (EAD) studies have shown that antennal preparation of Pterostichus melanarius Illiger adults could respond to odor chemicals originating from live or dead slugs, or even from the slug trails (McKemey et al., 2004). Likewise, antennal preparations of P. melanarius larvae produced detectable electrical signals when exposed to chemical odors of different prey types (Thomas et al., 2008). Strong electrical signals could also be recorded from labial palps of the ant-specialist carabid Siagona euro- paea Dejean when exposed to formic and acetic acid secretions from its ant prey (Talarico et al., 2010). Laboratory studies have also reported that some species of seed-eating carabids exhibit olfactory-oriented seed choice re- sponses (Law & Gallagher, 2015). Wheater, 1989). This uncertainty stems from reports showing diur- nal carabid species that actively hunt live mobile prey usually carry larger compound eyes and shorter antennae that house significantly fewer olfactory receptors compared to nocturnal species (Bauer & Kredler, 1993; Merivee et al., 2001, 2002). Such sensory differences seem to suggest that visual cues are likely to play more vital roles in guiding food searching behaviors in diurnal carabids compared to nocturnal ones (Wheater, 1989). The same studies showed that visually driven hunting behaviors in carabid predators broke down when prey items were immobilized. Therefore, it could be suggested that carabid visual receptors are more finely-tuned toward detecting movement and are not expected to be of much help when carabid predators are searching for immobile and often cryptic food items like weed seeds (Srinivasan et al., 1999; Oster et al., 2014). Still, a possible role for vision in seed foraging in carabid weed seed preda- tors cannot be decisively ruled out based on our current knowledge, and more studies are warranted. carabids, not the absence of visual cues due to seed burial (Evans & Forsythe, 1985; Wallin & Ekbom, 1988). While there may be some role for vision in seed foraging, we suggest that any such role is likely minor given the findings of seed burial experiments. Overall, evi- dence to date clearly suggests that chemoperception is likely the pri- mary mechanism guiding seed finding and seed selection responses in carabid weed seed predators. It is crucial to reiterate that carabid predators forage in com- plex environments. Weed seeds are imbedded in an intricate ma- trix of environmental variables that could influence the foraging decisions of carabid predators in unpredictable ways (De Heij & Willenborg, 2020; Sarabi, 2019). Under such conditions, there will be certain cases where predicting the suitable seed type based on chemical cues alone fails to explain the observed seed selection responses of carabid weed seed predators (Foffova et al., 2020). That is, there will be certain cases where other impactful ecological factors might interfere and sway weed seed preferences in certain directions (De Heij & Willenborg, 2020). Positive orientation responses to seed volatiles were reported for some omnivorous carabid species when seed masses of different brassicaceous weed species were placed into odor chambers of a four-arm olfactometer (Kulkarni et al., 2017b). The carabids used in the experiment also spent sig- nificantly more time in odor arms harboring the highly preferable weed species (i.e., Brassica napus L. seed in this case), indicating that they were able to discriminate among the seed species offered in the olfactometer based on odor alone. Moreover, the species of ca- rabid weed seed predators were able to excavate weed seeds bur- ied down to 1 cm below soil surface, without considerable loss in seed-finding efficiency (Kulkarni et al., 2015b; White et al., 2007). Of note here, efficiency for excavating seeds that were buried at the same depth differed between the carabid species under study, yet seeds were detected and dug out in most cases. One can hence infer that differences in seed excavation efficiency resulted from species- specific differences in soil-burrowing responses between the tested 3.2 \| Seed detection and location In other words, seed searching for carabid weed seed predators is a multilayered behavioral process fraught with challenges. Seed abundance is highly variable in both space and time (Dessaint et al., 1991; Henderson, 1990). Moreover, seeds are small in size and randomly scattered on the soil surface, or even buried underneath it (Menalled et al., 2007; White et al., 2007). This essentially renders any reliable cues for seed location and qual- ity assessment sparse and difficult to detect (Baldwin, 2010; Poisot et al., 2011). Hence, successful foraging requires individual carabids to engage in active seed-searching behaviors, vigorously scanning the environment for any seed-related cues. Unlike random encoun- ters, active foraging behaviors require the foraging animal to have highly developed motor abilities coupled with a sensitive sensory apparatus for picking up and recognizing cues of low detectability (Aarstma et al., 2019; Meiners, 2015). This assumption has been 13708 ALI and WILLENBORG mobilize a wide array of secondary metabolites to maturing seeds, many of which serve multiple defensive functions (Rattan, 2010; Trowbridge, 2014). While seed nutrients (i.e., primary metabo- lites) are likely to act as major feeding stimulants for carabid weed seed predators, the presence of defensive chemicals (i.e., second- ary metabolites) could act against the phago-stimulatory effects of nutrients (Chapman, 1999, 2003). Presumably, the acceptability of weed seeds would be determined by the overall balance between primary and secondary metabolites in seed tissues (Agrawal & Fishbein, 2006; Bernays et al., 2004; Seric Jelaska et al., 2014). That is, weed seeds with low levels of chemical defenses should be more preferable to carabid weed seed eaters, perhaps irrespective of nu- tritional content. However, the assumption that insects choose to feed on plant tissues based on chemistry alone does not hold true in all situations. A meta-analysis has shown that levels of secondary metabolites in plant tissues do not always determine the acceptabil- ity of those tissue to insect herbivores (Carmona et al., 2011). This should not be surprising because the levels of primary and secondary metabolites in plant tissues are determined by complex interactions and tradeoffs between multiple plant traits (Blate et al., 1998; Davis et al., 2016). In line with this, studies that tested the impact of sec- ondary metabolite levels on seed choice by rodent seed predators also produced conflicting results (Wang & Chen, 2012). Rodents, for example, avoided seeds with high levels of phenols in some cases (Gong et al., 2015; Henderson, 1990). In another more extensive study, effects arising from seed toxins on seed selection responses in rodent seed predators were totally absent (Rodgerson, 1998). More interestingly, studies investigating how the chemistry of weed seeds affect their persistence in seed banks showed that physical traits like seed mass, size, and coat hardness were much more important to long-term seed persistence than chemical traits (Davis et al., 2008). Similarly, for seeds of multiple weed species, the physical character- istics of the seed were more crucial for avoiding predation by car- abid predators than was seed chemistry (Foffova et al., 2020). We suggest that given the evidence to date, seed chemistry per se is unlikely to be the only driver of seed selection decisions in carabid weed seed predators. Instead, seed biophysical properties may pro- foundly interact with and perhaps even override the impacts of seed chemistry. considerable variations across plant families and genera (Bretagnolle et al., 2015). It remains unclear which nutrient or combination of nutrients, if any, brings about a higher vulnerability to seed pre- dation. Early studies with rodents reported that seeds contain- ing high levels of protein suffered elevated predation rates (Gong et al., 2015; Halkier & Gershenzan, 2006; Henderson, 1990; Herms & Mattson, 1992). However, similar data for arthropod seed preda- tors are scarce. Studies on ant-dispersed seeds, for example, showed that seeds with lipid-rich elaiosomes were usually picked up at higher rates by ants (Brew et al., 1986). Indirectly, this could be an indication of a lipid limitation in seed-feeding arthropods because ants usually eat only the elaiosome and leave the seed intact (Brew et al., 1986; Rodgerson, 1998). In line with this, some carabid preda- tors were found to interfere with ants and compete with them for seed elaiosomes (Ohara & Higashi, 1987). Carabid predators here consumed only the lipid-rich elaiosomes and left the seeds intact in much the same way as ants. This could be an indirect indication that carabid predators also suffer lipid limitations in their diets and thus seek out lipids in seeds. Recent field studies have indeed reported that weed seeds with high lipid content were usually more preferable to carabid weed seed predators than were seeds with low lipid contents (Petit et al., 2014). Laboratory feeding experiments also have shown that, within certain limits of seed size, weed seeds with high lipids suf- fered higher rates of attack by carabid weed seed predators (Gaba et al., 2019). Together, field and laboratory findings fall nicely in line with nutritional ecology data demonstrating that carabid species in arable fields usually forage under lipid-limited conditions (Jensen et al., 2012; Raubenheimer et al., 2007; Toft et al., 2019). This agrees with other studies reporting that arthropod predators, including spiders and other insects, also suffer considerable lipid limitations in their natural habitats as well (Wilder & Eubanks, 2010; Wilder et al., 2013). Plus, carabids seem to maintain a strong preference for weed seeds even when offered along with prey items (Blubaugh et al., 2016; Frank et al., 2011; Lundgren & Harwood, 2012). Taken to- gether, the totality of evidence thus far suggests that seeds of some weed species must contain certain lipids essential for carabid phys- iology, which might be absent from protein-rich insect prey tissues (Booij et al., 1994; Wilder et al., 2013). Seeds could be thus essential to the diet of carabid weed seed predators. While it is compelling to assume that lipid limitations are one primary reason carabid weed seed predators consume seeds of certain weed species, much more detailed studies are needed to ascertain if other important nutrients like protein or carbohydrates might also impact seed selection deci- sions and thus, foraging ecology (Denno & Fagan, 2003). 4.1.3 \| Seed size and mass Size is one physical trait of special interest to seed ecology (Baskin & Baskin, 1998; Dalling et al., 2020). Seed size directly relates to many seed quality parameters (Petit et al., 2014; Wang & Yang, 2020). Larger seeds, for instance, usually contain more energy and nutri- ents (Gong et al., 2015), but could also contain more chemical de- fenses (Agrawal & Fishbein, 2006; Wang & Chen, 2012). Also, larger seeds tend to have thicker and harder seed coats (Moles et al. 2003a; Moles et al. 2003b). Overall, the relationships between seed size, seed chemistry, and seed physical properties are quite complex and not well understood (Dalling et al., 2020; Wang & Chen, 2012). 4.1.1 \| Seed nutritional content Seeds of plants usually contain large amounts of essential nutri- ents that are vital for embryo survival (Agrawal, 2007; Wang & Yang, 2020). Nutrients in seeds are typically comprised of carbo- hydrates (starch), protein, and fatty acids (lipids), and these show ALI and WILLENBORG 13709 4.1.2 \| Seed chemical defenses Plants cannot compromise on the nutritional needs for embryo survival, so they deploy different layers of defense that make seed nutrients difficult to access and costly to extract by seed predators (Rees & Long, 1992; Dalling et al., 2011). Plants, including weeds, 13710 \| ALI and WILL Seed trait Vulnerability to seed predation References Seed nutrients (lipids) + Petit et al. (2014); Gaba et al. (2019) Seed chemical defenses 0 Foffova et al. (2020) Seed size and massa ± Lungren and Rosentrater (2007); Honek et al. (2007); Saska, Honek, and Martinkova (2019); Foffova et al. (2020); Saska et al. (2020) Seed coat hardness − Jorgensen and Toft (1997); Lungren and Rosentrater (2007); Noroozi et al. (2012); Foffova et al. (2020); Saska et al. (2020) Note: +, indicates a positive effect; −, indicates a negative effect; 0, indicates no documented effect. aNet effects depend on the seed-predator mass-ratio scaling relationship. TA B LE 2 Summary of potential seed traits that mediate seed vulne or avoidance to carabid weed pred 13710 \| Seed trait Vulnerability to seed predation References Seed nutrients (lipids) + Petit et al. (2014); Gaba et al. (2019) Seed chemical defenses 0 Foffova et al. (2020) Seed size and massa ± Lungren and Rosentrater (2007); Honek et al. (2007); Saska, Honek, and Martinkova (2019); Foffova et al. (2020); Saska et al. (2020) Seed coat hardness − Jorgensen and Toft (1997); Lungren and Rosentrater (2007); Noroozi et al. (2012); Foffova et al. (2020); Saska et al. (2020) Note: +, indicates a positive effect; −, indicates a negative effect; 0, indicates no documented effect. aNet effects depend on the seed-predator mass-ratio scaling relationship ALI and WILLENBORG 13710 TA B LE 2 Summary of potential weed seed traits that mediate seed vulnerability or avoidance to carabid weed predators TA B LE 2 Summary of potential weed seed traits that mediate seed vulnerability or avoidance to carabid weed predators TA B LE 2 Summary of potential weed seed traits that mediate seed vulnerability or avoidance to carabid weed predators Note: +, indicates a positive effect; −, indicates a negative effect; 0, indicates no documented effect. Note: +, indicates a positive effect; −, indicates a negative effect; 0, indicates no documented effect. aNet effects depend on the seed-predator mass-ratio scaling relationship. coat hardness on removal rates of weed seeds. 4.1.2 \| Seed chemical defenses More direct test- ing of the relationship between seed coat hardness and predation avoidance has found that seed coat hardness was crucial in deter- mining the susceptibility of weed seeds to predation by carabids (Foffova et al., 2020; Lungren & Rosentrater, 2007). This suggests that weed seed coat hardness could decisively determine vulnerabil- ity to seed predation if coat hardness differs considerably between the seed types in any given environment (Foffova et al., 2020). Still, it remains unclear if coat hardness acts alone or together with other seed traits as syndromes influencing predation avoidance (Agrawal & Fishbein, 2006; Dalling et al., 2011). A more plausible scenario is that the relative importance of any given seed trait with regard to predation avoidance is likely to be determined by how other seed traits also match or mismatch with the functional traits of the preda- tor (Dalling et al., 2020; Foffova et al., 2020). In other words, trait values for the different seed defensive traits are only one part of the interaction. Predation pressure would also be determined by how many seed defensive traits actually match or mismatch with the functional traits of carabid predators with regard to the ability of the carabid to neutralize seed defenses (Ball et al., 2015; Quieter et al., 2007). This appears crucial because morphological traits of the seed like seed mass, size, and coat hardness undergo consider- able physiological changes over time in the soil seed bank, and these changes are likely to affect seed vulnerability to carabid predation (Martinkova et al., 2016; Saska et al., 2019, 2020). For a summary of the different seed traits influential on weed seed vulnerability to carabid predators, see Table 2. Nonetheless, the size of weed seeds was observed to be among the factors determining which seeds of weed species would be more preferable to carabid weed seed predators in laboratory trials (Lungren & Rosentrater, 2007; Saska, Honek, & Martinkova, 2019). In a laboratory study, Honek et al. (2007) produced measurements of dry mass for 25 different weed species and the body mass of 30 carabid weed seed predators in laboratory experiments. The authors managed to establish seed-predator mass-ratio curvilinear scaling re- lationships that strongly influenced seed selection responses of the carabid predators under study. 4.1.4 \| Seed coat hardness Seed coat hardness is determined by the amount of sclerenchyma deposited in the palisade cells of seeds (Radchuck & Borisjuk, 2014). Weed seed coat hardness, in general, decreases with increasing seed mass (Janzen, 1969; Lungren & Rosentrater, 2007). Yet, the opposite relationship patterns were observed for some species (van der Meij & Bout, 2000). Functionally, the relationship between seed mass and seed coat hardness differs from one weed species to another and is more likely to follow phylogenetic patterns rather than sim- ple general linear patterns (Lovas-Kiss et al., 2020). Despite these species-specific differences, what seems to be of most consequence to the carabid weed seed predator is seed coat hardness (Jorgensen & Toft, 1997; Petit et al., 2014). The authors of two field studies ob- served that weed seeds with soft coats were much more suscep- tible to carabid weed seed predators than seeds with hard coats (Jorgensen & Toft, 1997; Noroozi et al., 2012). This piece of evidence remains anecdotal because neither study tested the effects seed 4.1.2 \| Seed chemical defenses Such mass-scaling relationships have been shown to be vital for determining the strength of predator– prey interactions in vertebrate systems (Freeman & Leman, 2008), but the mechanistic aspects of size-based prey choice remain poorly understood. Likewise, mechanisms of the size-based seed selection responses in carabid weed seed predators remain unknown. More studies are needed in this regard to elucidate the factors underly- ing mass-ratio scaling relationships and their impact on interaction strengths between weed seeds and carabid weed seed predators. Predator body mass allometry was found highly predictive of prey selection decision in carnivorous carabids preying on insects like aphids, collembola, and caterpillars in the field (Bell et al., 2008; Rusch et al., 2015; Schmitz, 2007). Similarly, predator body mass strongly affected seed selection decisions in carabid weed seed predators in laboratory studies (Honek et al., 2007; Martinkova et al., 2019; Saska, Honek, & Martinkova, 2019). Despite their strong impact, effects emerging from predator body mass on food choice remain poorly understood. Body mass is a complex trait with links to multiple other traits that directly relate to foraging behaviors, food handling capabilities, and functional responses of carabid preda- tors (Aljetlawi et al., 2004; Brose, 2010; Reum et al., 2018). In this respect, a positive scaling relationship was found between carabid body mass (but not body length) and size of the muscle mass that power the mandibles and control the strength of bite force (Evans & Forsythe, 1985; Wheater & Evans, 1989). The relationship between body mass, jaw musculature, and bite force has been found to shape vital aspects of feeding ecology in mammalian predators (Freeman & Leman, 2008; Wore et al., 2005), and granivorous birds (van der Meij & Bout, 2004). The importance of body mass and bite force for the feeding ecology of insects re- mains poorly understood, nonetheless. Emerging evidence in this regard suggests that body mass and bite force underlie funda- mental aspects of the feeding ecology of predatory insects as well (Weihmnann et al., 2015; Blanke et al., 2018). Recent studies, for instance, have shown that match between bite force and cuticular hardness of prey was the most powerful factor in predicting prey preferences when carabid species were offered different types of prey (Brousseau et al., 2018; Konuma & Chiba, 2007). There are no similar studies looking into the role of bite force in seed selection decisions in carabid weed seed predators as yet. Besides, some au- thors speculate that bite force should play a key role in weed seed selection as well (Brousseau et al., 2018). Here, it is tempting to infer that the strong effects of carabid body mass in shaping the feeding response of carabid predators are most likely derived from its inti- mate relationship with bite force. and lipids are the two major macronutrients that drive the forag- ing efforts of arthropod predators (Jensen et al., 2012; Schmidt et al., 2012; Wilder et al., 2019). Weed seeds offer both types of macronutrients, but to different extents (Bretagnolle et al., 2015). Given that the majority of carabid species generally suffer lipid limitations in their agricultural habitats, foraging for lipids could be, in fact, the main trophic link binding weed seeds and carabid seed predators together (Gaba et al., 2019; Raubenheimer et al., 2007; Toft et al., 2019). Although the principal goal of trophic interac- tions is obtaining nutrients from the environment, there are other aspects of nutrient foraging behaviors than food extraction per se (McArthur & Pianka, 1966; Ydenberg et al., 1994). Setting out on foraging bouts is associated with different risks and entails high costs related to food handling and processing (Schoener, 1971; Pyke et al., 1977). Carabid weed seed predators in this sense need not only to find weed seeds, the chosen seed type or seed patch must also offer high rewards at a low cost (Fawki et al., 2003; Sih, 1984). Clearly, the balance between reward and cost of differ- ent seed types is likely a major factor shaping seed selection deci- sions (Bretagnolle et al., 2015; Brousseau et al., 2018). Moreover, how rewarding a certain type of weed seed is to any given carabid is determined by the biophysical and biochemical functional traits of the predator itself (Brousseau et al., 2018; Evans & Forsythe, 1985; Forsythe, 1987). In other words, the carabid predator needs to be able to neutralize seed defenses in order to gain more sufficient nu- trition at a low cost. Should this not be the case, the carabid preda- tor will suffer great fitness costs (Emlen, 1966; Pyke et al., 1977). Therefore, the species-specific functional traits of carabid species are expected to strongly affect the types of weed seeds they come to accept for consumption. Among the various functional traits of carabid species, physiological and biochemical traits remain the least understood. Hence, it could be much more informative to ex- amine the feeding ecology of carabid predators from a biophysical standpoint. niches however, functional traits other than mouthpart structure seem to determine aspects of food choice and preference. niches however, functional traits other than mouthpart structure seem to determine aspects of food choice and preference. This reasoning may explain why the relationship between carabid body mass and metabolic rates provided only a partial explanation of the interaction strength be- tween carabid predators and prey (Brose et al., 2006, 2008; Brown et al., 2004; Reum et al., 2018). 4.2.1 \| The physiological traits of carabid predators Through feeding interactions, carabid predators obtain nutrients to address the nutritional limitations they face in their habitats and thus fulfill the nutritional needs for survival and reproduction (Frank et al., 2011; Potter et al., 2018). Generally speaking, protein ALI and WILLENBORG 13711 4.2.2 \| The biophysical traits of carabid predators The relationship between morphological traits and feeding habits is well established for insect herbivores, as well as for carabid predators (Bernays, 1991; Knapp & Knappova, 2013; Knapp & Uhnava, 2014). Curiously though, these relationships have remained largely over- looked in the study of carabid feeding ecology. For instance, there are generally strong functional links between mouthpart structure and the type of foods adult carabids can consume (Forsythe, 1982, 1983b). Recently, similar functional links between mouthpart mor- phology and the degree of feeding specialization have been un- covered for carabid larvae as well (Sasakawa, 2016). By and large, mouthpart morphology and structure of carabid species (both adults and larvae) seem to be among the fundamental functional traits that determine key aspects of the feeding specialization niche in terms of carnivory, omnivory, or granivory (Evans & Forsythe, 1985, Forsythe, 1987; Paarmann et al., 2006). Within each of these feeding The bio-morphological reasoning laid out above seems to apply to larval carabids as well. Larvae of carabid weed seed predators, for example, have been shown to deliver stronger bite forces compared to strictly carnivorous larvae (Brandmayr et al., 1998; Paarmann et al., 2006). This is essential adaptation for handling the hard coats of weed seeds. Moreover, the feeding niche of carabids larvae ex- pand as they grow and molt from one instar to another (Klimes & Saska, 2010; Saska, 2005). Such changes in feeding habits are likely due to increases in body mass and strength of mandibular muscles of the growing larvae as they advance from one instar to the other (Refeseth, 1984; Sasakawa, 2016). Overall, bio-morphological traits 13712 ALI and WILLENBORG weed seeds show significant differences in seed coat hardness, predators would select the seed type that is easier to crush and handle, regardless of the chemical quality of that chosen seed type (Ananthakrishan, 1990; Potter et al., 2018). It is now more informa- tive to build upon the trait-based seed selection discussion and ex- plore its aspects at the community level. are better in predicting the feeding ecology of carabid beetles com- pared to physiological traits (Bell et al., 2017). are better in predicting the feeding ecology of carabid beetles com- pared to physiological traits (Bell et al., 2017). 4.2.2 \| The biophysical traits of carabid predators Gape size should not be expected to constrain food choice in carabid species in general, including weed seed predators, because larval and adult carabids are fragmentary feeders and show no swallow feeding habits in general (Brousseau et al., 2018; Evans & Forsythe, 1985). Based on evidence discussed above, it is plausible to suggest that bio-morphology in terms of mouthpart structure and bite force are two traits that shape key aspects of feeding special- ization in carabid species, including carabid weed seed predators. Again, this accentuates further our pervious discussion as to why seed selection is unlikely to be based on seed chemistry alone. For a summary of carabid traits likely to affect ability of carabid predators to destroy weed seeds, see Table 3. Weed communities in arable fields are composed of several co- existing weed species (Booth & Swanton, 2002; Petit et al., 2011). Consequently, weedy plants in any given weed community produce seeds of different types (i.e., species) and shed them in various numbers each year (Bagavathiannan & Norsworthy, 2012; Dessaint et al., 1991). There are thus two main sources of variability in weed seeds of arable fields; seed type as defined by species-specific seed traits (i.e., trait-based seed quality rank) and seed density, as defined by seed numbers per unit area (Albrecht & Auerswald, 2009). The optimal foraging theory predicts carabid predators, when choosing weed seeds, should choose be based not only on seed types but also seed numbers (Sih & Christensen, 2001). 5 \| TRAIT-BASED SEED CHOICE, FORAGING STRATEGIES, AND EFFECTS OF CARABID WEED SEED PREDATION ON WEED COMMUNITIES The optimal seed foraging assumption predicts that carabid weed seed predators should keep track of any changes in abun- dance of seed types they prefer and adjust their foraging de- cisions accordingly (Hubbard & Cook, 1978; Pyke et al., 1977). Foraging decisions in carabid weed seed predators should there- fore be dynamic and show changes through time and/or space. This assumption has been borne out by data from laboratory and field studies as carabid weed seed predators were shown to re- spond to changes in weed seed abundance in their environments (Dudenhoffer et al., 2016; Frank et al., 2011). In many cases, the rates of weed seed removal by carabid predators increased when seeds were offered in higher numbers per unit area (Honek et al., 2003; Westerman et al., 2008). Technically, these patterns suggest that carabid predators destroy more weed seeds as seed numbers increase and are therefore expected to have a stabiliz- ing influence on weed populations as models of predator–prey dynamics suggests (e.g., Abrams, 2000; Sih, 1980, 1984). The mechanisms underlying the density-dependent responses exhib- ited by weed seed predators in arable fields remain unknown, nonetheless. The phenomenon could arise from individual carabid The discussions above have established that seed detection and quality assessment are most likely mediated by olfactory mecha- nisms in some way. Following weed seed localization by a carabid seed predator, seed feeding will commence only if traits of both the predator and seed overlap to large extents (Ananthakrishan, 1990; Saska, Honek, Foffova, et al., 2019). Seed suitability rank in such trait-based foraging scenarios will be determined by the ability of the predator to overcome the physical and/or chemical defenses of the seed (Foffova et al., 2020; Larios et al., 2017; Schmitz, 2007). The ability of the carabid predator to break through the seed coat is an essential first step before feeding on seed tissues can be initiated (Brousseau et al., 2018; Forsythe, 1982, 1983b). It can be expected therefore that in cases where predators are able to efficiently break through the seed coat of different seed types without consider- able costs, seed chemistry will likely rule and predators would se- lect seed types of superior chemical qualities (Blate et al., 1998; Moles et al. 2003a, 2003b). TA B LE 3 Summary of potential carabid traits that influence the ability of carabid predators to destroy weed seeds Note: +, indicates a positive effect; −, indicates a negative effect; 0: indicates no documented effect. This fits nicely with the core assumptions of optimal foraging models as highly preferable weed seed types should always be selected when they are present in the environ- ment (Sih & Christensen, 2001; Westoby, 1978). Less preferable types of weed seed would be consumed only when the highly preferable type is not available or is available only in small num- bers (Hassell & Southwood, 1978). It has been suggested that frequency-dependent functions rule seed foraging strategies in arable fields (Greenwood, 1985; Horst & Venable, 2018). The key factor that powers seed preda- tion dynamics in such case would be seed encounter rate (Kuang & Chesson, 2010 ). The seed type encountered most frequently in the environment will hence suffer the most attacks, whereas seed types that are less abundant will be largely ignored by seed pred- ators (Merilaita, 2006). Frequency-dependent models may help explain some aspects of weed seed selection in arable fields, but such explanations are based on seed numbers alone. Frequency dependence assumptions ignore that weed seeds shed in arable fields differ not only in number, but also in quality (Bagavathiannan & Norsworthy, 2012; Dessaint et al., 1991). Basically, frequency- dependent reasoning turns weed seed predation interactions into a game of numbers, leaving no leeway for predators to choose seed types that suit them best as optimal foraging models predict (Pyke et al., 1977; Merilaita, 2006). Given the above, it could be suggested that within certain ranges of seed densities, attack rates against the preferable weed seed type are likely to follow positive density-dependent patterns (Cardina et al., 1996; Westerman et al., 2008). When the density of the highly preferable weed seed species starts to run low, the feeding responses of carabid weed seed predators can be summa- rized under the following two scenarios. The first scenario would involve seed predators continuing to attack the highly prefer- able weed seed type until it is depleted or no longer detectable (Holling, 1959; Zhang et al., 1992). Then, switching to less prefer- able seed types would take place. As a result, the highly prefer- able weed seed type would suffer significantly higher predation pressures when scattered in the environment at low densities com- pared to moderate and high densities. In such cases, the predation dynamics would be powered by inverse-density-dependent func- tions (Zhang et al., 1992). predators consuming more weed seeds as they become more avail- able per unit area, perhaps giving rise to foraging strategies ruled by functional responses (Holling, 1959). Alternatively, the higher rates of weed seed predation at increasing seed densities could come about by larger numbers of individual carabid weed seed predators consuming the dense seed patches, giving rise to forag- ing strategies ruled by numerical responses (Hulme, 1997; Marino et al., 2005). Some evidence has documented that dense seed patches attract higher numbers of carabid weed seed predators (Honek & Martinkova, 2001), but the correlation between carabid numbers aggregating to a dense seed patch and consumption rates of the preferable seed type in the patch was quite poor in some cases (Honek et al., 2005). Numerical responses are thus unlikely to always explain seed removal rates in responses to changes in seed density. Instead, both functional and numerical responses are more likely to work together and create complex seed predation dynamics in the field (Lester & Harmsen, 2002; Kuang & Chesson, 2010). Hence, a deeper treatment of seed foraging strategies re- quires elucidating how functional and numerical responses are shaped by changes in density of the preferable seed types relative to densities of other seed types (Lester & Harmsen, 2002). Noroozi et al., 2012; Pufal & Klein, 2013). The absence of density- dependent effects in such situations suggests that factors other than seed numbers alone also play into seed selection decisions. Hence, weed seed predation interactions in arable fields are unlikely to be a mere numbers game. Density-dependent and selective (trait-based) seed foraging should not be mutually exclusive in weed seed predation interac- tions as frequency-dependent models presume (Baraibar, Carrion, et al. 2011; Mongel & Clark, 1986). Instead, the two strategies should work together, and abundance of preferable weed seed types would determine seed selection decisions in such cases (Pyke et al., 1977). One key difference from frequency depen- dence here is that upon an initial successful encounter with the preferable food type in the environment, the selective forager would alter its foraging behavior toward increasing the chances of coming across the preferable food type (Garay et al., 2018; Sih, 1980, 1984). Thus, food searching behavioral patterns exhib- ited by selective foragers should be directed rather than random, and not driven by encounter rates alone (Pyke et al., 1977; Hassell & Southwood, 1978). 5 \| TRAIT-BASED SEED CHOICE, FORAGING STRATEGIES, AND EFFECTS OF CARABID WEED SEED PREDATION ON WEED COMMUNITIES By contrast, if the different types of TA B LE 3 Summary of potential carabid traits that influence the ability of carabid predators to destroy weed seeds Carabid trait Ability for weed seed destruction References Mouthpart morphology + Forsythe (1982, 1983b, 1987), (1985); Sasakawa (2016) Body mass + Honek et al. (2007); Martinkova et al. (2019); Saska, Honek, and Martinkova (2019) Bite force + Evans and Forsythe (1985); Wheater and Evans (1989); Brousseau et al. (2018) Body length 0 Kulkarni et al. (2016) Gape size 0 Evans & Forsythe (1985); Brousseau et al. (2018) Note: +, indicates a positive effect; −, indicates a negative effect; 0: indicates no documented effect. Note: +, indicates a positive effect; −, indicates a negative effect; 0: indicates no documented effect. ALI and WILLENBORG 13713 Under such conditions, the highly prefer- able weed seed type would suffer strong negative effects when its abundance is low, and its population could be extinguished in some local environments (Murdoch & Oaten, 1975; Figure 2a). There are field data to support the assertion of this scenario, suggesting that events of inverse-density-dependent weed seed predation could unfold under some realistic situations (see e.g., Cardina et al., 1996; Pannwitt et al., 2017; Westerman et al., 2008). Similar inverse- density-dependent prey selection patterns were also reported for carabid predators foraging on colonies of soybean aphids (Firlej et al., 2013). It could be inferred therefore that inverse density dependence might be the primary response governing foraging Ignoring this important shortcoming in frequency-dependent models has led to wide contradictions in weed seed foraging data. For example, data from some field studies have shown that weed seed predation rates followed inverse-density-dependent patterns, and seed consumption rates declined as more seeds were offered per unit area (Cardina et al., 1996; Marino et al., 2005; Westerman et al., 2008). In such cases, weed seed predation would have a bio- regulatory effect on weed populations only when weed seeds are scattered at low densities in the field. Higher weed seed densities would overwhelm the environment and saturate the functional and/ or numerical responses of carabid weed seed predators, breaking down any bio-regulatory effects against weed populations (Pannwitt et al., 2017; Petit et al., 2014). Intriguingly, the density-dependent removal of weed seeds was totally absent in some feeding trials conducted under field conditions (Baraibar, Daedlow, et al., 2011; 13714 \| ALI and WILLENBORG FI G U R E 2 Possible scenarios of the foraging strategies of carabid weed seed predators and their effects on weed communities in arable fields. (a) the preferable type of weed seeds suffer higher attacks at lower densities, and switching to less preferable seed types takes place only carabid when the preferable seed type is depleted to large extents. Predation pressure is strongly directed against the preferable seed type leading to local extinctions in some case. (b) the preferable type of weed seeds suffer lower attacks at lower densities, and switching to less preferable seed types takes place when abundance of the preferable drops below a certain threshold. But given that the abundance of the highly preferable weed seed species is kept below certain thresh- olds, some suppression against its populations should be expected Predation pressure is more distrusted among the different seed types and no local extinctions take place Composition and population size of weed community Different types of weed seeds Community of carabid predators Magnitude of seed predation pressure Most preferable Less preferable Most preferable Less preferable (b) (a) ALI and WILLENBORG 13714 Composition and population size of weed community Different types of weed seeds Most preferable Most preferable Most preferable Magnitude of seed predation pressure Community of carabid predators Community of carabid predators (b) (a) FI G U R E 2 Possible scenarios of the foraging strategies of carabid weed seed predators and their effects on weed communities in arable fields. (a) the preferable type of weed seeds suffer higher attacks at lower densities, and switching to less preferable seed types takes place only carabid when the preferable seed type is depleted to large extents. Predation pressure is strongly directed against the preferable seed type leading to local extinctions in some case. (b) the preferable type of weed seeds suffer lower attacks at lower densities, and switching to less preferable seed types takes place when abundance of the preferable drops below a certain threshold. Predation pressure is more distrusted among the different seed types and no local extinctions take place (Holling, 1959; Lester & Harmsen, 2002; Figure 2b). Data in support of this scenario remain wanting; however, and studies testing this prediction are needed. In the other possible scenario, carabid weed seed predators would switch their foraging mode toward the less preferable seed type when the abundance of the preferable type drops below a cer- tain threshold level (Chernov, 1976; Pyke et al., 1977). Below that abundance threshold attacks against the preferable seed type starts to wane as predators start to seek more rewarding seed patches (Pyke, 1984; Sih, 1980, 1984). This would give the preferable seed type an “escape density” below which predation risks diminish (Sentis & Boukal, 2018). Local population extinctions would not be expected under such conditions. Instead, carabid weed seed pred- ators could promote coexistence between multiple weed species (Juliano, 2001; Kuang & Chesson, 2010). This is a less attractive sce- nario for weed biocontrol programs because the predation pressure is more distributed among the different weed species in the commu- nity (Kuang & Chesson, 2010). REFERENCES Aarstma, Y., Cusumano, A., de Babadilla, M. F., Rusman, Q., Vosteen, I., & Poleman, E. H. (2019). Understanding insect foraging in complex habitats by comparing trophic levels: Insights from specialist host- parasitoid-hyperparasitoid systems. Current Opinion in Science, 32, 54–60. https://doi.org/10.1016/j.cois.2018.11.001 Carabid communities in arable fields comprise numerous species, nonetheless. These communities show considerable changes in their species composition through space and time (Jacob et al., 2006; Pufal & Klein, 2013). Carabid species within those communities also are expected to interact, interfere, or even compete with one another in ways that remain poorly understood (Niemela, 1993; Niemela et al., 1997; Currie et al., 1996). Hence, the exact composition of the cara- bid community at any point in time or space would determine which exact weed species should incur the strongest predation pressures (Charalabidis et al., 2019; Petit et al., 2014). 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ACKNOWLEDGEMENTS We would like to thank Dr. Boyd Mori for reading the early drafts of the manuscript and providing valuable comments that helped improving the text. Funding was provided by a Discovery Grant awarded to CJW by the Natural Sciences and Engineering Research Council of Canada. We are also thankful for two anonymous review- ers who provided valuable comments that helped improve and refine the manuscript. Balnke, A., Pinheiro, M., Watson, P. J., & Fang, M. J. (2018). A biome- chanical analysis of prognathous and orthognathous insect head capsules: Evidence from a many-to-one mapping of from to func- tion. Journal of Evolutionary Biology, 31(5), 665–674. https://doi. org/10.1111/jeb.13251 Baraibar, B., Carrion, E., Recasens, J., & Westerman, P. R. (2011). Unravelling the process of weed seed predation: Developing op- tions for better weed control. Biological Control, 56(1), 85–90. https://doi.org/10.1016/j.biocontrol.2010.09.010 Authors declare no conflict of interest. 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https://openalex.org/W4285818456	https://www.ijfmr.com/papers/2022/4/606.pdf	English	null	Utilization of Cereals and Millets for Nutritious Rich Food Products	International Journal For Multidisciplinary Research	2,022	cc-by-sa	6,808	Rebba Phileshia Shanthi, Prof. Dr. Virginia Paul, Prof. Dr. Ranu Prasad, Dr. Alka Gupta Rebba Phileshia Shanthi, Prof. Dr. Virginia Paul, Prof. Dr. Ranu Prasad, Dr. Alka Gupta Department of Food, Nutrition and Public Health (FNPH), Ethelind College of Home Science (ECHS), Sam Higginbottom University of Agriculture, Technology and Sciences (SHUATS) International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 Keywords: Black Rice Flour, Foxtail Millet Flour, Moringa Leaves Powder, Anti-oxidants, Nutrition- rich Food Keywords: Black Rice Flour, Foxtail Millet Flour, Moringa Leaves Powder, Anti-oxidants, Nutrition- rich Food International Journal for Multidisciplinary Research (IJFMR) Introduction Black rice was initially grown in China before the Chinese dynastic period and was called the 'luck rice' because it was believed that people consuming black rice would live longer and also it can cure most diseases. Earlier people refused to consume black rice due to its black color as they considered it to be 'dirty' because of the black color. Later it was found that the color of the grain is determined by the accumulation of colored pigments. Black rice is black due to the presence of the anthocyanin pigment on the outer layers (bran) of the rice kernel. The purple color pigment anthocyanin is rich in antioxidants and is naturally present in many berries like blueberry, black currant, and vegetables like eggplant (brinjal) (Nitin Kumar, Roshini Deepika Murali, et al., 2020). The grain color of cereals is determined by the pigmentation of certain phytochemicals. the black grain color is caused by the deposition of anthocyanins (Rahman, et al., 2013) black color pigment called anthocyanin which is rich in antioxidants and poses a variety of health benefits such as anti-aging, anticancer, anti-diabetes, lowering the risk of obesity, etc. Black rice contains essential amino acids like lysine, tryptophan; vitamins such as vitamin B1, vitamin B2, folic acid; and it is a good source of minerals including iron, zinc, calcium, phosphorus, and selenium. It contains the highest amount of antioxidants, protein, and dietary fiber of all rice varieties, besides it has phenolics, flavonoids, and anthocyanins. Antioxidants are the first line of defense against free radical damage and are critical for maintaining optimum health and well-being. These antioxidant compounds have tremendous health benefits and can reduce the risk of developing various chronic diseases. On cooking, it turns into purple color with a shiny indigo finish and has a mild nutty flavor and its texture is smooth and firm Anthocyanins possess antioxidative and antimicrobial activities, improve visual and neurological health and also protects against non-communicable diseases. These all good health benefits are due to the antioxidant activity i.e., free radical scavenging activity which prevents the release of free radicals in the body. Anthocyanins are used to treat a wide variety of minor health issues like blood pressure, cold, and urinary tract infections. It can also cure major health problems like heart attack (CVD), cancer, obesity, and diabetes. Foxtail millet ranks second in the total world production of millets. International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 Abstract The present research entitled "Utilization of Cereals and Millets for Nutritious Rich Food Products" was undertaken to develop nutrition-rich food in which anti-oxidants and phenolic compounds, rich fiber content, vitamins, minerals, amino acids, and nutritional benefits of health. They fight against cardiovascular diseases, diabetes, obesity, and cancer, and reduce inflammation. Products like black rice, foxtail millets, and moringa leaves are utilized through baked products where the amount of nutritive value remains almost the same when compared with steamed or cooked and gives good color, and texture, and maintains its shelf life for many days. As in this experiment, the products like bread, muffins, and cookies are chosen for the preparation which helps every age group like to eat. The research was conducted during the year 2020 in the Food lab of the Department of Food Nutrition and public health, Ethelind College of Home Science, Sam Higginbottom University of Agriculture. Technology and Sciences, Prayagraj, U.P., India. A close-ended Evaluation schedule was prepared to evaluate the developed product prepared in the university lab. Data were analyzed using suitable statistical tools i.e. Analysis of variance (Two-way ANOVA or Two-way classification) technique. A significant difference between the treatments was determined by using the CD (Critical Difference) test. The test was performed for comparing the difference in the nutritional content between control and best treatment of the developed food products. Based on the findings, it is concluded that the black rice flour, foxtail millet flour, and moringa leaves powder enhance the nutritive value of “Bread, Cookies and Muffins”. Sensory evaluation of prepared products T2 (25:15:2:58) was highly acceptable. Nutritionally, it was found that the nutrient content of best treatment T2 of the three products, was significantly higher with regards to energy, protein, fat, carbohydrate, fiber, calcium, iron, and activity as compared to the control T0. The cost was increased marginally in all treatment of prepared products comparatively control. The cost of the prepared product per 100 gm of raw ingredient for “Bread” ranged from Rs. 30 to Rs. 35, “Cookies” from Rs. 25 to Rs. 35, and “Muffins” from Rs. 30 to Rs. 40. y rich Food Email : editor@ijfmr.com Website : www.ijfmr.com IJFMR2204005 56 International Journal for Multidisciplinary Research (IJFMR) oxidative stress due to the presence of antioxidants (Silva, Pereira, & Azevedo, et al., 2013). Additionally, owing to its low cost and excellent functional properties of flour and protein concentrate, foxtail millet can be considered a good candidate for replacing animal protein foods. Furthermore, there is huge potential for successfully developing low-cost, protein-rich functional food products helpful in the prevention and management of lifestyle-related chronic diseases. The less explored, commercially available foxtail millet-milled fractions like whole flour & bran-rich fractions were studied for their antioxidant potency. Phytochemicals like alkaloids, phenolic, reducing sugars, and flavonoids were found only in methanolic & aqueous extracts, while tannins and terpenoids were present in all the solvent extracts of whole flour & bran-rich fraction. Antioxidants were extracted using methanol, ethanol, and water. Metabolic extracts of whole flour and bran-rich fraction exhibited a significantly higher. The grain of Foxtail millet is used in China as an astringent and emollient in choleric affections and diarrhea. The seeds are used in India as a diuretic, to strengthen virility, and treat indigestion, dyspepsia, and rheumatism. It helps to treat food stagnancy, white seeds are useful for fever and cholera, and in India to enhance vigor and treat bone fractures. The cooked grains are used in Chhattisgarh as a cure for diarrhea. The paste is externally used Foxtail millet can fight diabetes and reduces the chances of blockages in the heart resulting in cardiac arrest. 100 grams of Foxtail millet contains 12 g of moisture, 351 calories, 11.2 g of protein, 4 g of total fat, 63.2 g of carbohydrate, and 6.7 g of crude fiber. It grants 803 g of isoleucine, 1764 g of leucine, 103 g of tryptophan, 328 g of threonine, 233 g of lysine, 0.6 mg of thiamin, 63.2 g of carbohydrate, 2.8 mg of iron, 11.2 mg of protein, 3.2 mg of niacin, 4 g of lipid fat, 0.1 mg of riboflavin, and 31 mg of calcium. Vitamin B1 assists in the formation of the neurotransmitter acetylcholine (Sharma, et al., 2017). Moringa oleifera is the most nutrient-rich plant yet discovered. This humble plant has been making strides in less-developed societies for thousands of years, and significant nutritional research has been conducted since the 1970s. Moringa provides a rich and rare combination of nutrients, amino acids, antioxidants, and anti-aging and anti-inflammatory properties used for nutrition and healing. Moringa is sometimes called "Mother's Best Friend". Introduction Foxtail millet is fairly tolerant of drought; it can escape some droughts. It has been reported that foxtail millet has many nutritional and medicinal properties and is recommended in ayurvedic and Unani products by practitioners. Foxtail millet grains are about 2 mm in length and the glumes can be white, red, yellow, brown, or black. Neither white nor red varieties were found to contain tannins. The granule size varies from 0.8 to 9.6 μm. Foxtail millet can be waxy (high in amylopectin), normal (low amylose), or non- waxy (high amylose) (John R.N. Taylor, M. Naushad, et al., 2008). Foxtail millet is non-glutinous, like buckwheat and quinoa, and is a non-acid-generating food, hence considered an easily digestible food also possesses a higher amount of proteins and minerals that act as a potential functional food ingredient and a supplementary protein source to most cereals, due to its high lysine content (Mohamed, Zhu, Issoufou, Fatmata, Zhou, et al., 2009). The availability of minerals increased during germination due to the catabolism of anti-nutrients like polyphenol and saponins which hinder the bioavailability of minerals described that germination of foxtail millet for 3 days resulted in the production of a high concentration of minerals. The consumption of sprouts as functional food can be considered very important in reducing human diseases related to Email : editor@ijfmr.com Website : www.ijfmr.com 57 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 oxidative stress due to the presence of antioxidants (Silva, Pereira, & Azevedo, et al., 2013). 3.4 Sensory Evaluation of Developed Products Sensory evaluation of the food products for their acceptability was done by a panel member of 5 judges. Panel members were selected based on their performance in initial evaluation trials. Sensory descriptors of the samples were appearance, color, aroma, taste, texture, and overall acceptability. The nine-point hedonic scale was used for sensory evaluation (Srilakshmi 2014). Materials And Methods The study entitled “Utilization of Cereals, Millets for Nutritious Rich Food Products” was conducted in the Department of Food Nutrition and Public Health, Ethelind College of Home Science, Sam Higginbottom University of Agriculture, Technology and Science, Prayagraj, U.P. India. The details of materials, experiments, procedures, and techniques adopted during the present investigation have been elaborated in this chapter under the following headings: Table 1: Showing Experimental design of Bread, Cookies and Muffins for 100 gms Value-added Food Product Treatments Replications T0 T1 T2 T3 4 Black Rice Flour (g) - 25 g 25 g 15 g Foxtail Millet Flour (g) - 23 g 15 g 25 g Moringa Powder (g) - 2 g 2 g 2 g All-purpose Flour/Refined Flour (g) 100 48 g 68 g 68 g Table 1: Showing Experimental design of Bread, Cookies and Muffins for 100 gms 3.3 Detail of Treatments and Replications tail of Treatments and Replications Four treatments were prepared for “Bread, Cookies and Muffins” as follows: nts were prepared for “Bread, Cookies and Muffins” as follows: Four treatments were prepared for Bread, Cookies and Muffins as follows: (Control) T0: The product was prepared using only 100 gms all-purpose flour. (Control) T0: The product was prepared using only 100 gms all-purpose flour. (Control) T0: The product was prepared using only 100 gms all-purpose flour. Treatment (T1): The product was prepared using 25% black rice powder, 25% of foxtail millet powder 2% of moringa powder, and 48% of all-purpose flour. Treatment (T1): The product was prepared using 25% black rice powder, 25% of foxtail millet powder 2% of moringa powder, and 48% of all-purpose flour. Treatment (T2): The product was prepared using 25% black rice powder, 15% of foxtail millet powder 2% of moringa powder, and 58% of all-purpose flour. Treatment (T2): The product was prepared using 25% black rice powder, 15% of foxtail millet powder 2% of moringa powder, and 58% of all-purpose flour. Treatment (T3): The product was prepared using 15% black rice powder, 25% of foxtail millet powder 2% of moringa powder, and 58% of all-purpose flour. Treatment (T3): The product was prepared using 15% black rice powder, 25% of foxtail millet powder 2% of moringa powder, and 58% of all-purpose flour. Replications: Control and treatments were replicated three times respectively to get the average value. Replications: Control and treatments were replicated three times respectively to get the average value. International Journal for Multidisciplinary Research (IJFMR) A large number of reports on the nutritional qualities of Moringa now exist in both the scientific and popular literature. Moringa has been in use for centuries for nutritional as well medicinal purposes. These include vitamin C, which fights a host of illnesses including colds and flu; vitamin A, which acts as a shield against eye disease, skin disease, heart ailments, diarrhea, and many other diseases; Calcium, which builds strong bones and teeth and helps prevent osteoporosis; Potassium, which is essential for the functioning of the brain and nerves, and Proteins, the basic building blocks of all our body cells. Another important point is that Moringa leaves contain all of the essential amino acids, which are the building blocks of proteins. It is very rare for a vegetable to contain all of these amino acids. And Moringa contains these amino acids in a good proportion so that they are very useful to our bodies. These leaves could be a great boon to people who do not get protein from meat. Moringa even contains arginine and histidine two amino acids especially important for infants. Arginine and histidine, are especially important for infants who are unable to make enough protein for their growth requirements. Experts tell us that 30% of children in subSaharan Africa are protein deficient. Moringa could be an extremely valuable food source (Khawaja Tahir Mahmood, et al., 2013). Email : editor@ijfmr.com Website : www.ijfmr.com 58 Nutrient / 100 g of product = Ingredient used ( g)× Nutritivevalue of the Ingredient 100 Nutrient / 100 g of product = Ingredient used ( g)× Nutritivevalue of the Ingredient 100 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 3.6 Determination of Cost The cost of the prepared products was determined based on the price of raw ingredients at the prevailing market price. 3.3 Statistical Analysis The data were statistically analyzed by using the Analysis of variance (Two-way ANOVA or Two-way Classification) technique. A significant difference between the treatments was determined by using the CD (Critical Difference) test (Appendix). "t-test" was performed for comparing the difference in the nutritional content between the control and best treatment of the developed food products (Chandel, 2006). 3.5 Determination of Nutrient Content of Prepared Products The nutritive value of the ingredients (black rice flour, foxtail millet flour, moringa leaves powder) used in the development of food products was calculated with the use of food composition tables by Gopalan et al. (2015). Nutrients such as energy, protein, carbohydrates, fat, iron, calcium and fiber were calculated. IJFMR2204005 Website : www.ijfmr.com Email : editor@ijfmr.com Website : www.ijfmr.com 59 Results and Discussion Table 2: Average Sensory Scores of Different Parameters in Control and Treated Samples of “Bread” Parameters Mean + SE Statistical Analysis T0 T1 T2 T3 Colour and Appearance 7.7 + 0.42 7.03 + 0.35 7.5 + 0.48 7.26 + 0.39 F = 4.59 (4.76) S* C.D. = 0.47 Body and Texture 7.1 + 0.35 7.6 + 0.39 7.5 + 0.38 7.06 + 0.11 F = 4.88 (4.76) S* C.D. = 0.24 Taste and Flavor 7.4 + 0.39 7.7 + 0.28 7.6 + 0.30 7.7 + 0.49 F = 27.30 (4.76) S* C.D. = 0.56 Overall Acceptability 7.2 + 0.31 7.4 + 0.30 7.4 + 0.14 7.7 + 0.25 F = 0.76 (4.76) S* C.D. = 0.42 Table 3: Average Sensory Scores of Different Parameters in Control and Treated Samples of “Cookies” Control and Treatments T0 T1 T2 T3 Colour and Appearance 7.5 7.1 7.6 7.3 Body and Texture 7.1 7.2 7.6 6.9 Taste and Flavour 7.4 7.5 7.8 7.3 Overall Acceptability 7.4 7.1 7.8 7.1 Table 4: Average Sensory Scores of Different Parameters in Control and Treated Samples of “Muffins” Parameters Mean + SE Statistical Analysis T0 T1 T2 T3 IJFMR2204005 Website : www.ijfmr.com Email : editor@ijfmr.com 60 Sensory Scores of Different Parameters in Control and Treated Samples of “Cookies” Table 4: Average Sensory Scores of Different Parameters in Control and Treated Samples of “Muffins” Parameters Mean + SE Statistical Analysis T0 T1 T2 T3 IJFMR2204005 Website : www.ijfmr.com Email : editor@ijfmr.com 60 Sensory Scores of Different Parameters in Control and Treated Samples of “Muffins” Table 4: Average Sensory Scores of Different Parameters in Control and Treated Sample Email : editor@ijfmr.com 60 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 Colour and Appearance 7.5 + 0.40 7.0 + 0.30 7.6 + 0.40 7.4 + 0.41 F = 4.59 (4.76) S* C.D. = 0.25 Body and Texture 7.2 + 0.30 7.0 + 0.17 7.7 + 0.28 7.3 + 0.02 F = 1.26 (4.76) S* C.D. = 0.13 Taste and Flavor 7.4 + 0.39 7.3 + 0.34 7.2 + 0.40 7.2 + 0.46 F = 27.30 (4.76) S* C.D. = 0.47 Overall Acceptability 7.4 + 0.63 7.1 + 0.12 7.4 + 0.51 7.0 + 0.43 F = 0.59 (4.76) S* C.D. Email : editor@ijfmr.com Results and Discussion = 0.21 International Journal for Multidisciplinary Research (IJFMR) nternational Journal for Multidisciplinary Research (IJFMR) Table 5: The Average Nutrient Content of “Bread” per 100 g Table 5: The Average Nutrient Content of Bread per 100 g Treatments Energy (kcal) Protein (g) Fat (g) Carbohydrate (g) Fiber (g) Iron (mg) Calcium (mg) T0 452.52 15.72 7.20 22.26 2.46 3.92 63.27 T1 453.86 17.33 7.88 78.66 4.21 3.66 69.07 T2 452.66 17.58 7.66 38.76 3.75 3.71 67.146 T3 454.26 17.75 7.75 38.70 2.59 3.11 66.51 Table 6: The Average Nutrient Content of “Cookies” per 100 g Table 6: The Average Nutrient Content of “Cookies” per 100 g Treatments Energy (kcal) Protein (g) Fat (g) Carbohydrate (g) Fiber (g) Iron (mg) Calcium (mg) T0 454.88 15.72 7.203 22.592 2.462 3.92 63.28 T1 456.74 17.331 7.881 79.36 4.21 3.66 69.08 T2 455.54 17.59 7.66 38.89 3.72 3.718 67.15 T3 457.14 17.76 8.701 38.55 2.59 3.11 66.52 Table 7: The Average Nutrient Content of “Muffins” per 100 g Table 7: The Average Nutrient Content of “Muffins” per 100 g Treatments Energy (kcal) Protein (g) Fat (g) Carbohydrate (g) Fiber (g) Iron (mg) Calcium (mg) T0 514.94 19.878 10.98 22.44 2.597 3.411 63.27 T1 516.8 25.213 11.66 79.41 3.462 6.145 69.31 T2 515.6 24.095 11.44 38.94 3.75 4.53 67.39 T3 517.2 24.479 12.48 38.68 2.59 3.84 66.74 Table 8: The Average Cost of “Bread” per 100 g of Raw Ingredients Ingredients T0 T1 T2 T3 Amt (g) Cost (Rs) Amt (g) Cost (Rs) Amt (g) Cost (Rs) Amt (g) Cost (Rs) Black Rice Flour @ Rs. 56 - - 25 14 25 14 15 8.4 Foxtail Millet Flour @ Rs. 23.50 - - 25 5.87 15 3.52 25 5.87 Moringa Leaves powder @ Rs. 49.90 - - 2 0.9 2 0.9 2 0.9 Maida @ Rs. 3.90 100 3.9 48 1.87 58 2.26 58 2.26 Yeast @ Rs. 129 1.5 1.9 1.5 1.9 1.5 1.9 1.5 1.9 IJFMR2204005 Website : www.ijfmr.com Email : editor@ijfmr.com 61 Table 8: The Average Cost of “Bread” per 100 g of Raw Ingredients Website : www.ijfmr.com 61 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 Sugar @ Rs. 3.80 3 0.1 3 0.1 3 0.1 3 0.1 Salt @ Rs. 1.70 1 0.01 1 0.01 1 0.01 1 0.01 Butter @ Rs. 32 1 0.4 1 0.04 1 0.04 1 0.04 Milk @ Rs. Results and Discussion 5.60 60 ml 3.36 60 ml 3.36 60 ml 3.36 60 ml 3.36 Baking Powder @ Rs. 30 2 0.6 2 0.6 2 0.6 2 0.6 Total Cost Rs./100gm 10.27 27.75 26.69 23.44 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 International Journal for Multidisciplinary Research (IJFMR) Table 8 shows that the cost of “Bread ” per 100 g of raw ingredients at the prevailing cost of the raw materials was T0 is Rs. 10.26 for treatment, T1 is Rs. 27.75, T2 is Rs. 26.69, T3 is Rs. 23.44. This shows that as the incorporation level of black rice flour increased the cost also increased marginally. Table 8 shows that the cost of “Bread ” per 100 g of raw ingredients at the prevailing cost of the raw materials was T0 is Rs. 10.26 for treatment, T1 is Rs. 27.75, T2 is Rs. 26.69, T3 is Rs. 23.44. This shows that as the incorporation level of black rice flour increased the cost also increased marginally. Table 9: The Average Cost of “Cookies” per 100 g of Raw Ingredients Ingredients T0 T1 T2 T3 Amt (g) Cost (Rs) Amt (g) Cost (Rs) Amt (g) Cost (Rs) Amt (g) Cost (Rs) Black Rice Flour @ Rs. 56 - - 25 14 25 14 15 8.4 Foxtail Millet Flour @ Rs. 23.5 - - 25 5.87 15 3.52 25 5.87 Moringa Leaves Powder @ Rs. 49.90 - - 2 0.9 2 0.9 2 0.9 Maida @ Rs. 32 100 1.95 48 1.87 58 2.26 58 2.26 Vanilla Essence @ Rs. 44.90 1 0.224 1 0.449 1 0.449 1 0.449 Sugar @ Rs. 3.80 50 1.25 50 2.5 50 2.5 50 2.5 Salt @ Rs. 1.70 1 0.01 1 0.017 1 0.017 1 0.017 Butter @ Rs. 32 80 19.8 80 39.6 80 39.6 80 39.6 Milk @ Rs. 5.60 3 ml 0.16 3 ml 0.16 3 ml 0.16 3 ml 0.16 Baking Powder @ Rs. 30 2 0.6 2 0.6 2 0.6 2 0.6 Total Cost Rs./100 g 23.60 32.98 32 30 Table 9: The Average Cost of “Cookies” per 100 g of Raw Ingredients Table 9 shows that the cost of “Cookies” per 100 g of raw ingredients at the prevailing cost of the raw materials was T0 is Rs. 23.60 for treatment, T1 is Rs. 32.98, T2 is Rs. 32, T3 is Rs. 30. Email : editor@ijfmr.com Results and Discussion This shows that as the incorporation level of black rice flour increased the cost also increased marginally. Table 9 shows that the cost of “Cookies” per 100 g of raw ingredients at the prevailing cost of the raw materials was T0 is Rs. 23.60 for treatment, T1 is Rs. 32.98, T2 is Rs. 32, T3 is Rs. 30. This shows that as the incorporation level of black rice flour increased the cost also increased marginally. Table 10: The Average Cost of “Muffins” per 100 g of Raw Ingredients Table 10: The Average Cost of “Muffins” per 100 g of Raw Ingredients Ingredients T0 T1 T2 T3 Amt (g) Cost (Rs) Amt (g) Cost (Rs) Amt (g) Cost (Rs) Amt (g) Cost (Rs) Black Rice Flour @ Rs. 28 - - 25 7 25 7 15 4.2 Foxtail Millet Flour @ Rs. 11.75 - - 25 2.93 15 1.76 25 2.93 Moringa Leaves Powder @ Rs. 24.95 - - 2 0.45 2 0.45 2 0.45 Maida @ Rs. 1.95 100 1.95 48 0.94 58 0.94 58 0.94 IJFMR2204005 Website : www.ijfmr.com Email : editor@ijfmr.com 62 62 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 Sugar @ Rs. 1.90 70 1.3 70 1.3 70 1.3 70 1.3 Egg @ Rs. 20 50 3 50 3 50 3 50 3 Salt @ Rs. 8.50 1 0.01 1 0.01 1 0.01 1 0.01 Vanilla Essence @ Rs. 22.45 1 0.224 1 0.224 1 0.224 1 0.224 Butter @ Rs. 16 70 17.3 70 17.3 70 17.3 70 17.3 Milk @ Rs. 2.80 10 2.31 10 2.31 10 2.31 10 2.31 Baking Powder @ Rs. 15 50 3 50 3 50 3 50 3 Total Cost Rs./100 g 28.77 38.45 37.47 35.85 Table 10 shows that the cost of “Muffins” per 100 g of raw ingredients at the prevailing cost of the raw materials was T0 is Rs. 28.77 for treatment, T1 is Rs. 38.45, T2 is Rs. 37.47, T3 is Rs. 35.85. International Journal for Multidisciplinary Research (IJFMR) International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 Table 10 shows that the cost of “Muffins” per 100 g of raw ingredients at the prevailing cost of the raw materials was T0 is Rs. 28.77 for treatment, T1 is Rs. 38.45, T2 is Rs. 37.47, T3 is Rs. 35.85. Results and Discussion The study entitled “Utilization of Cereals and Millets for Nutritious Rich Food Products” was carried out to formulate and standardized the product as well as to assess their sensory attributes and nutrient content as well as determine the cost of the prepared products based on raw ingredients. The three food products “Bread”, “Cookies” and “Muffins” were prepared by incorporation of cereals like black rice flour and millets like foxtail millets flour. The basic recipe of each product with and without incorporation of T2 (25:15:2:68, black rice flour + foxtail millet flour + moringa leave powder + Maida) followed by T0 (100% of all-purpose flour; Maida), T1 (25:25:2:48, black rice flour + foxtail millet flour + moringa leaves powder + Maida), T3 (15:25:2:68, black rice flour + foxtail millet flour + moringa leaves powder + Maida) respectively, and the basic recipe of product T2 (25:15:2:68, black rice flour + foxtail millet flour + moringa leave powder + Maida) followed by T0 (100% of all-purpose flour; Maida), T1 (25:25:2:48, black rice flour + foxtail millet flour + moringa leaves powder + Maida), T3 (15:25:2:68, black rice flour + foxtail millet flour + moringa leaves powder + Maida) respectively. T2 (25:15:2:68, black rice flour + foxtail millet flour + moringa leave powder + Maida) followed by T0 (100% of all-purpose flour; Maida), T1 (25:25:2:48, black rice flour + foxtail millet flour + moringa leaves powder + Maida), T3 (15:25:2:68, black rice flour + foxtail millet flour + moringa leaves powder + Maida) respectively. And the method of cooking was baking at 180o C for 10-20 minutes. The experimental trial was replicated three times and in each replication of products were Bread, Cookies and Muffins. The organoleptic evaluation of the products about attributes of color, texture, taste, flavor, and overall acceptability was done using the panel of five judges. “Nine-point hedonic scale” and nutritive value was calculated using the food composition tables given by Gopalan, et al., (2015). The findings of the entire study are reported as follows. The observations were recorded and tabulated and results were statistically analyzed by analysis of variance and critical difference technique and t-test. The costs of the products were calculated from the cost of raw materials used. Conclusion Based on the findings, it is concluded that the Black rice flour + Foxtail millet + Moringa leaves powder + Maida were successfully incorporated and enhanced the nutritive value of "Bread, Cookies and Muffins". Based on sensory evaluation T2 (25+15+2+68) was highly acceptable based on overall acceptability for “Bread, Cookies and Muffins” about color and appearance, body and texture, taste, and flavor, and overall acceptability. Nutritive value of prepared product indicates carbohydrate, protein, fiber, energy, calcium, iron content and also anti-oxidants, flavonoids, anthocyanins, and phenolic compounds increased in enriched “Bread, Cookies and Muffins” as compared to control bread T2 is Rs. 26.69, cookies T2 is Rs. 32, muffins T2 is Rs. 37.47 and their cost are as recorded. International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582–2160, Volume: 4, Issue: 4, July-August 2022 between the nutrient content of control (T0) and the best treatment (T2) about antioxidants, energy, carbohydrate, calcium, fat, protein. However, a non-significant difference in the 1.88 and 2.78 for energy and fat content respectively were found. Comparison between the nutrient content of control and best treatment of “Cookies” by using “t-test” the difference in the t- calculated value of 7.94 for energy content, 3.66 for fat content, 63.11 for carbohydrate content, 5.23 for calcium content, and 4.31 for iron content was higher than the tabulated value of “t” which is 2.96 at 5% probability level indicate that there is a significant difference between the nutrient content of control (T0) and the best treatment (T1) about energy, carbohydrate, calcium, fat, protein. However, a non-significant difference in the 0.60 and 0.42 for iron fiber content respectively were found. In "Muffins" the difference in the t-calculated value was 5.48 for protein content, 14.97 for carbohydrate content, 5.14 for calcium content, 10.04 for iron calcium and 26.77 for fiber content was higher than the tabulated value of "t" which is 2.96 at a 5% probability level indicating that there is a significant difference between the nutrient content of control (T0) and the best treatment (T2) about antioxidants, energy, carbohydrate, calcium, fat, protein. However, a non-significant difference in the 1.88 and 2.78 for energy and fat content respectively were found. Since to calculate the cost of prepared products it was found the highest price was of T2 and T0 of the products namely: “Bread, Cookies and Muffins” per 100 g for control and treatment. The average cost of the “Bread, Cookies and Muffins” per 100 g of raw ingredients was  The cost of the product “Bread” per 100 g of the raw ingredient, ranged from Rs. 25 to Rs. 30  The cost of the product “Cookies” per 100 g of raw ingredient ranged from Rs. 45 to Rs. 50  The cost of the product “Muffins” per 100 g of raw ingredient ranged from Rs.65 to Rs. 70 Since to calculate the cost of prepared products it was found the highest price was of T2 and T0 of the products namely: “Bread, Cookies and Muffins” per 100 g for control and treatment. Th t f th “B d C ki d M ffi ” 100 f i di t Results and Discussion In "Bread" the difference in the t-calculated value of 5.48 for protein content, 14.97 for carbohydrate content, 5.14 for calcium content, 10.04 for iron calcium, and 26.77 for fiber content was higher than the tabulated value of “t” which is 2.96 at 5% probability level indicate that there is a significant difference Email : editor@ijfmr.com Website : www.ijfmr.com 63 References 1. Reddy, V.S., Dash, S., Reddy, A.R. (1995). Anthocyanin pathway in rice (Oryza sativa L): Identification of a mutant showing dominant inhibition of anthocyanins in leaf and accumulation of proanthocyanidins in the pericarp. Theoretical and Applied Genetics, 91, 301-312. 1. Reddy, V.S., Dash, S., Reddy, A.R. (1995). Anthocyanin pathway in rice (Oryza sativa L): Identification of a mutant showing dominant inhibition of anthocyanins in leaf and accumulation of proanthocyanidins in the pericarp. Theoretical and Applied Genetics, 91, 301-312. 2. 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Incorporation of different proportions of more flours from cereals or any other agricultural-related product for value addition in traditional recipes can be encouraged and popularized to improve the intake of protein, fat, calcium, carbohydrate and iron, and most importantly also anti-oxidants, flavonoids, anthocyanins, and phenolic compounds are very high in these cereals and millets. 2. Black rice and foxtail millets are very high in anti-inflammatory agents which is one of the main responses in the immune system 2. Black rice and foxtail millets are very high in anti-inflammatory agents which is one of the main responses in the immune system 3. The three food products Black rice, foxtail millet, and moringa leaves are very high in iron, calcium antioxidants vitamins like B, E, C and A folic acids, pyridoxine, and nicotinic acids by which the nervous system, digestive system, and skin health are controlled and maintained 3. 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https://openalex.org/W2033902534	http://www.scirp.org/journal/PaperDownload.aspx?paperID=31758	English	null	Identification of Categorical Registration Data of Domain Names in Data Warehouse Construction Task	Intelligent control and automation	2,013	cc-by	4,780	Intelligent Control and Automation, 2013, 4, 227-234 Intelligent Control and Automation, 2013, 4, 227-234 http://dx.doi.org/10.4236/ica.2013.42027 Published Online May 2013 (http://www.scirp.org/journal/ica) ABSTRACT This work is dedicated to formation of data warehouse for processing of a large volume of registration data of domain names. Data cleaning is applied in order to increase the effectiveness of decision making support. Data cleaning is ap- plied in warehouses for detection and deletion of errors, discrepancy in data in order to improve their quality. For this purpose, fuzzy record comparison algorithms are for clearing of registration data of domain names reviewed in this work. Also, identification method of domain names registration data for data warehouse formation is proposed. Deci- sion making algorithms for identification of registration data are implemented in DRRacket and Python. Keywords: Domain; Domain Name System; Registrar; Registrant; Category Data; Data Warehouse; Data Clearing; Fuzzy Search Algorithms; Damerau-Levenstein Distance; Decision Tree Identification of Categorical Registration Data of Domain Names in Data Warehouse Construction Task Rasim Alguliev, Rena Gasimova Institute of Information Technology of ANAS, Baku, Azerbaijan Email: director@iit.ab.az, renakasumova@gmail.com Rasim Alguliev, Rena Gasimova Institute of Information Technology of ANAS, Baku, Azerbaijan Email: director@iit.ab.az, renakasumova@gmail.com Received January 31, 2013; revised March 1, 2013; accepted March 8, 2013 Received January 31, 2013; revised March 1, 2013; accepted March 8, 2013 Copyright © 2013 Rasim Alguliev, Rena Gasimova. This is an open access article distributed under the Creative Commons Attribu- tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2013 SciRes. 1. Introduction collection, organized to support management”, designed to act as “unique and only source of truth”, providing managers and analysts with trustworthy information ne- cessary for operative analysis and decision making [5,6]. Richard Hackathorn, another founder of this concept, wrote that the objective of DW—is to provide “unique image of existing reality” for organizations [7]. Ralph Kimball, one of DW authors, described the warehouse as “a place, where people can access their data”. He also formulated in [8,9] main requirements to data ware- houses. Decision support systems (DSS) have been used for for- mation of administration decisions, i.e. discovery of the best alternative for a lengthy period of time. Majority of commercial, social and governmental organizations no longer make serious decisions without using elements of computer analysis [1]. At all stages of complex adminis- trative decision making—from identification of the pro- blem to control of realization of made decision—the more accurate, timely and complete is the appropriate in- formation, the more effective will be the decision. In- formation support of the decision making process deter- mines the effectiveness of decision at all stages [2]. For correct comprehension of data concept, it is nec- essary to understand following principal moments: DW concept—is not a data analyzing concept; rather it is a concept of preparation for data analyzing; DW concept does not predetermine architecture of purposive analytic- cal system. It describes the processes to be implemented in the system, but not the where precisely and how these processes must be implemented. The main objective of warehouses—is creation of a single logical presentation of data, contained in multitype data bases (DB) or in other words, a single corporate data model. Modern approach to automation of decision making support is based on use of data warehouse (DW) concept. Rampant development of information technologies, and data collection, storage and procession means in particu- lar, allows to collect vast volumes of data which require analyzing. DW provides analysts, executives and top ma- nagers with capability to study large volumes of interde- pendent data using fast interactive information reflection on different levels of detailing from different points of view in accordance with notion of the user on subject field [3,4]. 2. Relevance Apparently, at modern development level of information technologies, simple relocation of data from one DW to Bill Inmon, author of DW concept, defined them as “subject oriented, integrated, unchanged, supporting data ICA ICA R. ALGULIEV, R. GASIMOVA 228 another does not cause difficulties. However if we simply relocate the data from all sources to a single DB, then we will obtain a “dump”, an uncoordinated set of data. In order to create something more accessible for analysis by the final user, it is necessary to coordinate the data en- tering the DB from sources of data warehouse. In other words, to solve the main task of DW construction: to create the most coordinated, subject-oriented, integrated, time dependant data set. with independence of data allocation, inclusion of infor- mation system in domain contents, presence of special information systems (DNS servers) containing data on domain names allocated in domain and carries out the function of domain name space organization [11]. Registration data of domain include: domain name (domain), registry identifier (registrar), full name of the physical person (person), contact address of the physical person (address), domain administrator identifier (admin- o), organization identifier for administrative communica- tion (admin-c), title of organization (organization), do- main registration time (created), domain free date (free- date), telephone numbers with international codes (phone), e-mail address (e-mail), list of DNS servers supporting domain (nserver), domain type (type), information source (source), domain registration payment time (paid-till) [12]. Filling of warehouses, as a rule, is carried out by in- formation from several data sources. Development or consequential support of qualitative information storage and processing systems is a complex problem. Human factor and partial absence of control at submittal lead to occurrence of distortions in data. Misprints and omis- sions are present almost in all details of saved objects, as well as in identification sets. During entrance stage of information into the data base, human factor is the main reason for occurrence of distortions. Damerau demon- strated that 95% of errors during typing by a person are transpositions [10]. Clearing of domain name registration data is carried out in the works and domain names registration data identification method is developed based on decision tree apparatus application. Decision trees are selected as the main algorithmic approach for construction of effective data integration system. Upon development of DW, very little attention is paid to cleaning of incoming information. 4.1. Algorithm Choice There are different kinds of errors. There are also er- rors characteristics to a certain subject field or task. Er- rors, which do not depend of task: contrariety of infor- mation; data omissions, anomalous values; noise; data entry errors etc. There are different kinds of solutions for each of these problems. Data omissions are a very seri- ous problem for majority of DW. Generalized string matching task which includes detec- tion of substrings of text strings is also called fuzzy string matching task. This task is important for cases where errors are taken into account. Review of fuzzy comparison algorithms is provided in work [13-17]. Fuzzy search algorithms (also known as fuzzy string search) are the foundation of spell check and complete search systems such as Google or Yandex. There are many methods of analysis [18-25] and fuzzy (inexact) string matching [26-29]. The most popular of these fuzzy string matching methods are the methods of calculation of editing spacing [30-32]. Generally, metrics numeri- cally calculating the value of transformation of one line to another is considered as spacing editing. There are different several operations, each of which can have a value of its own: character stuffing, deleting, replacement and transposition of proximate symbols. There are dif- Due to lack of information, as a matter of actual prac- tice, application of DW is realized poorly or with sig- nificant limitations. Quantity of errors during data input is excessive, for example misprints, deliberate data dis- tortions, inconsistence of formats, excluding typical er- rors related to specifics of data input application opera- tion. 2. Relevance Apparently, larger the volume of warehouse, the better it is considered. This is an erroneous practice and the best way to turn the data warehouse into a disposal dump. It is necessary to clean data. Information is heterogeneous and is collected from different sources. During construction of data warehouse, problems oc- cur related to misprints and omission of data. As registra- tion data of domain mainly consists of categorical data, it was decided to apply fuzzy search algorithms for clean- ing of these data. Following tasks are formulated for research purposes: Following tasks are formulated for research purposes: Exactly presence of point sets of information collec- tion makes the clearing process especially relevant. Gen- erally speaking, errors are always committed, and it is impossible to completely dispose of them. Possibly, sometimes it is more reasonable to accept them, rather than spend money and time to get rid of them. But, in general cases, it is necessary to aim to reduce the amount of errors to an acceptable level so ever. 1) Processing of domain name registration data, data clearing using Damerau-Levenstain algorithm; g g g 2) Identification of domain name registration data us- ing decision tree construction. Copyright © 2013 SciRes. 4.2. Symbol Fields for Registration Data Recording  Comparatively large distances are made upon re- arrangement of words or parts of words; As a rule, symbol fields consist of a string which con- tains one or several words, divided in gaps and punctua- tion marks. Nonstandard phrases are used in fields. Data is entered manually by an operator, often in distorted condition. In this regard, punctuation marks such as “.”, “,”, “:”, “ ” ”, “-” etc. that do not carry a functional sig- nificance, are replaced by “blank” sign.  Distances among completely different short words become small, while distance between very similar long words become significant. As a result of analysis of different DNS data bases, it is known that, contents of records of same fields of registration data of domain names can be expressed in forms different (not identical) in content. The reason for occurrence of difference of field value might be lack of information, misprints, use of abbreviations, duplication of records etc. Name of physical person (person), contact address of physical person (address), domain administrator identi- fier (admin-o), organization identifier for administrative communication (admin-c), organization title (organiza- tion) are the key fields upon identification of registration data. Upon entry of domain name registration data in DW, abbreviations, misprints, omissions, double recordings and other distortions are encountered. In order to increase the quality of input registration data such as “registrar”, person”, “address”, “organization”, “admin-о” etc, pre- vention of errors and inconsistencies of duplications in records is required. For example, in names of countries (cities) misprints can be as Kanata (Canada), Russian (Russia), Frankfrut Am Main (Frankfurt Am Main) etc; organization identifier registration data (registrar) “MONIKER ONLINE SERVICES, INC.” can be identi- fied as “MONIKER, INC.”, “MONIKER”, “MONIKERS ONLINE SERVICES”. In this case, words included in phrases must be processed separately. Despite difference of these strings, it is clear that, all of these titles stand for the same registrar. But let’s also note that, upon com- paring strings “MONIKER ONLINE SERVICES, INC.” and “MONIKER, INC.” using Levenstein metrics, we Components of these fields can be present in random order, which is a difficult task for automatic processing of such information. 3. Objective Domain is a domain namespace field and is characterized ICA R. ALGULIEV, R. GASIMOVA 229 ferent fuzzy string matching algorithms, which are based on different editing distances. Hamming distance—is a number of positions, in which corresponding symbols of two words of the equal length are different [33]. In more general cases, Hamming distance is applied to lines of same length of any q alphabets and serves as the differ- ence metrics of objects of equal dimensions. Hammings distance is usually used in bio-informatics and genomics. receive a larger value for editing distance. In cases where it is necessary to process the words contained in phrases separately from each other and/or apply algorithms that can compare similar lines for equivalence (for example, full entrance of one line into another as subsequence) and detect them as “similar”. In reviewed paper, let’s use Damerau-Levenstein dis- tance—difference measure of two strings of symbols, defined as minimal quantity of insertion, deletion, re- placement and transposition (rearrangement of two pro- ximate symbols) operations necessary for transfer of one string to another. It is the modification of Levenstein distance, and differs from it by addition of transposition operation. If matching of two strings of different lengths is al- lowed, then as a rule, insertion and deletion are also re- quired. If they are given the same weight as replacement, minimal general value of transformation will be equal to one of the metrics proposed by Levenstein [34]. As a result, a more general task for a voluntary alphabet was associated with his name. Gasfield made a significant contribution in study of this issue [35]. Editing distance determined in such way can be calcu- lated using dynamic programming method [36]. Also, there is an algorithms for this, requiring O(MN) opera- tions, where M and N—are lengths of compared lines, and it is required to calculate MN of elements of so called dynamic programming matrix in order to find the distance value. Levenstein distance and its generalization is actively applied for correction of errors in words (in search sys- tems, data bases, during text input, during automatic de- tection of scanned text or speech), for comparison of text files, in bio-informatics for comparison of genes, chro- mosomes and proteins. From application point of view, determination of distance between words or text fields according to Levenstein has following disadvantages: Copyright © 2013 SciRes. 90292-660, US. 90292-660, US. Difference measure of attributes’ values, for example such as contact address of a person will be calculated using Damerau-Levenstein distance Decision tree will be generated following way: for ex- act identification of the objects, we will use fields univocally identifying the object. Domain registration data will be identified by following attributes: registrar, person, address, admin-o, admin-c, organization, created, updated, free-date, phone, e-mail, nserver, type, source. Intermediate nodes of the tree correspond to these attrib- utes, and arches—to possible alternative comparison values of these attributes “+” (same), “±” (similar), and “–“ (different). Tree leaves are indicated as one of three classes as “=” (compared objects are identical), “≠” (ob- jects are different), “?” (unknown). The example of the tree for identification of registration data domain names is provided on Figure 1. 4.3. Decision Tree Decision trees is the most comfortable decision making method for record (object) identification, for their de- monstrativeness while use, minimal calculation resources and simplicity of realization. Decision tree—is one the methods of automatic analysis of vast amounts of data. Decision trees—is a method of rule presentation in hier- archic, consecutive structure, where each object corre- sponds to single knot that gives decision. Under rule, we understand a logical construction, presented in “if…then” form. Main advantages of decision trees are generation of rules in fields where experts formalize their knowledge with difficulty; extraction of rules in natural language; intuitively understandable classification model; high forecast precision, comparable to other methods (statis- tics, neural networks); construction of non-parametric models [37]. Following are the most significant among attributes: registration identifier (registrar), full name of the physi- cal person (person) contact address of the physical per- son (address), domain administrator identifier (admin-o), organization identifier for administrative communication (admin-c), title of organization (organization), list of DNS servers supporting domain (nserver). Suppose that we are given any educating set of T, con- taining objects, each of which is characterized by m at- tributes, while one of them points at affiliation of the object o a certain class. Construction idea of decision tree from T set, first expresses by Hunt, is demonstrated in accordance with R. Queenlan [38]. Less significant, but as informative attributes are: do- main registration time (created), domain update time (updated), domain free date (free-date), telephone num- bers with international codes (phone), e-mail addresses (e-mail), domain type (type), domain registration pay- ment time (paid-till), information source (source) etc. Let’s label classes (values of class marks) through {C1, С2,···, Ck}, then there are 3 situations: Decision tree is formed based on the knowledge of experts of the subject field. Depth of the tree is selected based upon objects necessary for precise identification.  Set of T contains one or more examples related to one class Ck. Then decision tress for T—is a list, deter- mining the Ск class. Copyright © 2013 SciRes. 4.2. Symbol Fields for Registration Data Recording In order to implement comparison of two lines containing such information, it is necessary to dismember each field to its contents, then compare only those which have identical meaning, for example in the address, compare the name of the city to the name of the city, name of the street to the name of the street. For content analysis, we enter so-called, samples. For exam- ple, sample address template is a combination of address components: [apartment], [block], [building], [street], [city], [dis- trict], [index], [country]. For example, for domain—azerbaijan.info, sample ad- dress template is important: , , 4676, Admiralty Way, Marina del Rey, California, Copyright © 2013 SciRes. ICA R. ALGULIEV, R. GASIMOVA 230 etc [39,40]. But following two are the most widespread and popular: CART (Classification and Regression Tree) and С4.5. 4.4. Software Implementation Distribution platforms of free and open software is spread under LGPL license [41-43]. implemented in Python language which is used for solu- tion of wide range of tasks of applied programming [44-47]. Same algorithm is presented in Python program on Figure 3. Conducted experiments with above described decision tree gave the identical positive results in DRRacket and Pythin programs. 4.4. Software Implementation  Set of T does not contain any examples, i.e. it is an empty set. Then again, the list and the class associ- ated with the list are selected from another set differ- ent from T, let’s say from sets associated with the parent; Software implementation of identification algorihms of registration data of domain names is implemented on person organization ? ≠ ≠ = = = + – ± + ± + – – ± – address +‚± e-mail registrar phone – ? – ? ± + ? ? + ± ? Figure. 1. Decision tree for identification of registration data of domain names.  The Set of T contains examples affiliated with differ- ent classes. In this case it is necessary to divide Set of T to some subsets. For this purpose, we choose one of the attributes that has two or more different values O1, О2,···, On. T is divided into subsets of T1, Т2,···Тn, where each subset Тi; contains all examples that have a value of Oi for selected attribute. This procedure will be recursively continued until the final set will consist of examples related to the same class. Above described procedure is the basis of many mod- ern construction algorithms of decision trees. Obviously, upon using given methodology, construction of decision tree will be implemented from top downward. Currently, there is a significant number of algorithms, realizing de- cision trees CART, C4.5, Newld, ITrule, CHAID, CN2 Figure. 1. Decision tree for identification of registration data of domain names. Copyright © 2013 SciRes. ICA R. ALGULIEV, R. GASIMOVA 231 DRRacket language. Racket (former PLT Scheme) is the multi-paradigm programming language in Lisp/Scheme family, which also serves as a platform for creation of languages, design and realization. Programming langua- ge is known due to its extensive macro system, which allows creating built-in and subject-oriented languages, language constructions, such as classes or modules, as well as separate Racket dialects with different semantics. Distribution platforms of free and open software is spread under LGPL license [41-43]. DRRacket language. Racket (former PLT Scheme) is the multi-paradigm programming language in Lisp/Scheme family, which also serves as a platform for creation of languages, design and realization. Programming langua- ge is known due to its extensive macro system, which allows creating built-in and subject-oriented languages, language constructions, such as classes or modules, as well as separate Racket dialects with different semantics. 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We use Racket, in order to demonstrate that without crochets and other unused symbols, it is easy to under- stand complex program codes in programming language. Despite the fact that Racket language is simple and easy for perception, it is quite difficult to find errors commit- ted in the program. a fraction of decision algorithm for identification of registration data in DRRacket language is provided in Figure 2. DW provides high speed of data reception, ability to receive and compare, as well as consistency, complete- ness and authenticity of data. After withdrawal from the source, data are loaded into the warehouse, in order to implement cleaning and simultaneously provide com- prehensive support of data cleaning [48]. For comparison of results, the identical algorithm is re 2. Decision algorithm for identification of registration data of domain names in DRRacket progra Figure 2. Decision algorithm for identification of registration data of domain names in DRRacket program. e 2. Decision algorithm for identification of registration data of domain names in DRRacket program. Figure 2. Decision algorithm for identification of registration data of domain names in DRRac ICA Copyright © 2013 SciRes. 232 R. ALGULIEV, R. GASIMOVA (а) (b) 3. (a, b) Decision algorithm for identification of registration data of domain names in Python (а) (b) Figure 3. (a, b) Decision algorithm for identification of registration data of domain names in Python p (b) 3. (a, b) Decision algorithm for identification of registration data of domain names in Python program. Copyright © 2013 SciRes. ICA R. ALGULIEV, R. GASIMOVA 233 Approximate String Matching,” Journal of Algorithms, Vol. 10, No. 2, 1989, pp. 157-169. doi:10.1016/0196-6774(89)90010-2 Domain names registration data category identification was reviewed in this work. Abbreviations, misprints, omissions, conscious data corruption, record duplicates etc which were allowed on information collection stage are considered as errors. 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https://openalex.org/W3138716284	http://real.mtak.hu/130762/1/paper.pdf	English	null	Constructing a universe for the setoid model	Lecture notes in computer science	2,021	cc-by	11,056	∗Supported by USAF grant FA9550-16-1-0029. § Supported by USAF grant FA9550-16-1-0029 and Swedish Research Council grant 2019-03765. † Supported by ERC Starting Grant CoqHoTT 637339. ‡ ∗Supported by USAF grant FA9550-16-1-0029. † Supported by ERC Starting Grant CoqHoTT 637339. ‡ Supported by the Bolyai Fellowship of the Hungarian Academy of Sciences (BO/00659/19/3) and by the “Application Domain Speciﬁc Highly Reliable IT Solu- tions” project that has been implemented with support from the National Research, Development and Innovation Fund of Hungary, ﬁnanced under the Thematic Excel- lence Programme TKP2020-NKA-06 funding scheme. § Supported by USAF grant FA9550-16-1-0029 and Swedish Research Council grant 2019-03765. 1 Introduction Intuitionistic type theory is a formal system designed by Per Martin-L¨of to be a full-ﬂedged foundation in which to develop constructive mathematics [23,24]. A central aspect of type theory is the coexistence of two notions of equality. On the one hand deﬁnitional equality, the computational equality that is built into the formalism. On the other hand “propositional” equality, the internal notion of equality that is actually used to state and prove equational theorems within the system. The precise balance between these two notions is at the center of type theory research; however, it is generally understood that to properly sup- port formalization of mathematics, one should aim for a notion of propositional equality that is as extensional as possible. Two extensionality principles seem particularly desirable, since they arguably constitute the bare minimum for type theory to be comparable to set theory as a foundational system for set-level mathematics, in terms of power and ergonomics. One is function extensionality (or funext), according to which functions are equal if point-wise equal. Another is propositional extensionality (or propext), that equates all propositions that are logically equivalent. Type theory with equality reﬂection, also known as extensional type theory (ETT) does support extensional reasoning to some degree, but unfortunately equality reﬂection makes the problem of type-checking ETT terms computa- tionally unfeasible: it is undecidable. On the other hand, intensional type theory (ITT) has nice computational properties like decidable type checking that can make it more suitable for com- puter implementation, but as usually deﬁned (for example, in [23]) it severely lacks extensionality. It is known from model constructions that extensional prin- ciples like funext are consistent with ITT. Moreover, ITT extended with the principle of uniqueness of identity proofs (UIP) and funext is known to be as powerful as ETT [19]. We could recover the expressive power of ETT by adding these principles to ITT as axioms, however destroying some computational prop- erties like canonicity. What we would like instead is a formulation of ITT that supports exten- sionality, while retaining its convenient computational behaviour. Unfortunately, canonicity for Martin-L¨of’s inductively deﬁned identity type says that if two terms are propositionally equal in the empty context, then they are also deﬁ- nitionally equal. This rules out function extensionality. The ﬁrst step towards a solution is to give up the idea of propositional equality as a single inductive deﬁnition given generically for arbitrary types. Constructing a universe for the setoid model Sattler and F. Sestini 2 Constructing a universe for the setoid model Thorsten Altenkirch1 ∗ B, Simon Boulier2†, Ambrus Kaposi3 ‡, Christian Sattler4§, and Filippo Sestini1 1 School of Computer Science, University of Nottingham, Nottingham, UK {psztxa,psxfs5}@nottingham.ac.uk 2 Inria, Nantes, France simon.boulier@inria.fr 3 E¨otv¨os Lor´and University, Budapest, Hungary akaposi@inf.elte.hu 4 Chalmers University of Technology, Gothenburg, Sweden sattler@chalmers.se 1 School of Computer Science, University of Nottingham, Nottingham, UK {psztxa,psxfs5}@nottingham.ac.uk 2 Inria, Nantes, France simon.boulier@inria.fr 3 E¨otv¨os Lor´and University, Budapest, Hungary akaposi@inf.elte.hu 4 Chalmers University of Technology, Gothenburg, Sweden sattler@chalmers.se Abstract. The setoid model is a model of intensional type theory that validates certain extensionality principles, like function extensionality and propositional extensionality, the latter being a limited form of uni- valence that equates logically equivalent propositions. The appeal of this model construction is that it can be constructed in a small, intensional, type theoretic metatheory, therefore giving a method to boostrap ex- tensionality. The setoid model has been recently adapted into a formal system, namely Setoid Type Theory (SeTT). SeTT is an extension of intensional Martin-L¨of type theory with constructs that give full access to the extensionality principles that hold in the setoid model. Although already a rich theory as currently deﬁned, SeTT currently lacks a way to internalize the notion of type beyond propositions, hence we want to extend SeTT with a universe of setoids. To this aim, we present the construction of a (non-univalent) universe of setoids within the setoid model, ﬁrst as an inductive-recursive deﬁnition, which is then translated to an inductive-inductive deﬁnition and ﬁnally to an inductive family. These translations from more powerful deﬁnition schemas to simpler ones ensure that our construction can still be deﬁned in a relatively small metatheory which includes a proof-irrelevant identity type with a strong transport rule. Keywords: type theory · function extensionality · univalence · setoid model · induction-recursion · induction-induction † Supported by ERC Starting Grant CoqHoTT 637339. ‡ Supported by the Bolyai Fellowship of the Hungarian Academy of Sciences (BO/00659/19/3) and by the “Application Domain Speciﬁc Highly Reliable IT Solu- tions” project that has been implemented with support from the National Research, Development and Innovation Fund of Hungary, ﬁnanced under the Thematic Excel- lence Programme TKP2020-NKA-06 funding scheme. § Supported by USAF grant FA9550-16-1-0029 and Swedish Research Council grant 2019-03765. § Supported by USAF grant FA9550-16-1-0029 and Swedish Research Council grant 2019-03765. T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. Sestini T. Altenkirch, S. Boulier, A. Kaposi, C. 1 Introduction Instead, equality should be spe- ciﬁc to each type former in the type theory, or in other words, every type former should be introduced alongside an explanation of what counts as equality for its elements. This idea of pairing types together with their own equality relation goes back to the notion of setoid or Bishop set. Setoids provide a quite natural and useful semantic domain in which to interpret type theory. The ﬁrst setoid model was constructed to justify function extensionality without relying on funext in the metatheory [18]. Moreover, it was shown by Altenkirch [4] that if the model Constructing a universe for the setoid model 3 construction is carried out in a type theoretic metatheory with a universe of strict (deﬁnitionally proof-irrelevant) propositions, it is possible to deﬁne a univalent universe of propositions satisfying propositional extensionality. The setoid model thus satisﬁes all the extensionality principles that we would like to have in a set- level type theory 5 . The question is whether there exists a version of intensional type theory that supports setoid reasoning, and hence the forms of extensionality enabled by it. This question was revisited and answered in Altenkirch et al. [5]. In this paper, the authors deﬁne Setoid Type Theory (SeTT), an extension of inten- sional Martin-L¨of type theory with constructs for setoid reasoning, where funext and propext hold by deﬁnition. SeTT is based on the strict setoid model of Altenkirch6, which makes it possible to show consistency via a syntactic trans- lation. This is in contrast with other type theories based on the setoid model, like Observational Type Theory [9] and XTT [28], which instead rely on ETT for their justiﬁcation. A major property of SeTT is thus to illustrate how to bootstrap extensionality, by translation into a small intensional core. SeTT as deﬁned in [5] is already a rich theory, but its introspection capabili- ties are currently lacking, as its universes are limited to propositions. We would like to internalise the notion of type in SeTT, thus extending the theory with a universe of setoids. This goal brings up several questions, one of which has to do with the notion of equality with which the universe should come equipped: the universe of setoids is itself a setoid (as any type is) so it certainly cannot be uni- valent, since setoids lack the necessary structure. 6 A strict model is one where every equation holds deﬁnitionally. 5 In the sense of HoTT we mean a type theory limited to h-sets. 6 1 Introduction Another issue is the way such universe can be justiﬁed by the setoid model, and in particular what principles are needed in the metatheory to do so. Contributions This paper documents our work towards the construction of a universe of setoids inside the setoid model, and tries to answer these and other questions related to the design and implementation of this construction. Our main contribution is the construction of the universe in the model; this is given in steps, ﬁrst as an inductive-recursive deﬁnition, which is then translated to an inductive-inductive deﬁnition, and subsequently to an inductive type. As a consequence, we show that we only need to assume indexed W-types and proof- irrelevant identity types in the metatheory (along with some obligatory basic tools like Σ and Π types) to construct the universe. The universe constructions presented in this paper are, to our knowledge, the ﬁrst examples of two kinds of data type reductions in an intensional metatheory: the ﬁrst involving an inductive-recursive type which includes strict propositions, and the second involving an inﬁnitary inductive-inductive type. Finally, the mathematical contents of this paper have been formalized in the proof-assistant Agda (see [10]). Structure of the paper We begin by describing the metatheory that we will use throughout the paper, in Section 2. In Section 3, after brieﬂy recalling cate- T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. Sestini 4 gories with families as an abstract notion of models of type theory, we outline Altenkirch’s setoid model as given in [5]. We then brieﬂy discuss the rules of Setoid Type Theory in Section 3.2. In Section 4 we discuss the setoid model and various design choices related to it. We then recall inductive-recursive universes, and the way they can be equiv- alently deﬁned as a plain inductive deﬁnition, in Section 4.1. We then provide, in Section 4.2, a ﬁrst complete deﬁnition of the setoid universe using a special form of induction-recursion. This form of induction-recursion is not known to be reducible to plain inductive types. Then we describe an alternative deﬁnition of the universe in Section 4.3, that does not rely on induction-recursion but in- stead on inﬁnitary induction-induction. This inductive-inductive encoding of the universe is obtained from the inductive-recursive one, inspired by the method of Section 4.1. 7 To quote one of the referees: the fact that the interval is a pretype is but the easiest part of the story. 1 Introduction We end the series of universe constructions with Section 4.4, where we outline a purely inductive deﬁnition of the setoid universe, obtained from the inductive-inductive one. 1.1 Related work The setoid model was ﬁrst described in [18] in order to add extensionality princi- ples to Type Theory such as function extensionality and propositional extension- ality. A strict variant of the setoid model was given in [4] using a deﬁnitionally proof-irrelevant universe of propositions. Recently, support for such a universe was added to the proof-assistants Agda and Coq [17], allowing a full formal- ization of Altenkirch’s setoid model. Setoid Type Theory (SeTT) is a recently developed formal system derived from this model construction [5]. Observational Type Theory (OTT) [9] is a syntax for the setoid model diﬀering from SeTT in the use of a diﬀerent notion of heterogeneous equality. Moreover, the consis- tency proof for OTT relies on Extensional Type Theory, whereas for SeTT it is obtained via a syntactic translation. XTT [28] is a cubical variant of OTT where the equality type is deﬁned using an interval pretype 7 . XTT’s universes support universe induction, whereas it is left open whether the construction presented here supports this principle. Palmgren and Wilander [27] construct a setoid universe using a translation into constructive set theory. Palmgren [26] constructs an encoding of ETT in ITT through Aczel’s encoding of set theory in type theory [3]. He uses type theory as a language for his formalisation but his construction is set-theoretic in nature. Setoids are utilized to encode sets as arbitrarily branching well-founded trees quotiented by bisimulation. His notion of family of setoids does not use strict propositions and it has a weaker form of proof irrelevance which seems to be not enough to obtain a model of SeTT. The principle of propositional extensionality in the setoid model is an in- stance of Voevodsky’s univalence axiom [29]. The cubical set model is a con- structive model justifying this axiom [11]. A type theory extracted from this model is Cubical Type Theory [13]. The relationship between the cubical set Constructing a universe for the setoid model 5 model and cubical type theory is similar to that between the setoid model and SeTT. Compared to cubical type theories, SeTT has the advantage that the equality type satisﬁes more deﬁnitional equalities. For instance, whereas in cu- bical type theory equality of functions is isomorphic to pointwise equality, in SeTT the isomorphism is replaced by a deﬁnitional equality. 1.1 Related work SeTT is also a syn- tactically straightforward extension of Martin-L¨of Type Theory, that does not require exotic objects like the interval pretype. In turn, the obvious advantage of cubical type theory is that it is not limited to setoids. An exceptional aspect of the metatheory used in this paper is the presence of a proof-irrelevant identity type with a strong transport rule allowing to elim- inate into arbitrary types. In [1], Abel gives a proof of normalization for the Logical Framework extended with a similar proof-irrelevant equality type. Abel and Coquand show in [2] that the combination of impredicativity with a strong transport rule results in terms that fail to normalize but this is irrelevant in our setting. 2 MLTTProp This section describes MLTTProp, our ambient metatheory. We employ Agda notation to write down MLTTProp terms throughout the paper. One of the main appeals of Altenkirch’s setoid model is that it can justify several useful extensionality principles while being deﬁned in a small intensional metatheory. We tried to stay true to this idea when ﬁguring out the necessary metatheoretical tools for the universe construction in this paper. In particular, we wanted to avoid having to assume strong deﬁnition schemas that go beyond inductive families. MLTTProp is thus an intensional type theory in the style of Martin-L¨of type theory. We have sorts Typei of types and Propi of strict propositions for i ∈{0, 1}. Here, i = 0 means “small” (and we will omit the subscript) and i = 1 means “large”. We have implicit lifting from i = 0 to i = 1, but do not assume type formers are preserved. Type1 has universes for Type and Prop. We do not distinguish notationally between universes and sorts. We continue to describe only the case i = 0; everything introduced has an analogue at level i = 1. Propositions lift to types via Lift : Prop →Type, with constructor lift : {P : Prop} →P →Lift P and destructor unlift : {P : Prop} →Lift P →P. p} { p} We have standard type formers Π, Σ, Bool, 0, 1 in Type. Σ-types are deﬁned negatively by pairing –, – and projections π1, π2. We have deﬁnitional η-rules for Π-, Σ-, 1-types. We also require indexed W-types, both in Type and Prop: W□: (S : I →Type) →((i : I) →S i →I →Type) →I →□where □∈{Type, Prop}. The elimination principle of WProp only allows deﬁning functions into elements of Prop. From WProp we can deﬁne propositional trun- cation ∥–∥: Type →Prop, with constructor \|–\| : {A : Type} →A →∥A∥ and eliminator elim∥–∥: {P : Prop} →(A →P) →∥A∥→P. ∥ ∥ { } ( ) ∥∥ In addition to type formers in Type, we will need the propositional versions of 0, 1, Π, and Σ. The latter three can be deﬁned from their Type counterparts T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. Sestini 6 via truncation. That is, given P : Prop and Q : P →Prop: via truncation. 2 MLTTProp That is, given P : Prop and Q : P →Prop: 1Prop :≡∥1∥ ΠProp P Q :≡∥Π (Lift P) (Lift ◦Q ◦unlift)∥ ΣProp P Q :≡∥Σ (Lift P) (Lift ◦Q ◦unlift)∥ We assume that we have 0Prop : Prop together with exfalsoProp : {A : Type} → 0Prop →A. p Finally, we will assume an identity type in the style of Martin-L¨of’s inductive identity type. The main diﬀerence is that our identity type is a Prop-valued relation. We have a transport combinator transp from which J is derivable. Id : {A : Type} →A →A →Prop reﬂ: {A : Type}(a : A) →Id a a Id : {A : Type} →A →A →Prop reﬂ: {A : Type}(a : A) →Id a a transp : {A : Type}(C : A →Type){a0 a1 : A} →Id a0 a1 →C a0 →C a1 eﬂ: {A : Type}(a : A) →Id a a { yp }( ) transp : {A : Type}(C : A →Type){a0 a1 : A} →Id a0 a1 →C a0 →C a1 with transp C {x} {x} e u ≡u. The transp combinator provides a strong elim- ination principle allowing to eliminate a strict proposition (the identity type) into arbitrary types. We only use this identity type in Section 4.4. For the rest of our constructions, the traditional Martin-L¨of’s identity type suﬃces. 2.1 Formalization A universe of strict propositions has been recently added to the Agda proof assis- tant [17], making most of MLTTProp a subset of Agda, with the exception of the proof-irrelevant identity type. Most of the universe constructions presented here have been formalized and proof-checked using Agda, with the proof-irrelevant identity type and the strong transport rule added via postulates and rewriting. The formalization can be found in [10]. For convenience, we slightly deviate from MLTTProp both in the paper and in the formalization, for instance by relying on pattern matching instead of elim- inators, and using primitive versions of Prop-valued Π and Σ types instead of deriving them from truncation. We operate under the assumption that every- thing can be equivalently carried out in MLTTProp, although we have not fully checked all the necessary details. Constructing a universe for the setoid model Constructing a universe for the setoid model Here we consider a similar model construction due to Altenkirch [4]. The peculiarity of this model is that it is presented in a type theoretic and intensional metatheory which includes a strict universe of propositions. The setoid model thus deﬁned validates function extensionality, a universe of propositions with propositional extensionality, and quotient types. Therefore, it provides a way to bootstrap and “explain” extensionality, since the model con- struction eﬀectively gives an implementation of various extensionality principles in terms of a small, completely intensional theory. 3 Setoid model By setoid model we mean a class of models of type theory where contexts/closed types are interpreted as setoids, i.e. sets with an equivalence relation, and de- pendent types are interpreted as dependent/indexed setoids. A setoid model was ﬁrst given for intensional type theory by M. Hofmann [18], in order to provide a semantics for extensionality principles such as function and propositional ex- tensionality. 7 3.1 Setoid model as a CwF The setoid model can be framed categorically as a category with families (CwF, [14]) with extra structure for the various type and term formers. The core struc- ture of a CwF can be given as the following signature: Con : Type Ty : (Γ : Con) →Type Sub : (Γ ∆: Con) →Type Tm : (Γ : Con) →Ty Γ →Type In our presentation of the setoid model, contexts are given by setoids, that is, types together with an equivalence relation. A key point of the model is that the equivalence relation is valued in Prop and is thus deﬁnitionally proof irrelevant. Γ : Con \|Γ\| : Type Γ ∼: \|Γ\| →\|Γ\| →Prop reﬂΓ : (γ : \|Γ\|) →Γ ∼γ γ sym Γ : ∀{γ0 γ1} →Γ ∼γ0 γ1 →Γ ∼γ1 γ0 trans Γ : ∀{γ0 γ1 γ2} →Γ ∼γ0 γ1 →Γ ∼γ1 γ2 →Γ ∼γ0 γ2 Γ : Con \|Γ\| : Type Γ ∼: \|Γ\| →\|Γ\| →Prop reﬂΓ : (γ : \|Γ\|) →Γ ∼γ γ sym Γ : ∀{γ0 γ1} →Γ ∼γ0 γ1 →Γ ∼γ1 γ0 trans Γ : ∀{γ0 γ1 γ2} →Γ ∼γ0 γ1 →Γ ∼γ1 γ2 →Γ ∼γ0 γ2 \|Γ\| : Type Γ ∼: \|Γ\| →\|Γ\| →Prop reﬂΓ : (γ : \|Γ\|) →Γ ∼γ γ sym Γ : ∀{γ0 γ1} →Γ ∼γ0 γ1 →Γ ∼γ1 γ0 trans Γ : ∀{γ0 γ1 γ2} →Γ ∼γ0 γ1 →Γ ∼γ1 γ2 →Γ ∼γ0 γ2 sym Γ : ∀{γ0 γ1} →Γ ∼γ0 γ1 →Γ ∼γ1 γ0 Types in a context Γ are given by displayed setoids over Γ with a ﬁbra- tion condition given by coe, coh. In the following, we sometimes omit implicit quantiﬁcations such as the ∀{γ0 γ1} in the type of sym Γ. 8 Semantically, this translation corresponds to a model construction, in particular a functor from the category of models of the target theory to the category of models of what will be Setoid Type Theory. Since the setoid translation is structural in the context component, we can work with models in the style of categories with families rather than contextual categories. A : Ty Γ \|A\| : \|Γ\| →Type A∼: {γ0 γ1 : \|Γ\|} →Γ ∼γ0 γ1 →\|A\|γ0 →\|A\|γ1 →Prop reﬂ* : {γ : \|Γ\|}(a : \|A\|γ) →A∼(reﬂΓ γ) a a sym* : ∀{γ0 γ1 a0 a1}{p : Γ ∼γ0 γ1} →A∼p a0 a1 →A∼(sym Γ p) a1 a0 trans* : A∼p0 a0 a1 →A∼p1 a1 a2 →A∼(trans Γ p0 p1) a0 a2 coe : Γ ∼γ0 γ1 →\|A\|γ0 →\|A\|γ1 coh : (p : Γ ∼γ0 γ1)(a : \|A\|γ0) →A∼p a (coe A p a) \|A\| : \|Γ\| →Type A∼: {γ0 γ1 : \|Γ\|} →Γ ∼γ0 γ1 →\|A\|γ0 →\|A\|γ1 →Prop reﬂ* : {γ : \|Γ\|}(a : \|A\|γ) →A∼(reﬂΓ γ) a a sym* : ∀{γ0 γ1 a0 a1}{p : Γ ∼γ0 γ1} →A∼p a0 a1 →A∼(sym Γ p) a1 a0 trans* : A∼p0 a0 a1 →A∼p1 a1 a2 →A∼(trans Γ p0 p1) a0 a2 coe : Γ ∼γ0 γ1 →\|A\|γ0 →\|A\|γ1 coh : (p : Γ ∼γ0 γ1)(a : \|A\|γ0) →A∼p a (coe A p a) This deﬁnition of types in the setoid model is diﬀerent from the one in [4], but it is equivalent to it [12, Section 1.6.1]. The main diﬀerence here is in the use of a heterogeneous equivalence relation A∼in the deﬁnition of types. 8 T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. Sestini Substitutions are interpreted as functors between the corresponding setoids, whereas terms of type A in context Γ are sections of the type seen as a se- toid ﬁbration Γ.A →Γ. Note that we only need to include components for the functorial action on objects and morphisms, since the functor laws follow from proof-irrelevance in the metatheory, and thus hold deﬁnitionally. σ : Sub Γ ∆ \|σ\| : \|Γ\| →\|∆\| σ∼: Γ ∼ρ0 ρ1 →∆∼(\|σ\|ρ0) (\|σ\|ρ1) t : Tm Γ A \|t\| : (γ : \|Γ\|) →\|A\| γ t∼: (p : Γ ∼γ0 γ1) →A∼p (\|t\|γ0) (\|t\|γ1) σ : Sub Γ ∆ \|σ\| : \|Γ\| →\|∆\| σ∼: Γ ∼ρ0 ρ1 →∆∼(\|σ\|ρ0) (\|σ\|ρ1) t : Tm Γ A \|t\| : (γ : \|Γ\|) →\|A\| γ t∼: (p : Γ ∼γ0 γ1) →A∼p (\|t\|γ0) (\|t\|γ1) We can show that the setoid model validates the usual basic type formers (Π, Σ, etc.), function extensionality and a universe of strict propositions with propositional extensionality [4]. Note that we do not need identity types or in- ductive types (W-types) for this. 3.2 Setoid Type Theory The setoid model presented in the previous section is strict, that is, every equa- tion of a CwF holds by deﬁnition in the semantics. One advantage of strict models is that they can be turned into syntactic translations, in which syntactic objects of the source theory are interpreted as their counterparts in another tar- get theory. In the case of the setoid model, this gives rise to a setoid translation, where source contexts are interpreted as target contexts together with a target type representing the equivalence relation, and so on.8 A setoid translation is used in [5] to justify Setoid Type Theory (SeTT), an extension of Martin-L¨of type theory (+ Prop) with equality types for contexts and dependent types that reﬂect the setoid equality of the model. We recall the rules of SeTT that extend regular MLTT below, but with a variation: whereas the equality types in [5] are stated as elements of SeTT’s internal universe of propositions, here we state the context equalities as elements of the external, metatheoretic universe Prop. This generalises the notion of model of SeTT thus making it easier to construct models. Equality on types is deﬁned as before in [5]. [ ] We have a universe of propositions Prop deﬁned as follows: Γ : Con Prop : Ty Γ P : Tm Γ Prop P : Ty Γ u : Tm Γ P v : Tm Γ P u ≡v Γ : Con Prop : Ty Γ P : Tm Γ Prop P : Ty Γ u : Tm Γ P v : Tm Γ P u ≡v Equality type constructors for contexts and dependent types internalize the idea that every context and type comes equipped with a setoid equivalence rela- tion. Note that Prop is the universe of the metatheory while Prop is the internal Constructing a universe for the setoid model 9 one. As in the model, equality for dependent types is indexed over context equal- ity. one. As in the model, equality for dependent types is indexed over context equal- ity. Γ : Con ρ0, ρ1 : Sub ∆Γ Γ ∼ρ0 ρ1 : Prop A : Ty Γ ρ01 : Γ ∼ρ0 ρ1 a0 : Tm ∆A[ρ0] a1 : Tm ∆A[ρ1] A∼ρ01 a0 a1 : Tm ∆Prop We have rules witnessing that these are indeed equivalence relations. 3.2 Setoid Type Theory We only recall reﬂexivity: ρ : Sub ∆Γ R ρ : Γ ∼ρ ρ A : Ty Γ ρ : Sub ∆Γ a : Tm ∆A[ρ] R a : Tm Γ A∼(R ρ) a a In addition, we also have rules representing the fact that every construction in SeTT respects setoid equality, so that we can transport along any such equality: A : Ty Γ ρ0, ρ1 : Sub ∆Γ p : Γ ∼ρ0 ρ1 a : Tm ∆A[ρ0] coeA p a : Tm ∆A[ρ1] cohA p a : Tm ∆A∼p a (coeA p a) Notably, equality types in SeTT compute deﬁnitionally on concrete type formers. In particular, they compute to their obvious intended meaning, so that an equality of pairs is a pair of equalities, an equality of functions is a map of equalities, and so on. From this, we get deﬁnitional versions of function and propositional extensionality. We can easily recover the usual Martin-L¨of identity type from setoid equality, with transport implemented via coercion. A : Ty Γ a0, a1 : Tm Γ A IdA a0 a1 :≡A∼(R Γ) a0 a1 : Tm Γ Prop P : Ty (Γ.A) p : Tm Γ (Id A a0 a1) t : Tm Γ P[a0] transp P p t :≡coe P (R id, p) t : Tm Γ P[a1] A : Ty Γ a0, a1 : Tm Γ A IdA a0 a1 :≡A∼(R Γ) a0 a1 : Tm Γ Prop P : Ty (Γ.A) p : Tm Γ (Id A a0 a1) t : Tm Γ P[a0] transp P p t :≡coe P (R id, p) t : Tm Γ P[a1] We can also derive Martin-L¨of’s J eliminator for this homogeneous identity type. The only caveat is that transp and the J eliminator do not compute deﬁ- nitionally on reﬂexivity. 4 Universe of setoids As pointed out in the introduction, SeTT is seriously limited by the lack of a universes internalizing the notion of setoid. Our goal is to extend SeTT with a universe of setoids; since SeTT is a direct syntactic reﬂection of the setoid model, this essentially amounts to showing that a universe of setoids with the necessary structure and equations can be constructed within the setoid model. This opens several questions and possible design choices. A ﬁrst fundamental consideration has to do with the very deﬁnition of the setoid universe: as any type in the setoid model, this universe must be a setoid T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. Sestini 10 and thus come equipped with an equivalence relation. However, unlike the uni- verse of propositions, a universe of setoids cannot be univalent, since this would force it to be a groupoid. The obvious choice is therefore to have a non-univalent universe, and instead deﬁne the universe’s relation so that it reﬂects a simple syntactic equality of codes rather than setoid equivalence. Another question has to do with the metatheoretic tools required to carry out the construction of the universe. In fact, one of the main aspects of the setoid model construction recalled in Section 3 and shown originally in [4] is that it can be carried out in a very small type theoretic metatheory, thus providing a way to reduce extensionality to a small intensional core. We would like to stay faithful to this ideal when constructing this setoid universe. A known and established method for deﬁning universes in type theory relies on induction-recursion (IR), a deﬁnition schema developed by Dybjer [15,16]. Inductive-recursive deﬁnitions can be found throughout the literature, from the already mentioned type theoretic universes, including the original formulation `a la Tarski by Martin-L¨of [24], to metamathematical tools like computability predicates. Although universe constructions in type theory—including our own setoid universe—are naturally presented as inductive-recursive deﬁnitions, they may not necessarily require a metatheory with induction-recursion. In fact, it is pos- sible to reduce some instances of induction-recursion to plain induction (more speciﬁcally, inductive families), including some universe deﬁnitions. We recall this reduction in Section 4.1. Other design choices on the setoid universe are less essential, but still require careful consideration. For instance, one question is whether the setoid universe should support universe induction, thus exposing the inductive structure of the codes. 4 Universe of setoids Such an elimination principle is known to be inconsistent with univalence, although this is not an issue in our case; nevertheless it is not immediately clear if the elimination principle can be justiﬁed by the semantics, that is, if our encod- ing of the setoid universe in the model allows to deﬁne such a universe eliminator. The question arises because our ﬁnal encoding of the setoid universe only sup- ports a weak form of elimination, for reasons that are explained in Section 4.4. Although not currently needed, a stronger eliminator might be necessary to jus- tify universe induction. This problem should not arise in the other encodings of the setoid universe (as given in Section 4.2 and Section 4.3). Another design choice has to do with how the setoid universe relates to the other universes. One could provide a code for Prop in the setoid universe. Moreover, the setoid universes could form a hierarchy, possibly cumulative. Yet another choice is whether to have two separate sorts, one for propositions and one for sets (with propositions convertible to sets) or a single sort of types (sets), with propositions given by elements of a universe of propositions, which is a (large) type. We have chosen to present the second option to ﬁt with the standard notion of (unisorted) CwF. However, this has downsides: to even talk about propositions, we need to have a notion of large types. The ﬁrst option is more symmetric: we can have parallel hierarchies for propositions and sets. Constructing a universe for the setoid model 11 4.1 Inductive-recursive universes An inductive-recursive universe is given by a type of codes U : Type, and a family El : U →Type that assigns, to each code corresponding to some type, the meta-theoretic type of its elements. The resulting deﬁnition is inductive- recursive because the inductive type of codes is deﬁned simultaneously with the recursive function El. An example is the following deﬁnition of a small universe with bool and Π. data U : Type bool : U pi : (A : U) →(El A →U) →U El : U →Type El bool :≡2 El (pi A B) :≡(a : El A) →El (B a) data U : Type bool : U pi : (A : U) →(El A →U) →U El : U →Type El bool :≡2 El (pi A B) :≡(a : El A) →El (B a) Induction-recursion is arguably a nice and natural way to deﬁne internal universes in type theory, however it is not always strictly required. We can translate basic instances of induction-recursion into inductive families using the equivalence of I-indexed families of types and types over I (that is, A : Type with A →I) [22]. ) [ ] In our case, we can encode U as an inductive type inU that carves out all types in Type that are in the image of El. In other words, inU is a predicate that holds for any type that would have been obtained via El in the inductive- recursive deﬁnition. As El is indexed by the type of codes, the deﬁnition of inU quite expectedly reﬂects the inductive structure of codes. data inU : Type →Type1 inBool : in-U 2 inPi : inU A →((a : A) →inU (B a)) →inU ((a : A) →(B a)) U and El can be given by U :≡Σ (A : Type) (in-U A) and El :≡π1. Note that this construction gives rise to a universe in Type1, rather than Type, since the deﬁnition of U quantiﬁes over all possible types in Type. Hence this kind of construction requires a metatheory with at least one universe. El : U →Type El : U →Type – ⊢– ∼El – : {a a′ : U} →a ∼U a′ →El a →El a′ →Prop Note that – ⊢– ∼El – is indexed over equality on the universe, because El is a displayed setoid over U, hence in particular it must respect the setoid equality of U. We also require data and proofs that make sure we get setoids out of El: reﬂEl : (A : U)(x : El A) →reﬂU A ⊢x ∼El x symEl : p ⊢x ∼El x′ →symU p ⊢x′ ∼El x transEl : p ⊢x ∼El x′ →q ⊢x′ ∼El x′′ →transU p q ⊢x ∼El x′′ coeEl : A ∼U B →El A →El B cohEl : (p : A ∼U A′) (x : El A) →p ⊢x ∼El coeEl p x We give an inductive deﬁnition of U, mutually with a recursive deﬁnition of the 4 functions – ∼U –, reﬂU, El and – ⊢– ∼El –. The other functions are then recursively deﬁned: reﬂEl alone, symU and symEl mutually, transU, transEl, coeEl and cohEl mutually. The whole construction is quite long, below we only show the more interesting deﬁnitions of U and El: data U : Type1 bool : U pi : (A : U)(B : El A →U) →({x x′ : El A} →reﬂU A ⊢x ∼El x′ →B x ∼U B x′) →U El bool :≡2 El (pi A B h) :≡ Σ (f : (a : El A) →El (B a)) (∀{x x′}(p : reﬂU A ⊢x ∼El x′) →h p ⊢f x ∼El f x′) El bool :≡2 El (pi A B h) :≡ Σ (f : (a : El A) →El (B a)) (∀{x x′}(p : reﬂU A ⊢x ∼El x′) →h p ⊢f x ∼El f x′) El bool :≡2 El (pi A B h) :≡ Σ (f : (a : El A) →El (B a)) (∀{x x′}(p : reﬂU A ⊢x ∼El x′) →h p ⊢f x ∼El f x′) Note that in the deﬁnition of U we require that the family B : El A →U be a setoid morphism, respecting the setoid equalities involved. This choice is crucial for the deﬁnition of El to go through, in particular since we eliminate the code for Π types into the setoid of functions that map equal elements to equal results. 4.2 Inductive-recursive setoid universe In this section we give a ﬁrst deﬁnition of the setoid universe, as a direct general- ization of the simple inductive-recursive deﬁnition just shown. We only consider a very small universe with bool type 2 and Π for simplicity; a more realistic uni- verse that includes more type formers can be found in the Agda formalization. To construct the universe of setoids in the setoid model, we ﬁrst of all need to deﬁne a type U : Ty Γ for every Γ : Con, and for every A : Tm Γ U a type El A : Ty Γ. Recalling Section 3, these are essentially record types made of several components. Since U is a closed type, it requires the same data of a setoid; in particular, we need a type of codes together with an equivalence relation reﬂecting equality of codes, in addition to proofs that these are indeed equivalence relations: reﬂU : (A : U) →A ∼U A data U : Type1 – ∼U – : U →U →Prop1 data U : Type1 – ∼U – : U →U →Prop1 data U : Type1 symU : A ∼U B →B ∼U A – ∼U – : U →U →Prop1 transU : A ∼U B →B ∼U C →A ∼U C transU : A ∼U B →B ∼U C →A ∼U C 12 T Alt ki h S B li A K i C S ttl d F S ti i 12 T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. Sestini El is given by a family of setoids indexed over the universe, that is, a way to assign to each code in the universe a carrier set and an equivalence relation. El : U →Type El : U →Type To state this mapping property we need to compare elements in diﬀerent types, coming from applying f to diﬀerent arguments x and x′. We know that x and x′ are equal, but to conclude B x ∼U B x′ we need to know that B respects setoid equality. This is exactly what we get from our deﬁnition of U. We can now give a full deﬁnition of the setoid universe, and of El A for any A : Tm Γ U: \|U\| :≡λ γ. U U∼:≡λ p x y. x ∼U y reﬂU :≡reﬂU . . . coe U :≡λ p a. a coh U :≡λ p. reﬂU \|El A\| :≡λ γ. El (\|A\| γ) (El A)∼:≡λ p x y. A∼p ⊢x ∼El y reﬂ(El A) :≡reﬂEl . . . coe (El A) :≡λ p. coeEl (A∼p) coh (El A) :≡λ p. cohEl (A∼p) coe U :≡λ p a. a coh U :≡λ p. reﬂU 13 Constructing a universe for the setoid model We can show that U is closed under Π types and booleans, and satisﬁes El (pi A B) ≡Π (El A) (El B) and El bool = Bool. The universe can be closed under more constructions if more codes are added to U. This gives a complete deﬁnition of a universe of setoids, which is, however, inductive-recursive. More- over, the kind of recursion involved in this deﬁnition is particularly complex, and not obviously reducible to well-understood notions of induction-recursion like the one described in [16]. In any case, we would like to avoid extending the metatheory with any form of induction-recursion in order to keep the metatheory as small and essential as possible. In the next section we transform our current inductive-recursive deﬁnition to one that does not use induction-recursion. The way this is done is inspired by the well-known trick to eliminate induction-recursion described in Section 4.1, but modiﬁed in a novel way to account for the presence of Prop-valued types. To our knowledge, this is the ﬁrst time this reduction method is applied to an inductive-recursive type of this kind. 4.3 Inductive-inductive setoid universe Since these types are mutually deﬁned, they form an instance of induction-induction, a schema that allows the deﬁnition of a type mutually with other types that contain the ﬁrst one in their signature [25].9 As in the universe example in Section 4.1, we now deﬁne U as a Σ type, and El as the corresponding ﬁrst projection. U : Type1 El : U →Type U :≡Σ (X : Type) (inU X) El :≡π1 El : U →Type El :≡π1 U : Type1 What is left now is to deﬁne the setoid equality relation on the universe, as well as the setoid equality relation on El A for any A in U. Two codes A, B in the universe U are equal when there exists a setoid equivalence relation on their respective sets El A and El B. Intuitively, since elements of a setoid are only ever compared to elements of the same setoid, this should only be possible if A and B are codes for the same setoid, that is, if A ∼U B. Existence and well-formedness of such relations is expressed via the type inU∼just deﬁned, hence we would expect A ∼U B to be deﬁned as follows: (A, a) ∼U (B, b) :≡Σ (R : A →B →Prop) (inU∼a b R) Unfortunately this deﬁnition only manages to capture the idea, but does not actually typecheck. In fact, – ∼U – should be a Prop1-valued relation, so A ∼U B should be a proposition. However, the Σ type shown above clearly is not, since it quantiﬁes over a type of relations, which is not a proposition. One possible solution is actually quite simple, and it just involves truncating the Σ type above to force it to be in Prop1. – ∼U – : U →U →Prop1 (A, a) ∼U (B, b) :≡∥Σ (R : A →B →Prop) (inU∼a b R)∥ We are now left to deﬁne the indexed equivalence relation on El: – ⊢– ∼El – : {A B : U} →A ∼U B →El A →El B →Prop p ⊢x ∼El y :≡? e now left to deﬁne the indexed equivalence relation on El: – ⊢– ∼El – : {A B : U} →A ∼U B →El A →El B →Prop p ⊢x ∼El y :≡? – ⊢– ∼El – : {A B : U} →A ∼U B →El A →El B →Prop p ⊢x ∼El y :≡? 4.3 Inductive-inductive setoid universe We will follow the method outlined in Section 4.1. In addition to inU for deﬁning U, we also introduce a family inU∼of binary relations between types in the universe, from which we then deﬁne – ∼U –. data inU : Type →Type1 bool : inU 2 π : inU∼a a A∼→(∀{x0 x1}(x01 : A∼x0 x1) →inU∼(b x0) (b x1) (B∼x01)) →inU (Σ (f : (x : A) →B x) ((x0 x1 : A)(x01 : A∼x0 x1) →B∼x01 (f x0) (f x1))) π : inU∼a a A∼→(∀{x0 x1}(x01 : A∼x0 x1) →inU∼(b x0) (b x1) (B∼x01)) →inU (Σ (f : (x : A) →B x) ((x0 x1 : A)(x01 : A∼x0 x1) →B∼x01 (f x0) (f x1))) data inU∼: {A A′ : Type} →inU A →inU A′ →(A →A′ →Prop) →Type1 data inU∼: {A A′ : Type} →inU A →inU A′ →(A →A′ →Prop) →Type1 bool∼: inU∼bool bool (λx0 x1 . x0 ?=2 x1) π∼: {b0 : (x0 : A0) →inU (B0 x0)}{b1 : (x1 : A1) →inU (B1 x1)} {a0∼: inU∼a0 a0 A0∼}{a1∼: inU∼a1 a1 A1∼} {b0∼: ∀{x0 x1}(x01 : A0∼x0 x1) →inU∼(b0 x0) (b0 x1) (B0∼x01)} {b1∼: ∀{x0 x1}(x01 : A1∼x0 x1) →inU∼(b1 x0) (b1 x1) (B1∼x01)} →inU∼a0 a1 A01∼ →(∀{x0 x1}(x01 : A01∼x0 x1) →inU∼(b0 x0) (b1 x1) (B01∼x01)) →inU∼(π a0 a0∼b0 b0∼) (π a1 a1∼b1 b1∼) (λf0 f1 . ∀(x0 x1) →A01∼x0 x1 →B01∼x01 (π1 f0 x0) (π1 f1 x1)) π∼: {b0 : (x0 : A0) →inU (B0 x0)}{b1 : (x1 : A1) →inU (B1 x1)} {a0∼: inU∼a0 a0 A0∼}{a1∼: inU∼a1 a1 A1∼} {b0∼: ∀{x0 x1}(x01 : A0∼x0 x1) →inU∼(b0 x0) (b0 x1) (B0∼x01)} {b1∼: ∀{x0 x1}(x01 : A1∼x0 x1) →inU∼(b1 x0) (b1 x1) (B1∼x01)} Just as the role of inU is, as before, to classify all types that are image of El, in the same way inU∼a a′ classiﬁes all relations of type A →A′ →Prop that are image of – ⊢– ∼El –, given proofs a : inU A, a′ : inU A′. In particular, this deﬁnition of inU∼states that the appropriate equivalence for boolean elements is the obvious syntactic equality – ?=2 –, whereas functions are to be compared T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. Sestini 14 pointwise. Note that inU appears in the sort of inU∼. 9 The main example of induction-induction is the intrinsic deﬁnition of a dependent type theory in type theory [6]. 4.3 Inductive-inductive setoid universe In the deﬁnition above, p has type ∥Σ (R : El A →El B →Prop) (. . .)∥. If the type was not propositionally truncated, we could deﬁne p ⊢x ∼El y by extracting the relation out of the ﬁrst component of p, and apply it to x, y. That is, p ⊢x ∼El y :≡π1 p x y. This would make the deﬁnition of – ∼U – and – ⊢– ∼El – in line with how we deﬁned U and El. However, this does not work in our case, since the type of p is propositionally truncated, hence it cannot be eliminated to construct a proof-relevant object. Fortunately, we can work around this limitation by deﬁning p ⊢x ∼El y by induction on the codes A B : U, in a way that ends up being logically equivalent to the proposition we would have obtained by π1 p x y if there were no truncation. 15 Constructing a universe for the setoid model More precisely, we need to construct proofs that for any concrete R and inR, the types \|(R, inR)\| ⊢x ∼El y and R x y are logically equivalent. These in turn need to be deﬁned mutually with – ⊢– ∼El –. We direct the interested reader to the Agda formalization for the full details of these deﬁnitions, as they are quite involved. More precisely, we need to construct proofs that for any concrete R and inR, the types \|(R, inR)\| ⊢x ∼El y and R x y are logically equivalent. These in turn need to be deﬁned mutually with – ⊢– ∼El –. We direct the interested reader to the Agda formalization for the full details of these deﬁnitions, as they are quite involved. The full deﬁnition of the universe is concluded with the remaining deﬁnitions, like reﬂU, reﬂEl, etc., which can be adapted from their IR counterparts more or less straightforwardly. The ﬁnal result does not use induction-recursion, but it is nevertheless an instance of inﬁnitary induction-induction. The ability to deﬁne arbitrary, inﬁnitary inductive-inductive types clashes, again, with our objective of keeping the metatheory as small and simple as possible. The next step is therefore to reduce this inductive-inductive universe to one that does not require (inﬁnitary) induction-induction. 4.4 Inductive setoid universe Sestini 16 •0 : Con0 –,0 – : Con0 →Ty0 →Con0 ι0 : Con0 →Ty0 Π0 : Con0 →Ty0 →Ty0 →Ty0 •1 : Con1 •0 –,1 – : ∀{Γ0 A0} →Con1 Γ0 →Ty1 Γ0 A0 →Con1 (Γ0 ,0 A0) ι1 : ∀{Γ0} →Con1 Γ0 →Ty1 Γ0 (ι0 Γ0) Π1 : ∀{Γ0 A0 B0} →Con1 Γ0 →Ty1 Γ0 A0 →Ty1 (Γ0 ,0 A0) B0 →Ty1 Γ0 (Π0 Γ0 A0 B0) •0 : Con0 –,0 – : Con0 →Ty0 →Con0 ι0 : Con0 →Ty0 Π0 : Con0 →Ty0 →Ty0 →Ty0 •1 : Con1 •0 –,1 – : ∀{Γ0 A0} →Con1 Γ0 →Ty1 Γ0 A0 →Con1 (Γ0 ,0 A0) ι1 : ∀{Γ0} →Con1 Γ0 →Ty1 Γ0 (ι0 Γ0) Π1 : ∀{Γ0 A0 B0} →Con1 Γ0 →Ty1 Γ0 A0 →Ty1 (Γ0 ,0 A0) B0 →Ty1 Γ0 (Π0 Γ0 A0 B0) •1 : Con1 •0 •1 : Con1 •0 –,1 – : ∀{Γ0 A0} →Con1 Γ0 →Ty1 Γ0 A0 →Con1 (Γ0 ,0 A0) ι1 : ∀{Γ0} →Con1 Γ0 →Ty1 Γ0 (ι0 Γ0) Π1 : ∀{Γ0 A0 B0} →Con1 Γ0 →Ty1 Γ0 A0 →Ty1 (Γ0 ,0 A0) B0 →Ty1 Γ0 (Π0 Γ0 A0 B0) •0 : Con0 •0 : Con0 –,0 – : Con0 →Ty0 →Con0 ι0 : Con0 →Ty0 Π0 : Con0 →Ty0 →Ty0 →Ty0 –,0 – : Con0 →Ty0 →Con0 ι0 : Con0 →Ty0 We can recover the original inductive-inductive type as Con :≡Σ (Γ0 : Con0) (Con1 Γ0) and Ty Γ :≡Σ (A0 : Ty0) (Ty1 (π1 Γ) A0). Recovering the constructors is straightforward: • :≡(•0, •1) (Γ0, Γ1), (A0, A1) :≡((Γ0 ,0 A0), (Γ1,1 A1)) ι (Γ0, Γ1) :≡(ι0 Γ0, ι1 Γ1) Π {Γ0, Γ1}(A0, A1)(B0, B1) :≡(Π0 Γ0 A0 B0, Π1 Γ1 A1 B1) • :≡(•0, •1) (Γ0, Γ1), (A0, A1) :≡((Γ0 ,0 A0), (Γ1,1 A1)) ι (Γ0, Γ1) :≡(ι0 Γ0, ι1 Γ1) Π {Γ0, Γ1}(A0, A1)(B0, B1) :≡(Π0 Γ0 A0 B0, Π1 Γ1 A1 B1) Finally, we can deﬁne eliminators/induction principles for Con and Ty as just deﬁned, by induction on the well-typing predicates. Following [25], we distinguish two versions of the eliminator: the simple and the general one. Note that this is orthogonal to the distinction between non- dependent and dependent eliminators, from which we only consider the latter. 4.4 Inductive setoid universe This section encodes the inductive-inductive universe of setoids from the pre- vious section without assuming arbitrary inductive-inductive deﬁnitions in the metatheory. Before turning our attention to the setoid universe, we recall the known, sys- tematic method to reduce ﬁnitary inductive-inductive types to inductive families. Reducing ﬁnitary induction-induction It is known that ﬁnitary inductive- inductive deﬁnitions can be reduced to inductive families [8,7,21]. To illustrate the idea, let us consider a well-known example of a ﬁnitary inductive-inductive type, the intrinsic encoding of type theory in type theory itself. Actually, we only consider the type of contexts Con : Type and the type of types Ty : Con → Type; since the latter is indexed over the former, this is already an example of induction-induction. Contexts in Con are formed out of empty contexts • and context extension –, –. Types in Ty are either the base type ι or Π types. ι : (Γ : Con) →Ty Γ Π : {Γ : Con}(A : Ty Γ) →Ty (Γ, A) →Ty Γ • : Con ι : (Γ : Con) →Ty Γ –, – : (Γ : Con) →Ty Γ →Con Π : {Γ : Con}(A : Ty Γ) →Ty (Γ, A) →Ty Γ • : Con ι : (Γ : Con) →Ty Γ –, – : (Γ : Con) →Ty Γ →Con Π : {Γ : Con}(A : Ty Γ) →Ty (Γ, A) →Ty Γ • : Con The general method to eliminate induction-induction is to split the original inductive-inductive types into a type of codes and associated well-formedness predicates. In our Con/Ty example, these would be respectively given by codes Con0, Ty0 : Type and predicates Con1 : Con0 →Type, Ty1 : Con0 →Ty0 → Type. The deﬁnition of the codes and predicate types follows that of the original inductive-inductive type, and can be derived systematically from it. More im- portantly, they can be deﬁned without induction-induction, since although Con0 and Ty0 are deﬁned mutually, their sorts are not indexed. T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. 4.4 Inductive setoid universe The motives for the simple eliminator are C′ : Con →Type, T ′ : (Γ : Con)(A : Ty Γ) →Type and the eliminators themselves have the following signatures: elim′ Con : (Γ : Con) →C′ Γ elim′ Ty : ∀{Γ}(A : Ty Γ) →T ′ Γ A elim′ Ty : ∀{Γ}(A : Ty Γ) →T ′ Γ A In the case of the general eliminator, the motive for Ty depends on the motive for Con, making the two eliminators recursive-recursive functions. For motives C : Con →Type and T : (Γ : Con) →Ty Γ →C Γ →Type the signatures are: In the case of the general eliminator, the motive for Ty depends on the motive for Con, making the two eliminators recursive-recursive functions. For motives C : Con →Type and T : (Γ : Con) →Ty Γ →C Γ →Type the signatures are: elimCon : (Γ : Con) →C Γ elimTy : ∀{Γ}(A : Ty Γ) →T Γ A (elimCon Γ) In the case of the general eliminator, the motive for Ty depends on the motive for Con, making the two eliminators recursive-recursive functions. For motives C : Con →Type and T : (Γ : Con) →Ty Γ →C Γ →Type the signatures are: elimCon : (Γ : Con) →C Γ elimTy : ∀{Γ}(A : Ty Γ) →T Γ A (elimCon Γ) elimTy : ∀{Γ}(A : Ty Γ) →T Γ A (elimCon Γ) The general eliminators can be derived from our encoding of Con and Ty via untyped codes and well-typing predicates. The way to do it is to ﬁrst deﬁne the graph of the eliminators in the form of inductively-generated relations: data R-Con : (Γ : Con) →C Γ →Type data R-Ty : {Γ : Con}(A : Ty Γ)(γ : C Γ) →T Γ A γ →Type The next step is to prove that these relations are functional, by induction on the untyped codes Con0 and Ty0 [21]. From this result, deﬁning the eliminators is immediate. Reducing the setoid universe The reduction described in the previous sec- tion works generically for an arbitrary ﬁnitary inductive-inductive type, thus Constructing a universe for the setoid model 17 giving a systematic way to reduce ﬁnitary inductive-inductive deﬁnitions to in- ductive families. However, it is not clear whether this method extends to in- ﬁnitary induction-induction, of which the setoid universe deﬁned in Section 4.3 is an instance. 4.4 Inductive setoid universe Of course, the absence of a general reduction method does not mean that we cannot reduce particular concrete instances of inﬁnitary induction- induction, which is exactly what we hope for our universe construction. The obvious challenge in successfully completing this reduction is to avoid the need for extensionality in the metatheory. In fact, consider the simple in- ﬁnitary inductive-inductive type obtained from the previous Con/Ty example by replacing the ﬁnitary constructor Π with an inﬁnitary one: Π : {Γ : Con} → (N →Ty Γ) →Ty Γ. Already with this simple example, we run into prob- lems as soon as we try to deﬁne the eliminator. One issue is that the deﬁnition of the eliminator relies on a proof that the well-typing predicates inU1, inU∼1 are propositional, that is, any two of their elements are equal. Without further assumptions this proof can only be done by induction, and requires function extensionality since these predicates include higher-order constructors. y g One way to get around this is to deﬁne the well-typing predicates as Prop- valued families, rather than in Type: data inU0 : Type →Type1 data inU∼0 : {A A′ : Type} →(A →A′ →Prop) →Type1 data inU1 : (A : Type) →inU0 A →Prop1 data inU∼1 : {A A′ : Type} →(R : A →A′ →Prop) →inU∼0 R →Prop1 data inU0 : Type →Type1 data inU∼0 : {A A′ : Type} →(A →A′ →Prop) →Type1 data inU1 : (A : Type) →inU0 A →Prop1 data inU∼1 : {A A′ : Type} →(R : A →A′ →Prop) →inU∼0 R →Prop1 Using Prop avoids the issue of proving propositionality altogether, since the predicates are now propositional by deﬁnition. However, it introduces a diﬀerent issue: inU1 and inU∼1 give rise to equational constraints on their indices, in the form of proofs of the Prop-valued identity type. The deﬁnition of the eliminators for inU and inU∼relies on the ability to transport along these proofs, hence the need to extend our metatheory with a primitive, strong form of transport for Id.10 Having Prop and a strong transport principle does help to some extent. However, we would still need extensionality to derive the general eliminators for inU and inU∼. In fact, as explained in the previous section, to derive the general recursive-recursive eliminators we need to prove that the corresponding graph relations are functional, which cannot be done without funext. 10 Note that this issue cannot be solved by expressing the equational constraints with an identity type in Type, since the well-typing predicates force it to necessarily be in Prop. 4.4 Inductive setoid universe Luckily, the simple elimination principle is suﬃcient for our purposes: all functions described in Section 4.3 can be deﬁned just using the simple elimina- tor without recursion-recursion. The simple eliminator itself can be deﬁned by pattern matching on the untyped codes, and does not require extensionality or any extra principles beyond strong transport. Once the inductive encoding of the inductive-inductive universe is done, the setoid universe can be deﬁned just as in Section 4.3. 10 Note that this issue cannot be solved by expressing the equational constraints with an identity type in Type, since the well-typing predicates force it to necessarily be in Prop. T. Altenkirch, S. Boulier, A. Kaposi, C. Sattler and F. Sestini 18 1. Andreas Abel. Extensional normalization in the logical framework with proof irrele- vant equality. In Olivier Danvy, editor, Workshop on Normalization by Evaluation, aﬃliated to LiCS 2009, Los Angeles, 15 August 2009, 2009. 5 Conclusions and further work We have described the construction of a universe of setoids in the setoid model of type theory; this is given in several steps, ﬁrst as an inductive-recursive deﬁni- tion, then as an inductive-inductive deﬁnition, and ﬁnally as an inductive type. Every encoding is obtained from the previous by adapting known data type transformation methods in a novel way that accounts for the peculiarities of our construction. In [5] we present rules for SetTT, clearly these rules need to be extended by the rules for a universe reﬂecting the semantics presented here. It is known that ﬁnitary IITs can be reduced to inductive types in an exten- sional setting [21]. In our paper we reduce an inﬁnitary IIT to inductive types in an intensional setting. In the future, we would like to investigate whether this reduction can be generalised to arbitrary inﬁnitary IITs. In contrast to the inductive-recursive and inductive-inductive versions of the universe, the inductive deﬁnition relies on a metatheory with a strong transport rule. As future work, we would like to prove normalization for this metatheory since previous work in this respect [2] seems to suggest that is represents a non-trivial addition. Another question regards the relationship between SeTT [5] and XTT [28]. Both systems are syntactic representations of the setoid model with similar de- sign choices, like deﬁnitional proof-irrelevance. We would like to know whether their respective notions of models are equivalent, that is, if we can obtain an XTT model from a SeTT model, and vice versa. Since XTT universes support universe induction, for one direction we would need to extend our own universe with the same principle (see discussion in Section 3 and the previous paragraph). Thus a related question is whether our encodings of the setoid universe can sup- port universe induction. A further question is whether this mapping of models is functorial. Groupoids can be regarded as generalized setoids. In the future we would like to design a type theory internalizing the groupoid model of type theory [20], in the same way that SeTT represents a syntax for the setoid model. 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https://openalex.org/W2922730194	http://journal.ipb.ac.id/index.php/jpsl/article/download/16096/16728	Indonesian	null	Ethics of Domestic Tourist to Beach Conservation (Case Study of Balekambang Beach, Malang District)	Jurnal pengelolaan sumberdaya alam dan lingkungan	2,019	cc-by	5,072	Ethics of Domestic Tourist to Beach Conservation (Case Study of Balekambang Beach, Malang District) Atok Miftachul Hudhaa, Husamahb aPusat Studi Lingkungan dan Kependudukan, Universitas Muhammadiyah Malang 65144  atok1964@gmail.com bPusat Studi Lingkungan dan Kependudukan, Universitas Muhammadiyah Malang 65144 Abstract. Conservation of biodiversity and the development of marine tourism destinations can be categorized as synergic efforts. But the evidence in the field shows different things, because the behavior of domestic tourists have not awareness of biodiversity conservation. This is indicated by the behavior of domestic tourists who often take various tidal biotas without educational purposes. It is so not educational, because at this time of nature conservation is very necessary, so that no damage to the environment, especially in the marine environment due to imbalance of living creatures in it by human activities. This is an observational research by conducting survey method to the behavior of domestic tourists while traveling on the coast of tidal Balekambang Malang regency. The findings indicate that the various ages of domestic tourist entering tidal zones during low tide take a variety of tidal biota, such as Echinoidea, Holothuroidea, Ophiuroidea, Anthozoa (corals) and other biota without educational purposes, since the biota referred to is taken and then disposed of in the mainland. It can be concluded that the bad behavior shown by the domestic tourists by taking biota is not for education, but for fun can be categorized as violation behavior of environmental ethics. Keyword: behavior of visitors, biodiversity, conservation, domestic tourist, environmental ethic (Diterima: 25-05-2017; Disetujui: 11-12-2017) Jurnal Pengelolaan Sumberdaya Alam dan Lingkungan Vol. 9 No. 1 (Maret 2019): 9-16 Jurnal Pengelolaan Sumberdaya Alam dan Lingkungan Vol. 9 No. 1 (Maret 2019): 9-16 1. Pendahuluan Berkaitan dengan potensi lingkungan kelautan yang prospektif bagi pengembangan wisata bahari, maka strategi pengembangannya harus diarahkan untuk meningkatkan sosial ekonomi masyarakat. Bertambahnya jumlah penduduk Indonesia tentu berdampak pada meningkatnya kebutuhan hidup yang bergantung pada produk-produk kelautan. Hal ini mengingat laut memiliki berbagai macam sumberdaya alam yang menjadi kebutuhan manusia sehari-hari, mulai biota pantai, berbagai jenis ikan, mulai ikan yang dikonsumsi hingga ikan hias, berbagi jenis rumput laut, dan berbagai jenis biota laut lainnya. Secara geografis Indonesia merupakan negara kepulauan terbesar di dunia, karena memiliki lebih dari 17,480 pulau-pulau besar dan kecil yang tersebar dari ujung pulau sumatera hingga pulau papua. Selain itu menurut Santoso (2011), Indonesia memiliki panjang garis pantai sejauh 95,181 km. Luas daratan hanya 1.9 juta km2, maka berarti 75% wilayah Indonesia merupakan lautan, yang terdiri dari 3.1 juta km2 wilayah laut teritorial dan 2.7 juta km2 zona ekonomi eksklusif. Lebih lanjut Santoso (2011), menjelaskan bahwa dengan kondisi seperti ini, maka Indonesia memiliki potensi sumberdaya kelautan yang terdiri dari sumberdaya alam dapat pulih atau dapat diperbaiki (renewable resources), sumberdaya alam tidak dapat pulih atau tidak dapat diperbaiki (non- renewable resources), sumber energi kelautan, dan jasa-jasa lingkungan yang sangat besar. Sumber daya kelautan dapat pulih diantaranya ekosistem terumbu karang, padang lamun, hutan mangrove dan berbagai jenis ikan. Sumber daya kelautan tidak dapat pulih meliputi minyak bumi dan gas, mineral dan bahan tambang atau galian. Potensi sumber energi kelautan dapat berasal dari pasang surut, angin, gelombang, dan ocean thermal energy conversion (OTEC), sedangkan salah satu jasa lingkungan kelautan yang sangat prospektif mendukung perekonomian masyarakat adalah pengembangan pariwisata bahari dan jasa perhubungan laut. Tingginya kebutuhan hidup manusia yang bergantung pada potensi laut telah berpotensi terhadap tingginya eksploitasi sumberdaya alam laut dan berdampak negatif terhadap upaya konservasi kelautan. Eksploitasi sumber daya alam laut semakin mengkhawatirkan dengan hadirnya teknologi yang semakin maju, sebab kemajuan teknologi tidak diarahkan untuk upaya konservasi, akan tetapi dijadikan alat untuk mengeruk kekayaan laut secara eksploitatif. Menyoal masalah konservasi, maka Undang- undang RI Nomor 5 Tahun 1990 tentang Konservasi Sumber daya Alam Hayati dan Ekosistemnya pasal 1 butir 2 menyebutkan, bahwa konservasi adalah pengelolaan sumber daya alam hayati yang pemanfaatannya dilakukan secara bijaksana untuk menjamin kesinambungan persediaannya dengan tetap memelihara dan meningkatkan kualitas doi: 10.29244/jpsl.9.1.9-16 9 ISSN 2086-4639 \| e-ISSN 2460-5824 JPSL Vol. 9 (1): 9-16 3. Mengidentifikasi biota laut yang banyak diambil oleh para wisatawan domestik di Pantai Balekambang Kabupaten Malang. 2. Metode Jenis penelitian yang dilakukan adalah penelitian deskriptif kualitatif dengan metode survey, melalui observasi dan wawancara, serta studi kepustakaan. Deskriptif kualitatif digunakan untuk menganalisa perilaku wisatawan domestik terhadap keberadaan hewan di zona pasang surut. Teknik observasi dan wawancara digunakan untuk mengungkap hal-hal terkait dengan lingkungan hidup dan konservasi keaneragaman hayati biota laut di zona pasang surut Pantai Balekambang Kabupaten Malang, dan studi kepustakaan digunakan untuk telaah pemikiran guna memberikan kontribusi terhadap upaya mengidentifikasi perilaku wisatawan domestik di Pantai Balekambang dan temuan biota di zona pasang surut Pantai Balekambang Kabupaten Malang. Sampel adalah wisatawan domestik yang berada di zona pasang surut Pantai Balekambang Kabupaten Malang dengan teknik accidental sampling. Berdasarkan definisi dan tujuan konservasi, maka ada lima manfaat konservasi terhadap ekosistem, sebagaimana dinyatakan oleh Sora (2015), yaitu 1) Untuk melindungi kekayaan ekosistem alam dan memelihara proses-proses ekologi maupun keseimbangan ekosistem secara berkelanjutan, 2) Untuk melindungi spesies flora dan fauna yang langka atau hampir punah, 3) Untuk melindungi ekosistem yang indah, menarik dan juga unik, 4) Untuk melindungi ekosistem dari kerusakan yang disebabkan oleh faktor alam, mikro organisme dan lain-lain, 5) Untuk menjaga kualitas lingkungan supaya tetap terjaga, dan lain sebagainya. Kelima manfaat konservasi ekosistem yang bertujuan melindungi dan menjaga ekosistem dapat saja terganggu dan gagal mencapai tujuan karena tidak adanya interaksi antara pemerintah dan masyarakat, antara pecinta lingkungan dengan masyarakat, antara dunia pendidikan atau lembaga pendidikan yang menyosialisasikan kesadaran dan gerakan konservasi dengan masyarakat, akibat masyarakat belum tersadarkan akan pentingnya konservasi terhadap lingkungan dan sumber daya alam. 3.1. Karakteristik Pantai Balekambang Pantai Balekambang merupakan salah satu destinasi wisata dengan status taman wisata yang memiliki luas area kurang lebih 10 ha dengan wilayah meliputi kawasan hutan lindung, hutan produksi, taman bermain, dan pantai. Semula taman wisata balekambang dikelola oleh Desa Srigonco, namun sejak 1985 pengelolaan Pantai Balekambang dialihkan ke Perusahaan Daerah (PD) Jasa Yasa milik pemerintah daerah kabupaten Malang. Meskipun pembenahan fisik terus dilakukan, namun pembenahan aspek sosial budaya, kelestarian alam dan lingkungan belum terkelola dengan baik. Minimnya pengetahuan wisatawan domestik terhadap biota laut di zona pasang surut serta perilaku yang tidak mendukung upaya konservasi sumber daya alam sebagaimana yang telah ditunjukkan mendorong pentingnya sosialisasi pengetahuan tentang biota laut dan upaya konservasi ekosistem pantai. Kesadaran untuk menumbuhkan pentingnya konservasi (pelestarian dan perlindungan) sumber daya alam, khususnya pada ekosistem pantai memang harus dilakukan secara sinergis, kolaboratif, dan integratif dari berbagai lembaga terkait dan berbagai lapisan masyarakat, agar dikemudian hari upaya pemeliharan dan perlindungan biota laut, secara keberlanjutannnya dapat menumbuhkan sikap etika berlingkungan atau lebih spesifik disebut ber bioetika lingkungan (environmental bioethics). Oleh karena itu penulisan artikel ini adalah untuk: 1. Pendahuluan keanekaragaman dan nilainya dan pada UU Nomor 23 Tahun 1997 pasal 1 butir 15 disebutkan, bahwa konservasi adalah pengelolaan sumberdaya alam tak terbaharui untuk menjamin pemanfatannya secara bijaksana dan sumber daya yang terbaharui untuk menjamin kesinambungan ketersediaannya dengan tetap memelihara dan meningkatkan kualitas nilai dan keanekaragamannya. 3. Mengidentifikasi biota laut yang banyak diambil oleh para wisatawan domestik di Pantai Balekambang Kabupaten Malang. 4. Mengidentifikasi perilaku wisatawan domestik terhadap biota di zona pasang surut Pantai Balekambang Kabupaten Malang. Lebih lanjut dinyatakan dalam pasal 5 Undang- undang Nomor 5 tahun 1990, bahwa tujuan konservasi, yaitu: 1) perlindungan sistem penyangga kehidupan; 2) pengawetan keanekaragaman jenis tumbuhan dan satwa beserta ekosistemnya; 3) pemanfaatan secara lestari sumber daya alam hayati dan ekosistemnya. 3.2. Karakteristik Wisatawan Domestik Karakteristik wisatawan domestik di Pantai Balekambang Kabupaten Malang 95% berasal dari Jawa Timur dan tersebar dari berbagai usia (anak-anak hingga lansia), laki-laki perempuan, dan dari berbagai jenis profesi (pelajar, mahasiswa, profesional, TNI- POLRI, PNS, wiraswasta) dan masyarakat umum lainnya. Karakteristik pengunjung tempat wisata kawasan pantai sebagaimana di Pantai Balekambang Kabupaten Malang ini tidak jauh berbeda dengan penelitian Hasan et al. (tanpa tahun), yang menyatakan, bahwa karakteristik usia pengunjung obyek wisata di Kota Tidore Kepulauan adalah laki- 1. Mendeskrispsikan karakteristik wisatawan domestik di Pantai Balekambang Kabupaten Malang. 2. Menginventarisasi berbagai biota laut di zona pasang surut pantai Balekambang Kabupaten Malang. 10 JPSL Vol. 9 (1): 9-16 Maret 2019 JPSL Vol. 9 (1): 9-16 Maret 2019 laki 63% perempuan 37%, usia dibawah 20 tahun 9%, usia 21-30 tahun 19%, usia 31-40 tahun 27%, usia 41- 50 tahun 36%. Asteroidea dan Kelas Crinoidea tidak ditemukan. Hasil biota laut yang ditemukan sebagaimana Tabel 1. Asteroidea dan Kelas Crinoidea tidak ditemukan. Hasil biota laut yang ditemukan sebagaimana Tabel 1. Tabel 1. Hasil inventarisasi Biota Laut Phylum echinodermata Tabel 1. Hasil inventarisasi Biota Laut Phylum echinodermata Dilihat dari kehadirannya di Pantai Balekambang para pengunjung (wisatawan domestik), memiliki tujuan yang bervariasi, 90% bertujuan untuk berlibur (rekreasi) bersama keluarga dan kerabat, 8% bertujuan untuk berjualan, 1% bertujuan untuk ritual, 1% lain- lain. y Kelas Nama Spesies Keterangan Echinoidea 1. Diadema setosum 2. Echinometra mathaei Jumlah cukup banyak dan tersebar Jumlah terbatas Holothuroidea 1. Holothuria scabra 2. Holothuria atra Jumlah terbatas Jumlah terbatas Ophuiroidea 1. Ophiotrichoides nereidina 2.Ophiothrix hybrid Jumlah cukup banyak dan tersebar Jumlah terbatas (Sumber: Data Penelitian, 2016) Demikian juga hasil penelitian Hasan et al. (Tanpa Tahun), bahwa pengunjung daerah wisata di kepulauan Tidore berdasarkan tujuan berkunjung, didominasi oleh tujuan berlibur sebanyak 34%, kemudian disusul oleh tujuan perjalanan dinas sejumlah 9%, selanjutnya tujuan mengunjungi keluarga 6%, tujuan mengunjungi kerabat 9%, untuk tujuan studi sebanyak 17% dan yang terakhir untuk lain-lain termasuk tujuan ekonomi sejumlah 5%). Berdasarkan Tabel 1 dapat dijelaskan, bahwa keanekaragaman biota pasang surut pada Phylum echinodermata di Pantai Belekambang Kabupaten Malang, didominasi oleh tiga kelas hewan, yaitu Echinoidea, Holothuroidea, dan Ophiuroidea dan untuk hewan kelas Asteroidea dan Crinoidea tidak ditemukan sama sekali. Hal ini didukung oleh kerusakan pantai akibat perilaku manusia, khususnya wisatawan domestik dan pencari lobster yang sering kali memecah batu karang untuk mencari lobster atau gurita. 3.3. Biota Laut Zona Pasang Surut yang Ditemukan di Lokasi Berdasarkan hasil penelitian yang telah dilakukan di zona pasang surut Pantai Balekambang Kabupaten Malang, maka berbagai biota laut yang ditemukan dari Phylum echinodermata adalah: (1) Kelas Echonoidea (Landak Laut); (2) Kelas Ophiuroidea (Bintang Ular Laut); (3) Kelas Holothuroidea; adapun kelas Gambar 1. Lokasi kerusakan habitat biota pasang surut di pantai balekambang (dok. penelitian, 2016) Gambar 1. Lokasi kerusakan habitat biota pasang surut di pantai balekambang (dok. penelitian, 2016) ISSN 2086-4639 \| e-ISSN 2460-5824 JPSL Vol. 9 (1): 9-16 Pada umumnya landak laut mempunyai jerohan atau vicera tersimpan dalam cangkok, yang tersusun menurut 10 jajaran lempengan kapur yang tersambung bersama membentuk bola. Seperti halnya bintang laut memiliki 5 jalur kaki ambulakral yang terselang oleh daerah inter ambulakral yang agak lebar tanpa kaki. Pada cangkok terdapat tonjolan atau tuberculum sebagai tempat persembunyian duri-duri. Tiap-tiap duri merupakan bentuk kristal dari CaCO3 yang ujung pangkalnya agak melebar tempat sendi dengan tuberculum. Pangkal duri itu terikat dengan otot sehingga duri dapat digerakkan. a b Gambar 4. (a) Holothuria scabra, (b) Holothuria atra (Dokumentasi Penelitian, 2016) b a b Gambar 4. (a) Holothuria scabra, (b) Holothuria atra (Dokumentasi Penelitian, 2016) Anus terletak di pusat tubuh pada permukaan aboral, terletak diantara lempengan kapur yang besar yang mengandung lima, empat atau dua lubang genital. Mulut yang besar terletak di daerah oral dikelilingi oleh 5 buah gigi yang kuat dan tajam. Gigi tersebut disokong oleh 5 rangka samping disebelah dalam cangkok yang terkenal sebagai “Lentera Aristoteles”. Teripang merupakan komponen penting dalam rantai makanan di terumbu karang dan ekosistem asosiasinya pada berbagai tingkat struktur pakan. Jenis hewan laut ini tersebar luas di lingkungan laut di seluruh dunia, mulai dari zona pasang surut sampai laut dalam, sehingga mudah ditemukan. Banyak sekali jenis teripang yang ada, tidak kurang ada sekitar 29 jenis teripang yang saat ini dan menjadi komoditas perdagangan global yang memiliki nilai jual dan nilai ekonomis tinggi. Holothuroidea (teripang) seperti yang sudah kita tahu memiliki banyak sekali kandungan khasiat dan manfaat yang berguna bagi kesehatan tubuh kita, akan tetapi ada satu jenis teripang laut yang mempunyai keunikan dan hanya dimiliki oleh teripang jenis ini, dimana teripang jenis ini bernama teripang emas (Golden stichopus variegatus). Salah satu keunggulan dari teripang emas jenis ini, yaitu memiliki kandungan Gamapeptide, kandungan ini tidak dimiliki oleh jenis teripang lain. Kandungan tersebut berkhasiat untuk: 1) mengaktifkan pertumbuhan sel, 2) mampu meregenerasi sel dengan sangat cepat, 3) anti inflamasi atau peradangan, 4) dapat mengurangi rasa nyeri bahkan di katakan memiliki anti analgesik 3 kali lebih kuat dari pada paracetamol (obat golongan analgesik-antipiretik/anti nyeri dan anti panas), 5) memelihara sirkulasi darah agar tetap lancar dan meningkatkan sistem kekebalan tubuh. Gambar 3. Menjelaskan morfologi Echinoidea ke mahasiswa (Dokumentasi Penelitian, 2016) Gambar 3. a. Kelas Echinoidea (Landak Laut) duri-duri yang dapat digerakkan. Sebagai contoh yaitu: Arbacic, Stronglyocentrotus, yang berbentuk bola, Spatangus bentuk oval, dan Echinarachinus, Dendraster berbentuk seperti uang logam atau terkenal dengan sebutan uang kepingan pasir atau kue laut. Kelas Echinoidea adalah kelompok hewan yang memiliki tonjolan-tonjolan pendek yang bulat, yaitu tempat menempel duri yang tersusun dari zat kapur. Hewan-hewan yang masuk kelas Echinoidea berbentuk bundar tidak berlengan, tetapi mempunyai a b Gambar 2. (a) Diadema setosum, (b) Echinometra mathaei (Dokumentasi Penelitian, 2016) b a b a b Gambar 2. (a) Diadema setosum, (b) Echinometra mathaei (Dokumentasi Penelitian, 2016) 11 c. Kelas Ophiuroidea (Bintang Ular Laut) Kelompok Ophiuroidea dapat hidup menempati berbagai macam habitat dan kedalaman, seperti zona rataan terumbu karang, daerah pertumbuhan algae, padang lamun, koloni karang hidup dan karang mati, dan berbagai macam kedalaman mulai dari kedalaman 1 meter sampai ribuan meter seperti dilaporkan oleh banyak pakar (Yusron, 2010). ISSN 2086-4639 \| e-ISSN 2460-5824 JPSL Vol. 9 (1): 9-16 Menjelaskan morfologi Echinoidea ke mahasiswa (Dokumentasi Penelitian, 2016) Makanan hewan ini bermacam-macam, misalnya hewan lain yang telah mati, organisme kecil lainnya yang ada di laut, kecuali itu hewan ini juga mencerna lumpur atau pasir yang mengandung bahan-bahan organis. Didalam pencernaan hewan ini banyak mengandung cilliata yang komersal atau yang parasit. Ikan, Bintang laut, Udang-udangan, Burung dan Mamalia yang predator merupakan musuh Echinoidea. b. Kelas Holothuroidea (Teripang) Teripang atau disebut timun laut merupakan istilah yang diberikan untuk hewan invertebrata Holothuroidea yang dapat dikonsumsi. Teripang merupakan jenis hewan laut yang bergerak lambat, hidup pada dasar substrat pasir, lumpur pasiran maupun dalam lingkungan terumbu. 3.4. Biota Laut yang Banyak Diambil Wisatawan Domestik Biota laut pasang surut yang sering diambil wisatawan domestik untuk tujuan kesenangan sesaat oleh wisatawan domestik yang berkunjung di Pantai Balekambang sebagaimana Hudha (2012, 2013, 2014, 2015, 2016), antara lain: Kelas Holothuroidea, Kelas Asteroidea, Kelas Ophiuroidea, dan beberapa ganggang laut seperti Sargassum, Halimeda, Padina, dan Fucus.Hal ini sebagaimana tertuang di Tabel 2. g Berbagai biota laut laut di zona pasang surut yang banyak ditemukan adalah Phylum echinodermata kelas Echinoidea, kelas Asteroidea, kelas Holothuroidea, kelas Ophiuroidea, kelas Crinoidea. Sebagaimana dinyatakan Hudha (2002), bahwa phylum Echinodermata adalah hewan invertebrata yang memiliki karakteristik, antara lain: (1) Simetri radial pada hewan yang telah dewasa, memiliki 5 bagian, sedang larvanya simetri bilateral; memiliki 3 jaringan dasar; sebagian alatnya bersifat tidak memiliki kepala dan otak, tidak bersegmen. (2) Permukaan tubuh yang umumnya simetri radial, memiliki kaki buluh atau kaki amburakral; (3)Tubuh terbungkus oleh epidermis yang halus dengan disokong oleh penguat berupa kepingan kapur yang disebut laminae atau ossicula yang mudah digerakkan atau tidak mudah digerakkan, dengan pola yang tetap, sering memiliki duri-duri kapur yang halus; (4) Saluran pencernaan yang sederhana, biasanya lengkap / beberapa jenis tidak memiliki anus; (5) Memiliki sistem sirkulasi radial yang mengalami reduksi; coelom dilapisi oleh perritonium bersilia; rongga coelom biasanya luas dan berisi amoebocyt- amoebocyt bebas. Pada tingkat larva coelom ini berfungsi sebagai sistem vaskular air dengan kaki-kaki ambulakral yang banyak digunakan untuk berjalan, menangkap mangsa atau respirasi; (6) Respirasi dilakukan dengan insang kecil atau papulae yang tertimbul dari coelom, beberapa jenis Echinodermata bernapas dengan kaki ambulakral; sedang pada Holothurroidea menggunakan batang-batang seperti pohon yang terdapat dalam clocoa; (7) Sistem syaraf dengan batang cincin yang bercabang-cabang ke arah radial; (8) Seks terpisah dengan beberapa pengecualian. Gonad yang relatif besar terletak di Tabel 2. Inventarisasi biota laut yang banyak diambil wisatawan untuk tujuan kesenangan di pantai balekambang Tabel 2. Inventarisasi biota laut yang banyak diambil wisatawan untuk tujuan kesenangan di pantai balekambang No Jenis Biota Lain-lain Hewan Tumbuhan 1 Kelas Echinoidea Alga: Sargassum Acropora 2 Kelas Ophiuroidea Alga: Halimeda 3 Kelas Holothuroidea Alga: Padina 4 Kelas Asteroidea Alga: Fucus 5 Ikan Hias (Sumber: Data Penelitian, 2016) Berdasarkan Tabel 2 diketahui, bahwa biota laut pada zona pasang surut yang diambil wisatawan domestik di Pantai Balekambang Kabupaten Malang, dominan pada Phylum echinodermata, khususnya Echinoidea, ophiuroidea dan beberapa jenis Holothuroidea. Jenis biota lain dari phylum Echinodermata yang tidak ditemukan adalah kelas Asteroidea (bintang laut) dan kelas Crinoidea pada kawasan yang luas di pantai Balekambang. b. Kelas Holothuroidea (Teripang) Teripang atau disebut timun laut merupakan istilah yang diberikan untuk hewan invertebrata Holothuroidea yang dapat dikonsumsi. Teripang merupakan jenis hewan laut yang bergerak lambat, hidup pada dasar substrat pasir, lumpur pasiran maupun dalam lingkungan terumbu. 12 JPSL Vol. 9 (1): 9-16 Maret 2019 a b Gambar 5. (a)Ophiothrix fragilis (habitas.org.uk), (b)Ophiothrix fragilis (Dokumentasi Penelitian, 2016) b a b Gambar 5. (a)Ophiothrix fragilis (habitas.org.uk), (b)Ophiothrix fragilis (Dokumentasi Penelitian, 2016) a b Gambar 5. (a)Ophiothrix fragilis (habitas.org.uk), (b)Ophiothrix fragilis (Dokumentasi Penelitian, 2016) ar mempunyai tubuh bola cakram kecil ngan bulat panjang. Tiap-tiap lengan as-ruas yang sama yang masing-masing garis tempat meletaknya ossicula yang 4 lempeng Di bagian lateral terdapat sebelah luar dengan pembuluh yang sederhana; jum ova banyak selai dan pembuahan terjadi didalam larva mikroskopis, bersilia dan transparan, d biasanya hidup bebas dengan berenang-renang dal air, bermetamorfosis yang kompleks. f g sebelah luar dengan pembuluh yang sederhana; jumlah ova banyak selai dan pembuahan terjadi didalam air; larva mikroskopis, bersilia dan transparan, dan biasanya hidup bebas dengan berenang-renang dalam air, bermetamorfosis yang kompleks. Bintang ular mempunyai tubuh bola cakram kecil dengan 5 lengan bulat panjang. Tiap-tiap lengan terdiri atas ruas-ruas yang sama yang masing-masing terdapat dua garis tempat meletaknya ossicula yang terliputi oleh 4 lempeng. Di bagian lateral terdapat duri, sedang bagian dorsal serta ventral tidak terdapat duri. Bagian dalam dari ruas sebagian besar terisi ossicula yang silindris dan tertanam pada bagian proksimal dan cembung pada bagian distal, sehingga penyokong tubuh itu bersendi dengan lainnya dengan sistem sendi peluru. Empat otot antara dua ossicula silindris itu memungkinkan lengan dapat dibengkokkan. 3.5. Identifikasi Perilaku Wisatawan Domestik di Zona Pasang Surut Pantai Balekambang Kabupaten Malang Temuan perilaku pengunjung yang mengambil Echinoidea untuk tujuan ekonomi, atau perilaku pengunjung yang mengambil berbagai jenis biota pasang surut adalah bentuk ketidaksadaran masyarakat terhadap pentingnya menjaga kelestarian kehidupan makhluk hidup. Hal ini sangat korelatif dengan asumsi, bahwa pendidikan etika berbasis lingkungan pada masyarakat belum tumbuh dengan baik, sehingga temuan perilaku wisatawan domestik terhadap kehidupan biota di Pantai Balekambang menjadi tolok ukur bahwa masyarakat belum mengeteahui pentingnya etika lingkungan. Perilaku wisatawan domestik saat berada di zona pasang surut Pantai Balekambang Kabupaten Malang seakan tidak menampakkan dampak ekologis. Namun jika diamati, maka perilaku wisatawan domestik yang mengambil biota laut yang ditemukan selanjutnya dibuang di pasir pantai adalah tindakan merusak keanekaragaman jenis bioata pasang surut. Selain itu juga ditemukan perilaku pengunjung yang mengambil berbagai jenis landak laut (echinoidea) dan dikumpulkan. Hasil wawancara dengan pengunjung Pantai Balekambang yang diketahui sedang mengambil berbagai jenis Echinoidea di ketahui, bahwa kegiatan yang dilakukan berlatar belakang ekonomi. Ketika ditanya mulai kapan kegiatan mengambil Echinoidea ini dilakukan, hasil petikan wawancara yang telah dilakukan sebagai berikut: “Sudah bertahun-tahun saya melakukan pengambilan landak laut” (W.1/R.1/III/2016) dengan keterangan (W1=Wawancara pertanyaan ke-1; R.1-Responden ke-1, III=bulan 3 (Maret), 2016=tahun 2016). Etika lingkungan merupakan hal baru di masyarakat luas, bahkan dalam lingkup internasional, sebagaimana Wapner dan Matthew (2009), menyatakan bahwa refleksi etis tentang urusan lingkungan internasional masih dalam tahap awal. Fakta di lapangan yang dideskripsikan dalam penelitian ini menunjukkan, bahwa perilaku wisatawan domestik yang merusak kehidupan makhluk hidup, khususnya biota pasang surut di Pantai Balekambang Kabupaten Malang sangat berkaitan erat dengan kesadaran melakukan hal baik dan tidak melakukan hal buruk terhadap lingkungan atau dengan kata lain berkaitan dengan tindakan etis. Oleh karena itu kelangkaan bintang laut (Asteroidea) di Pantai Balekambang Kabupaten Malang, juga akibat perilaku manusia yang mengeksploitasi bintang laut (Asteroidea) dalam waktu lama. Ketika responden ditanya untuk apa mengambil Echinoidea begitu banyak? Responden menjawab untuk mencari uang, berikut jawaban responden: “Landak laut-landak laut yang saya kumpulkan ini untuk saya jual agar dapat uang” (W.2/R.1/III/2016) Ketika responden ditanya untuk apa mengambil Echinoidea begitu banyak? Responden menjawab untuk mencari uang, berikut jawaban responden: “Landak laut-landak laut yang saya kumpulkan ini untuk saya jual agar dapat uang” (W.2/R.1/III/2016) Ketika ditanya bagaimana mendapatkan uang dari menjual aneka landak laut tersebut? Responden menyatakan, bahwa landak laut diperolehnya dijadikan asesoris agar laku dijual, berikut jawaban responden: “Yang saya jual dari landak laut ini cangkangnya. 3.4. Biota Laut yang Banyak Diambil Wisatawan Domestik Hasil telusur kepada salah satu pengunjung terhadap tindakan yang dilakukan, didasari oleh faktor ekonomi, yaitu untuk menghasilkan pendapatan Penulis memberikan atribut kepada salah satu pengunjung yang dijumpai sedang melakukan eksploitasi kelas Echinoidea (landak laut) sebagai responden 1. 13 ISSN 2086-4639 \| e-ISSN 2460-5824 JPSL Vol. 9 (1): 9-16 a b c d Gambar 6. (a) Sargassum, sp, (b) Halimeda, sp, (c) Padina, sp (d) Fucus, sp a a b yang saya ambil ini saya masukan ke dalam karung plastik, lalu saya kubur di dalam pasir di tepi pantai yang jauh dari air pasang sedalam 1 meter, dan saya biarkan selama kurang lebih satu minggu. Setelah satu minggu saya ambil dan sudah membusuk, sehingga duri-durinya sudah lepas dan bagian dalam sudah membusuk, lalu saya bawa ke laut untuk membuang duri-duri dan organ dalamnya yang membusuk itu sampai bersih. Setelah itu tinggal cangkangnya. Cangkangnya itu lalu saya jemur sampai kering terus saya cat warna-warni mengikuti tonjolan-tonjolan atau alur yang ada di cangkang.” (W.4/R.1/III/2016) b c d Gambar 6. (a) Sargassum, sp, (b) Halimeda, sp, (c) Padina, sp (d) Fucus, sp d Saat ditanya berapa harga setiap cangkang yang dijual? Responden menjawab: “Ya, yang besar saya tawarkan 10,000 rupiah tapi kadang lakukadang tidak, terutama yang bentuknya besar, untuk yang bentuknya kecil saya jual 5,000 rupiah” (W.5/R.1/III/2016) d Gambar 6. (a) Sargassum, sp, (b) Halimeda, sp, (c) Padina, sp (d) Fucus, sp Ketidaktahuan masyarakat terhadap pentingnya menjaga kelestarian lingkungan hidup harus terus ditumbuhkan, sebab menurut Ramadoss dan Moli (2011), melindungi keanekaragaman hayati lokal merupakan tujuan penting untuk pendidikan pembangunan berkelanjutan. 3.5. Identifikasi Perilaku Wisatawan Domestik di Zona Pasang Surut Pantai Balekambang Kabupaten Malang 3.5. Identifikasi Perilaku Wisatawan Domestik di Zona Pasang Surut Pantai Balekambang Kabupaten Malang 4. Ilustrasi Perilaku masyarakat, khususnya wisatawan domestik terhadap kehidupan biota pasang surut di Pantai Balekambang Kabupaten Malang, sebagaimana dinyatakan Hudha (2013, 2014, 2015, 2016), bahwa diantara para wisatawan domestik yang berkunjung ke Pantai Balekambang Kabupaten Malang mengambil berbagai bioata pasang surut tidak untuk tujuan penelitian, namun untuk tujuan kesenangan, kemudian dibiarkan mati dan dibuang menunjukkan belum dimilikinya kesadaran melestarikan lingkungan, khususnya kehidupan biota pasang surut di Pantai Balekambang. Disinggung tentang peran etika lingkungan dalam upaya konservasi pantai, Responden 2 menjawab: “Pantai adalah sumber potensial untuk menghasilkan banyak sumberdaya alam, karena itu konservasi perairan laut harus dimulai dari konservasi lingkungan pantai. Dan masyarakat kita belum memahami pentingnya ekosistem pantai, pantai masih dijadikan tempat rekreasi belum dimanfaatkan untuk pengembangan kekayaan laut, misalnya: pembudidayaan rumput laut dan sebagainya. Padahal banyak pantai di Indonesia yang bisa dikembangkan untuk mendapatkan penghasilan besar dengan berbudidaya runput laut, seperti yang telah dilakukan oleh banyak masyarakat nelayan di Sumenep Madura” (W.3/R.2/V/2016). Perilaku lain yang dinyatakan oleh Hudha (2012, 2013, 2014, 2015, 2016), terhadap perilaku wisatawan domestik adalah melakukan perusakan habitat biota laut dengan cara menghancurkan batuan karang yang menjadi tempat perlindungan biota laut, seperti: Landak Laut (Echinoidea), Bintang Ular Laut (Ophiuroidea), dan ikan-ikan hias, karena berburu hewan lain yang diinginkannya (gurita dan lobster). Perilaku pengunjung (wisatawan domestik) di Pantai Balekambang Kabupaten Malang dengan mengeksploitasi alam melalui pengambilan berbagai biota laut di zona pasang surut pantai balekambang untuk tujuan kesenangan sesaat maupun tujuan ekonomi dalam jangka panjang berdampak pada menurunnya populasi biota laut di zona pasang surut, khususnya pada Phylum echinodermata dan juga phylum lainnya (Mollusca), Coelenterata (anthozoa dan acropora) dan jenis biota lainnya. Hal ini menjadi bukti semakin langkanya temuan biota laut pada zona pasang surut di Pantai Balekambang Kabupaten Malang adalah akibat perilaku pengunjung (wisatawan domestik) Pantai Balekambang Kabupaten Malang. y g g y g Berdasarkan temuan fakta demikian ini, penulis menggaris bawahi, bahwa masalah etika lingkungan belum dimiliki oleh wisatawan domestik, dan menurut Aminatun (Tanpa Tahun), bahwa etika lingkungan merupakan refleksi kritis tentang norma dan nilai atau prinsip moral yang selama ini dikenal dalam komunitas manusia. Hal ini menjadi tugas berat semua lapisan masyarakat untuk menempatkan etika lingkungan (Environmental Bioethics) menjadi bagian penting dalam upaya melestarikan kehidupan yang seimbang dan lestari. 3.5. Identifikasi Perilaku Wisatawan Domestik di Zona Pasang Surut Pantai Balekambang Kabupaten Malang Setelah tinggal cangkangnya akan saya cat warna-warni dan saya jual” (W.3/R.1/III/2016) y j g p g ( ) Ketika ditanya bagaimana mendapatkan uang dari menjual aneka landak laut tersebut? Responden menyatakan, bahwa landak laut diperolehnya dijadikan asesoris agar laku dijual, berikut jawaban responden: “Yang saya jual dari landak laut ini cangkangnya. Setelah tinggal cangkangnya akan saya cat warna-warni dan saya jual” (W.3/R.1/III/2016) Bentuk perilaku wisatawan domestik yang ditemukan lebih ekstrim selain mengeksploitasi biota yang ditemukan dan dicari adalah merusak dan menghancurkan habitat biota dengan memecah bebatuan, mencongkel bebatuan, sehingga menghilangkan “rumah” bagi biota pasang surut termasuk “rumah”berbagai ikan hias. Ketika ditanya bagaimana mendapatkan cangkangnya saja, sementara duri-diri landak laut begitu banyak? Responden menjawab: “Landak laut 14 JPSL Vol. 9 (1): 9-16 Maret 2019 Menyikapi perilaku wisatawan pantai sebagaimana hasil temuan, peneliti melakukan wawancara dengan sumber ahli (Responden 2) untuk minta tanggapannya terhadap perilaku masyarakat yang belum memahami etika lingkungan, responden 2 menjelaskan: “Masyarakat kita sangat membutuhkan teladan dari orang-orang berpengaruh di lingkungan sekitarnya untuk taat dalam menjaga dan melestarikan lingkungannya” (W.1/R.2/V/20016). bahwa (1) terdapat hubungan positif yang cukup signifikan antara pengetahuan lingkungan hidup dengan sikap menjaga kelestarian lingkungan, (2) terdapat hubungan positif yang cukup signifikan antara pengetahuan etika lingkungan dengan sikap menjaga kelestarian lingkungan, (3) terdapat hubungan positif yang cukup signifikan antara pengetahuan lingkungan hidup dan pengetahuan etika lingkungan secara bersama-sama dengan sikap menjaga kelestarian lingkungan, dan (4) terdapat hubungan positif yang cukup signifikan antara sikap menjaga kelestarian lingkungan dengan perilaku menjaga kelestarian lingkungan. Siapa orang-orang berpengaruh dimaksud? Responden 2 menjawab: “Kalau masyarakat itu berada di lingkungan yang agamis, maka tokoh-tokoh agama, Kyai, Ustad yang ada di masyarakat itulah yang harus di bidik dan memberi contoh kepada warga atau umat di masyarakat tersebut. Jika para tokoh agama, tokoh masyarakat, dan orang yang ditokohkan sudah melakukan dan memberi contoh, pasti pada masyarakat itu mudah untuk digerakkan ke arah yang lebih baik. Kita harus paham, bahwa kultur masyarakat kita masih harus diberi contoh, belum memiliki kesadaran penuh untuk berbuat dan bertindak secara mandiri” (W.2/R.2/V/2016). Daftar Pustaka Azhar, M. D. Basyir, Alfitri, 2015. Hubungan pengetahuan dan etika lingkungan dengan sikap dan perilaku menjaga kelestarian lingkungan. Jurnal Ilmu Lingkungan 13 (1), pp. 36–41. Hasan, H., M. Asdar, Jusni. Tanpa Tahun. Faktor-faktor yang mempengaruhi keputusan wisatawan dalam melakukan kunjungan wisata di Kota Tidore Kepulauan. [Online]. http://pasca.unhas.ac.id/jurnal/files/1d64662f5dc278778fa42c9e 570effd6.pdf. Edukasi Etika Lingkungan Hudha, A. M., 2002. Invertebrata. Buku Pegangan Mahasiswa. Prodi Pendidikan Biologi-FKIP UMM, Malang. Hudha, A. M., 2012. Membentuk Pribadi yang Unggul Membangun Peradaban Utama. Materi Penunjang Pembentukan Kepemimpinan dan Kepribadian Mahasiswa Periode IX. Aditya Media Publishing, Yogyakarta. KERUSAKAN DAN KEPUNAHAN BIOTA Gambar 7. Bagan kerusakan dan kepunahan biota laut dan upaya solusinya Hudha, A. M., 2015. Inventarisasi Biota Pasang Surut di Pantai Balekambang Kabupaten Malang. Laporan Penelitian. Prodi Pendidikan Biologi FKIP Universitas Muhammadiyah, Malang. Berdasarkan gambar 7 dapat dinyatakan, bahwa upaya mencegah kerusakan dan kepunahan biota pada zona pasang surut, dapat dilakukan melalui proses edukasi kepada masyarakat dalam bentuk edukasi etika lingkungan. Bentuk edukasi etika lingkungan yang sesuai untuk wisatawan domestik Pantai Balekambang dalam upaya konservasi pantai dapat beranekaragam, antara lain melalui kampanye konservasi lingkungan di lokasi pantai, pemasangan baliho penyelamatan dan peduli lingkungan pantai, game-game konservasi penyelamatan lingkungan, aksi bersama masyarakat umum dan lembaga pendidikan untuk konservasi pantai, penyebarluasan booklet tentang konservasi pantai, lomba-lomba bertemakan konservasi pantai dan bentuk kegiatan kampanye lainnya. Hudha, A. M., 2016. Inventarisasi Biota Pasang Surut di Pantai Balekambang Kabupaten Malang. Laporan Penelitian. Prodi Pendidikan Biologi FKIP Universitas Muhammadiyah, Malang. Kementerian Kelautan dan Perikanan Direktorat Jenderal Kelautan, Pesisir dan Pulau-pulau Kecil Direktorat Konservasi Kawasan dan Jenis Ikan. (Tanpa Tahun) Konservasi Untuk Kesejahteraan. [Online]. https://surajis.files.wordpress.com/2014/08/konservasi-untuk- kesejahteraan.pdf [29 November 2016]. Ramadoss, A., G. P. Moli, 2011. Biodiversity conservation through environmental education for sustainable development - a case study from Puducherry, India. International Electronic Journal of Environmental Education 1 (2), pp. 97–111. Santoso, H. A., 2011. Progres pengembangan sistem kawasan konservasi perairan indonesia. [Online]. http://coraltriangleinitiative.org/sites/default /files/resources/1_ProgresPengembangan%20 Sistem%20Kawasan%20. Sora, N., 2015. Pengertian Konservasi Dan Tujuannya Serta Manfaatnya Terjelas. [Online]. http://www.pengertianku.net/2015/08 /pengertian-konservasi- dan-tujuannya-serta-manfaatnya.html_Konservasi_Bahasa.pdf [29 November 2016]. 4. Ilustrasi Hal ini didasarkan pada teori tentang persepsi, sebagaimana dinyatakan oleh Hudha (2012, 2013), bahwa kehidupan manusia dibangun oleh persepsinya yang membentuk pola pikir, yaitu jika pola pikir seseorang positif, maka perasaan dan tindakannya akan positif, dan jika pola pikirnya negatif, maka seluruh perasaan dan tindakannya negatif. Hal yang dimaksud dapat digambarkan sebagaimana gambar 7. Kontribusi pemikiran yang dapat diberikan secara berkelanjutan kepada para wisatawan domestik untuk sadar terhadap kegiatan pelestarian lingkungan dengan tidak mengambil dan mengekploitasi biota laut apalagi merusak habitat biota laut adalah perlu dilakukan sosialisasi terus menerus terhadap pentingnya pengetahuan lingkungan dan pentingnya etika lingkungan, melalui edukasi oleh pemerintah dan lembaga pendidikan. Fakta empiris terhadap hal ini ditunjukan oleh penelitian Azhar et al. (2015), yang menyimpulkan, 15 15 ISSN 2086-4639 \| e-ISSN 2460-5824 JPSL Vol. 9 (1): 9-16 Gambar 7. Bagan kerusakan dan kepunahan biota laut dan upaya solusinya Kerusakan Habitat Biota Pasang Surut Laut Faktor Manusia Faktor Alam Kegiatan Ekonomi Eksploitasi Tanpa Tujuan Edukasi Etika Lingkungan KERUSAKAN DAN KEPUNAHAN BIOTA adalah melakukan eksploitatif biota laut dan merusak habitat biota laut. Kerusakan Habitat Biota Pasang Surut Laut 5. Kesimpulan Kesimpulan dari penelitian ini adalah: a. Karakteristik wisatawan domestik di Pantai Balekambang Kabupaten Malang 95% wisatawan domestik berasal dari Jawa Timur, usia anak-anak sampai lansia, dengan latar belakang pekerjaan yang heterogen, antara lain: pelajar, mahasiswa, profesional (guru, dosen, dokter, dan lainnya), TNI-POLRI, PNS, wiraswasta, petani dan nelayan, dan masyarakat umum lainnya. Tujuan berwisata ke Balekambang heterogen, yaitu: 90% berlibur, 8% tujuan ekonomi,1% ziarah/ritual, 1% lain-lain; b. Berbagai biota yang dominan ditemukan dan di ambil wisatawan domestik adalah: (1) Hewan: a) Ikan hias, b) Phylum Echinodermata: kelas Echinoidea, yaitu: Diadema setosum, dan Echinometra mathaei, kelas Holothuroidea, yaitu: Holothuria scabra, kelas Ophiuroidea, yaitu: Ophiotrichoides nereidina dan Ophiothrix hybrid (2) Alga: Sargassum, sp., Halimeda, sp., Padina, sp., Fucus, sp. (3) Acropora; c. Perilaku wisatawan domestik terhadap biota di zona pasang surut Pantai Balekambang Kabupaten Malang Undang-Undang Republik Indonesia Nomor 5, 1990 Tentang Konservasi Sumber Daya Alam Hayati Dan Ekosistemnya Dengan Rahmat Tuhan Yang Maha Esa Presiden Republik Indonesia. Yusron, E., 2010. Keanekaragaman jenis ophiuroidea (bintang mengular) di Perairan Wori, Minahasa Utara, Sulawesi Utara. MAKARA, SAINS 14 (1), pp. 75–78. 16
https://openalex.org/W3044872633	https://www.frontiersin.org/articles/10.3389/fped.2020.00416/pdf	English	null	A Survey on Cannabinoid Treatment of Pediatric Epilepsy Among Neuropediatricians in Scandinavia and Germany	Frontiers in pediatrics	2,020	cc-by	3,123	A Survey on Cannabinoid Treatment of Pediatric Epilepsy Among Neuropediatricians in Scandinavia and Germany Claus Klingenberg 1,2, George Mouslet 1, Helle Hjalgrim 3 and Thorsten Gerstner 4 1 Paediatric Research Group, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway, 2 Department of Paediatrics and Adolescent Medicine, University Hospital of North-Norway, Tromsø, Norway, 3 Clinic for Children, Værløse, Denmark, 4 Department of Child Neurology and Rehabilitation (HABU-A), Sørlandet Hospital, Arendal, Norway Objectives: There is an increasing interest in cannabinoid-based products for the treatment of refractory pediatric epilepsy. However, a licensed cannabidiol (CBD) product was ﬁrst approved for use by the European regulatory authorities in 2019. We aimed to obtain knowledge about clinical experience and attitudes toward cannabinoid use for epilepsy treatment among neuropediatricians in Scandinavia and Germany in the era before a CBD-product was commercially licensed and available. Citation: Klingenberg C, Mouslet G, Hjalgrim H and Gerstner T (2020) A Survey on Cannabinoid Treatment of Pediatric Epilepsy Among Neuropediatricians in Scandinavia and Germany. Front. Pediatr. 8:416. doi: 10.3389/fped.2020.00416 Edited by: Pasquale Striano, University of Genoa, Italy Reviewed by: Simona Lattanzi, Marche Polytechnic University, Italy Nicola Pietrafusa, Bambino Gesù Children Hospital (IRCCS), Italy Pasquale Parisi, Sapienza University of Rome, Italy Correspondence: Claus Klingenberg claus.klingenberg@unn.no Edited by: Pasquale Striano, University of Genoa, Italy Study design: An internet-based questionnaire (Survey Monkey) was distributed by email to members of neuropediatric societies in Sweden, Germany, Denmark, and Norway between February and April 2018. One reminder email was sent. Reviewed by: Simona Lattanzi, Marche Polytechnic University, Italy Nicola Pietrafusa, Bambino Gesù Children Hospital (IRCCS), Italy Pasquale Parisi, Sapienza University of Rome, Italy Reviewed by: Simona Lattanzi, Marche Polytechnic University, Italy Nicola Pietrafusa, Bambino Gesù Children Hospital (IRCCS), Italy Pasquale Parisi, Sapienza University of Rome, Italy Results: Eighty-six responded. Only 10 of 86 (12%) respondents had personal experience with off-label prescription of cannabinoid-based products, mainly for severe refractory pediatric epilepsies like Dravet syndrome and Lennox-Gastaut syndrome. However, 49 respondents (57%) had been exposed to relatives of patients that had requested or wanted to discuss cannabinoid therapy, and 32 (37%) respondents knew about cannabinoid self-medication. The knowledge regarding cannabinoid-based therapy among the respondents was overall limited. Main reasons for not prescribing cannabinoid-based therapy were concerns about law regulations and lack of an available product. Specialty section: This article was submitted to Pediatric Neurology, a section of the journal Frontiers in Pediatrics Conclusion: Off-label cannabinoid-based therapy for pediatric epilepsy was not widely prescribed by neuropediatricians in Scandinavia and Germany in 2018. Received: 25 April 2020 Accepted: 16 June 2020 Published: 24 July 2020 Keywords: cannabinoid, refractory epilepsy, Dravet syndrome, Lennox-Gastaut syndrome, survey ORIGINAL RESEARCH published: 24 July 2020 doi: 10.3389/fped.2020.00416 Keywords: cannabinoid, refractory epilepsy, Dravet syndrome, Lennox-Gastaut syndrome, survey INTRODUCTION Over the last decade there has been an increasing interest in cannabinoid-based products for the treatment of refractory pediatric epilepsy (1). In animal studies cannabidiol (CBD) shows an anticonvulsant proﬁle largely devoid of the adverse psychoactive eﬀects which is mainly related to the tetrahydrocannabinol compound of cannabinoids (1). Anti-seizure properties of CBD are July 2020 \| Volume 8 \| Article 416 Frontiers in Pediatrics \| www.frontiersin.org A Survey on Cannabinoid Treatment of Pediatric Epilepsy Klingenberg et al. TABLE 1 \| Response from 86 neuropaediatricians from Sweden (n = 26), Denmark (n = 17), Norway (n = 23), and Germany (n = 20) on aspects related to cannabinoid therapy for epilepsy. Respondents who had prescribed cannabinoid-therapy (n = 10) Indications x N (%) • Lennox-Gastaut syndrome 5 (50) • Dravet syndrome 4 (40) • Infantile spasms 2 (20) • Tuberous sclerosis 2 (20) Perceived efﬁcacy x • Good seizure burden reduction (> 90%) 0 (0) • Moderate seizure burden reduction (> 50%) 4 (40) • Shortening of seizures 1 (6) • Less “severe seizures” 4 (40) • Better life quality 4 (40) • Improved cognition 2 (20) • No obvious effects 4 (40) Respondents who had not prescribed cannabinoid-therapy (n = 76) Reasons for not prescribing x • No licensed product available 25 (33) • National law regulations prohibits prescription 14 (18) • Skepticism toward efﬁcacy 21 (28) • Lack of knowledge/experience 39 (51) • Other 22 (29) xMultiple answers possible. TABLE 1 \| Response from 86 neuropaediatricians from Sweden (n = 26), Denmark (n = 17), Norway (n = 23), and Germany (n = 20) on aspects related to cannabinoid therapy for epilepsy. probably mediated by modulation of intracellular calcium levels, through inhibition of the G-protein coupled receptor GPR55 and activation of the transient receptor channel TRPV1, and the inhibition of adenosine re-uptake (2). Until a few years ago most data on the use of CBD-enriched extracts to treat pediatric epilepsy had been based on observational studies, mainly from the USA. Since 2016, three randomized placebo-controlled adjunctive- therapy trials of puriﬁed CBD-products including children and adolescents with refractory epilepsy have been published (3– 5). In these trials, CBD was superior to placebo in reducing the frequency of convulsive seizures in patients with Dravet syndrome (3), and in reducing the frequency of drop-seizures in patients with Lennox-Gastaut syndrome (LGS) (4, 5). INTRODUCTION The objective of this survey was to obtain knowledge about clinical experience and attitudes toward cannabinoid use for epilepsy treatment among neuropediatricians in Scandinavia and Germany in the era before a licensed product was available. MATERIALS AND METHODS Between February and April 2018 we distributed via email an internet-based survey (Survey Monkey, Portland, OR, USA) to members of neuropediatric societies in Sweden, Denmark, and Norway. We assume that around 250–300 active neuropediatricians work in Scandinavia, but the sub- specialization is not uniformly formalized and the number of members of the neuropediatric societies is therefore uncertain and may not reﬂect all neuropediatricians treating children with epilepsy. We also sent the survey to a random group of 140 neuropediatricians in Germany. Email addresses were obtained from society web sites or email lists, and distributed by designated persons (see acknowledgment section). CBD-formulation. The indications for using cannabinoids and the perceived eﬃcacy are shown in Table 1. We also asked for adverse eﬀects when cannabinoids were prescribed. Lethargy/drowsiness, gastrointestinal symptoms, and decreased appetite where those most frequently reported. We did not ask for liver function abnormalities and none of the respondents spontaneously reported this side eﬀect. The survey consisted of 16 questions using a combination of open and ﬁxed-response questions (Appendix in Supplementary Material). We asked speciﬁc questions around knowledge, experience and attitude regarding cannabinoid use for pediatric epilepsy. Moreover, we asked whether parents or relatives had asked for these treatments for their child, and to what extent they knew about self-medication with cannabinoids. Follow-up for non-responders was by one-1 reminder email. Data are presented as number and percentages. The 76 respondents who had not prescribed cannabinoids for pediatric epilepsy gave several reasons for not using this therapy (Table 1). However, 49/76 (64%) had been exposed to parents or relatives of patients that had requested or wanted to discuss cannabinoid therapy. Moreover, 32 of all 86 respondents (37%) were aware of cannabinoid self-medication, not prescribed by a doctor. Frontiers in Pediatrics \| www.frontiersin.org RESULTS This is the ﬁrst survey on clinical experience and attitudes toward use of cannabinoid therapy for refractory epilepsy among neuropediatricians in North-Europe. A recent, similar online survey reported data from 155 physicians treating children or adolescents for epilepsy within eight countries in Central- and South-Europe (7). In that survey 45% of the respondents prescribed oﬀ-label cannabinoids (mainly CBD) for epilepsy, but the response rate was not presented (5). Moreover, the authors reported that individual experience with cannabinoid therapy among responders was limited, and there were diverse opinions about the use of CBD and how to manage the CBD treatment (7). Eighty-six neuropediatricians responded; 42 (49%) were females (Table 1). Fifty-six respondents (65%) had more than 10 years of clinical experience in neuropediatrics, and 57 (66%) answered that they treat children with epilepsy at least every week. Eighty-one respondents (94%) had heard about of the use of cannabinoids for treating epilepsy in children. Forty-seven (55%) knew that CBD, and not THC, is the compound of cannabinoids that is suggested to be most important for anti-epileptic activity. Only 10 respondents (12%) had personally prescribed oﬀ- label cannabinoids for treatment of children with epilepsy. These caregivers had mostly prescribed a CBD oil or another puriﬁed July 2020 \| Volume 8 \| Article 416 2 A Survey on Cannabinoid Treatment of Pediatric Epilepsy Klingenberg et al. same time, clobazam may increase an active metabolite of CBD, which could lead to better seizure control. Overall, there are still many unanswered questions regarding the impact of interactions between CBD and other AEDs. However, due to an increased risk of side eﬀects such as somnolence and increased liver enzymes, close clinical and biochemical monitoring is recommended (6, 10, 11). Moreover, it has to be emphasized that clinicians need to report all possible severe side eﬀects, preferably by enrolling patients in post-authorization Phase IV safety studies. The main ﬁnding of the current survey was that very few neuropediatricians in Scandinavia and Germany had prescribed oﬀ-label cannabinoid therapy for pediatric epilepsy. However, those who had prescribed this therapy followed the indications used in recent clinical trials on CBD-based therapy for refractory epilepsies (3–5). p p Respondents that had not prescribed cannabinoid-based therapy gave diﬀerent reasons. Concerns about law regulations and lack of an available product (oﬀ-label use) was commonly cited. RESULTS This may change since a puriﬁed CBD-formulation (Epidiolex R⃝/Epidyolex R⃝, GW Pharma) is approved by both the American Food and Drug Administration (June 2018) and the European Medicine Agency (September 2019). Recent clinical trials support a possible adjunctive role of CBD-based therapy for selected refractory epilepsies such as Dravet syndrome and LGS, two of the most severe and diﬃcult-to-treat forms of childhood onset epilepsy (3–5). Long-term CBD treatment has showed an acceptable safety proﬁle and a sustained reductions in seizure frequency in patients with these two epileptic encephalopathies (2, 8–10). This was conﬁrmed in two recently published systematic reviews on CBD therapy including nearly 800 patients (children and adults) with Dravet syndrome (2) and LGS (9). These systematic reviews concluded that adjunctive add- on CBD therapy resulted in a signiﬁcant reduction in seizure frequency compared to placebo, but also in a higher rate of adverse events (AEs). Typical AEs were somnolence, sedation, decreased appetite, pneumonia, diarrhea, and increased liver enzymes. Nevertheless, the overall conclusion was that add- on CBD is an eﬃcacious long-term treatment option for these group of patients and that CBD is well-tolerated. CBD has relatively few side eﬀects, but interactions with other medications should be monitored carefully (2, 9, 10). It also has to be acknowledged that only a puriﬁed, pharmaceutical formulation of cannabidiol has been authorized, and data from RCTs included in these systematic reviews cannot be extrapolated to other cannabinoid products. The greatest limitation with our survey, and the one published by Klotz et al. (7), is that that we do not have the exact number of neuropediatricians that received the survey. We can therefore not give an exact response rate. Certainly many neuropediatricians on our email lists may in their daily practice focus on other pediatric neurology subspecialties (e.g., congenital neuromuscular disorders, cerebral palsy, and neurohabilitation) and thus do not have the same focused interest in or experience with therapy of refractory epilepsies. However, those who responded were overall very experienced neuropediatricians and around 2/3 were treating children with epilepsy on a weekly basis. We speculate that many of those who did not respond were less interested or familiar with the topic of CBD therapy and refractory epilepsies. In conclusion, this survey indicates that oﬀ-label cannabinoid- based therapy for pediatric epilepsy in children was not widely prescribed by neuropediatricians in Scandinavia and Germany in 2018. RESULTS However, self-medication and parental request for this therapy was common. This may reﬂect a previously reported disparity in opinion on the use of cannabinoids in epilepsy treatment with fewer medical specialists supporting its use compared with patients and the public (12). Evidence from recent clinical trials and improved availability of an approved CBD formulation will most likely change the situation regarding CBD-based therapy for pediatric refractory epilepsy in the future. The lack of clinical experience, but also theoretical knowledge, with CBD therapy for epilepsy in children was striking in our survey. In other countries, and in particular in the USA, oﬀ- label use of cannabinoids has increased over the last decade (1). Before initiating CBD-therapy clinicians must be aware of the most common side eﬀects such as gastrointestinal problems, drowsiness, and liver function abnormalities (1, 3–5, 7, 8). Moreover, it is of great importance that interactions with commonly used anti-epileptic drugs (AEDs) are appreciated by doctors prescribing CBD-based therapy (1, 5, 6, 11). It has been shown that CBD may exhibit numerous interactions with other AEDs, both enzyme inducers (such as carbamazepine or phenytoin) and inhibitors (such as valproic acid), even if the clinical signiﬁcance not yet is clear. CBD may have an impact on liver metabolism with signiﬁcant elevation of liver enzymes in up to 10% of treated patients, of whom more than three quarter had received concomitant valproic acid therapy. In addition, there are clinically signiﬁcant interaction between CBD and clobazam, an AED widely used in patients with both Dravet and LGS. The use of CBD (by inhibition of CYP2C19) may lead to a signiﬁcant increase in clobazam metabolites, leading to toxicity. At the DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS CK and GM had primary responsibility for the idea and design of this survey, survey distribution in Norway, data analysis and writing of the manuscript. TG participated in the design of the survey, distribution of the survey in Germany and contributed with writing of the manuscript. HH participated in the design of the survey, distribution of the survey in Denmark and writing of the manuscript. All authors have read and approved the manuscript before submission. Frontiers in Pediatrics \| www.frontiersin.org REFERENCES 9. Lattanzi S, Brigo F, Cagnetti C, Trinka E, Silvestrini M. Eﬃcacy and safety of adjunctive cannabidiol in patients with Lennox-Gastaut syndrome: a systematic review and meta-analysis. CNS Drugs. (2018) 32:905– 16. doi: 10.1007/s40263-018-0558-9 1. Perucca E. Cannabinoids in the treatment of epilepsy: hard evidence at last? J Epilepsy Res. (2017) 7:61–76. doi: 10.14581/jer.17012 1. Perucca E. Cannabinoids in the treatment of epilepsy: hard evidence at last? J Epilepsy Res. (2017) 7:61–76. doi: 10.14581/jer.17012 2. Lattanzi S, Brigo F, Trinka E, Zaccara G, Striano P, Del Giovane C, et al. Adjunctive cannabidiol in patients with Dravet syndrome: a systematic review and meta-analysis of eﬃcacy and safety. CNS Drugs. (2020) 34:229– 41. doi: 10.1007/s40263-020-00708-6 10. Chesney E, Oliver D, Green A, Sovi S, Wilson J, Englund A, et al. Adverse eﬀects of cannabidiol: a systematic review and meta- analysis of randomized clinical trials. Neuropsychopharmacology. (2020). doi: 10.1038/s41386-020-0667-2. [Epub ahead of print]. 3. Devinsky O, Cross JH, Laux L, Marsh E, Miller I, Nabbout R, et al. Trial of cannabidiol for drug-resistant seizures in the Dravet syndrome. N Engl J Med. (2017) 376:2011–20. doi: 10.1056/NEJMoa1611618 3. Devinsky O, Cross JH, Laux L, Marsh E, Miller I, Nabbout R, et al. Trial of cannabidiol for drug-resistant seizures in the Dravet syndrome. N Engl J Med. (2017) 376:2011–20. doi: 10.1056/NEJMoa1611618 11. Arzimanoglou A, Brandl U, Cross JH, Gil-Nagel A, Lagae L, Landmark CJ, et al. Epilepsy and cannabidiol: a guide to treatment. Epileptic Disord. (2020) 22:1–14. doi: 10.1684/epd.2020.1141 12. Mathern GW, Beninsig L, Nehlig A. Fewer specialists support using medical marijuana and CBD in treating epilepsy patients compared with other medical professionals and patients: result of Epilepsia’s survey. Epilepsia. (2015) 56:1– 6. doi: 10.1111/epi.12843 4. Thiele EA, Marsh ED, French JA, Mazurkiewicz-Beldzinska M, Benbadis SR, Joshi C, et al. Cannabidiol in patients with seizures associated with Lennox-Gastaut syndrome (GWPCARE4): a randomised, double- blind, placebo-controlled phase 3 trial. Lancet. (2018) 391:1085–96. doi: 10.1016/S0140-6736(18)30136-3 5. Devinsky O, Patel AD, Cross JH, Villanueva V, Wirrell EC, Privitera M, et al. Eﬀect of Cannabidiol on drop seizures in the Lennox-Gastaut syndrome. N Engl J Med. (2018) 378:1888–97. doi: 10.1056/NEJMoa1714631 5. Devinsky O, Patel AD, Cross JH, Villanueva V, Wirrell EC, Privitera M, et al. Eﬀect of Cannabidiol on drop seizures in the Lennox-Gastaut syndrome. N Engl J Med. (2018) 378:1888–97. ACKNOWLEDGMENTS The publication charges for this article have been funded by a grant from the publication fund of UiT the Arctic University July 2020 \| Volume 8 \| Article 416 3 A Survey on Cannabinoid Treatment of Pediatric Epilepsy Klingenberg et al. SUPPLEMENTARY MATERIAL of Norway. We thank Björn Bjurulf for input to design of the survey, Jakob Bie Granild-Jensen for distributing the survey in Denmark and Martin Jägervall/Roland Flink for distributing the survey in Sweden. We also thank all participants of this survey. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped. 2020.00416/full#supplementary-material Frontiers in Pediatrics \| www.frontiersin.org REFERENCES doi: 10.1056/NEJMoa1714631 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. 6. Lattanzi S, Brigo F, Trinka E, Zaccara G, Cagnetti C, Del Giovane C, et al. Eﬃcacy and safety of cannabidiol in epilepsy: a systematic review and meta- analysis. CNS Drugs. (2018) 78:1791–804. doi: 10.1007/s40265-018-0992-5 Copyright © 2020 Klingenberg, Mouslet, Hjalgrim and Gerstner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2020 Klingenberg, Mouslet, Hjalgrim and Gerstner. 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. 7. Klotz KA, Schulze-Bonhage A, Antonio-Arce VS, Jacobs J. Cannabidiol for treatment of childhood epilepsy-a cross-sectional survey. Front Neurol. (2018) 9:731. doi: 10.3389/fneur.2018.00731 7. Klotz KA, Schulze-Bonhage A, Antonio-Arce VS, Jacobs J. Cannabidiol for treatment of childhood epilepsy-a cross-sectional survey. Front Neurol. (2018) 9:731. doi: 10.3389/fneur.2018.00731 8. Devinsky O, Nabbout R, Miller I, Linda Laux L, Zolnowska M, Wright S, et al. Long-term cannabidiol treatment in patients with Dravet syndrome: an open- label extension trial. Epilepsia. (2019) 60:294–302. doi: 10.1111/epi.14628 July 2020 \| Volume 8 \| Article 416 Frontiers in Pediatrics \| www.frontiersin.org 4
https://openalex.org/W2975669305	https://www.matec-conferences.org/articles/matecconf/pdf/2019/41/matecconf_cscc2019_01003.pdf	English	null	Personalized Tour Recommender through Geotagged Photo Mining and LSTM Neural Networks	MATEC web of conferences	2,019	cc-by	4,169	1 Introduction recommendations based on visit histories and timestamps in order to compute an estimate of the visiting time and transportation time from one place to another. [6] developed a collaborative filtering system based on contextual information of geotagged photos. Their framework considers 5 aspects: the user information, the geotags, the timestamps, the tags and visual information. They make use of these elements to calculate user similarity, attraction and popularity scores among users and locations as well as the most popular visiting times. Many social media websites, such as Instagram or Foursquare, serve as platforms to gather the data that their users share. With the help of these valuable data, it is possible to discover hidden travel patterns from the user’s behaviour. The patterns can help us have insight about particular characteristics on different users, and make prediction or classification of future events under an acceptable margin of error [1]. Therefore, there has been an increasing interest and research in developing intelligent tour recommendation systems (or recommenders) to help travellers arrange their trips. g [ 7 ] proposed a trip planner by comparing the performance of different heuristic algorithms to optimize the time, the total travelled distance, and the overall satisfaction of users on an unfamiliar city. [8] established a topic based context-aware travel recommendation method based on geotagged photos. Their method is built on top of a topic model which mines the interest distribution of users, and is then used to build a user– user similarity model to make travel recommendations. [9] leveraged geotagged photos obtained from Flickr; they inferred landmarks by means of clustering and then generated recommended routing from separate roads instead of historic trajectories of users. [ 10 ] used clustering to extract landmarks and applied topic model to characterize these landmarks. The objective was of obtaining areas of interest and their corresponding landmarks. Furthermore, the landmark vector resulting from the topic model was used to calculate a subsequent user vector. Typically, the recommenders can be distinguished into three main approaches: collaborative filtering, content-based filtering, and hybrid recommender systems. Among them, collaborative filtering is one of the most popular ones and has been applied in many tour recommenders with different purposes. [2] proposed a framework that takes the user’s visit history to make recommendations after categorizing each tags included on each picture. Personalized Tour Recommender through Geotagged Photo Mining and LSTM Neural Networks Chieh-Yuan Tsai1,, Gerardo Paniagua1, Yu-Jen Chen1, Chih-Chung Lo2 and Liguo Yao3 1Department of Industrial Engineering and Management, Yuan Ze University, Taoyuan City, 32003, Taiwan, R.O.C. 2Department of Applied Informatics, Fo Guang University, Yilan County 26247, Taiwan, R.O.C. 3Key Laboratory of Advanced Manufacturing Technology, Ministry of Education, Guizhou University, Guiyang 550025, China Abstract. In this study, a tour recommendation system based on social media photos is proposed. The proposed recommendation system can generate trip tours considering both the user’s current location and interests. First, we exploited the geotagged photo dataset from social media websites, which includes photo related information such as user ID numbers, timestamps, hashtags, and GPS coordinates. With this information, the second step is to group photos and identify those places that could be considered relevant for travellers using clustering algorithms. The third step characterizes the resulting clusters by grouping them into different categories using latent dirichlet allocation (LDA) topic modelling approach. The last step is the generation of tours using a long-short term memory neural network (LSTM). The experiments show that the proposed system can be efficient to advise future travellers about the places they would be more likely to visit and arrange trips for them. , 0 (2019) MATEC Web of Conferences 292 100 CSCC 2019 3 , 0 (2019) MATEC Web of Conferences 292 100 CSCC 2019 3 https://doi.org/10.1051/matecconf/2019292010033 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Corresponding author: cytsai@saturn.yzu.edu.tw DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) 1 Introduction [3] used user generated GPS traces in combination with a tree based hierarchical graph model in order to extract location histories and then propose a HITS (Hypertext Induced Topic Search)-based model to infer the interest level of a location and a user’s travel experience. [ 4 ] used RFID sensors to train a recommender for tourists in a theme park to deliver recommendations for different areas, including the sequential orders to visit them. [5] collected several check-in datasets to make time sensitive tours , 0 (2019) MATEC Web of Conferences 292 100 CSCC 2019 3 , 0 (2019) MATEC Web of Conferences 292 100 CSCC 2019 3 https://doi.org/10.1051/matecconf/2019292010033 numbers. All this information was stored to later cleaned it, pre-processed it, and fed it into the model we trained. Although the above researches have demonstrated themselves to be useful at recommending new places for people to visit in an unfamiliar city, we found some problems that they still need to solve. Firstly, most of them fail to transform their recommended venues from a list format to a tour. Even after getting a complete list with the best touristic attractions on a city, many users may find it tedious to plot an itinerary by themselves including the many point of interests (POI) to visit. Secondly, those lists are ranked based on the opinion of the majorities of the users. People have different preferences, hence not everyone will be satisfied with just the most popular recommendation. Thirdly, many of these recommenders ignore some important considerations such as the user’s interests and current location. 2.3 Location clustering The second step is to group photos and identify those places that could be considered relevant for travellers using clustering algorithms. The clustering algorithm can help us to separate the area of a city into several small neighbouring areas according to every photo’s geographic positions, their frequency density, and the distances among each point. In this study, mean shift clustering algorithm is adopted for landmark extraction [ 11 ]. Mean shift clustering algorithm is a non-parametric algorithm can map the probabilistic distribution from a spatial dataset outlining landmarks based on clusters with higher photo density. This can be achieved by seeding this procedure from many initial points iterating this process until a convergence. The mean shift is defined as: To solve the above problems, a recommendation system based on a social media photo dataset is proposed. The proposed recommendation system can generate trip tours considering both the user’s current location and interests. First, we exploited the geotagged photo dataset from social media websites. With this information, we can transform the users’ travel log histories into trajectories and later use them to train a recommendation system to advise future travellers about the places they would be more likely to visit and arrange trips for them. 𝑚ℎ,𝐺(𝑥) = ∑ 𝑥𝑖\|\|(𝑥−𝑥𝑖)/ℎ\|\|2 𝑛 𝑖=1 ∑ 𝑔\|\|(𝑥−𝑥𝑖)/ℎ\|\|2 𝑛 𝑖=1 −𝑥 (1) (1) where g is the weight assigned to each point regarded to the kernel function chosen, and h represents the bandwidth parameter. The given location is represented as x, and xi represents the neighbouring points. For analysis involving landmark extraction, a Gaussian distribution is commonly set up in the kernel function G. 2.4 Landmark characterization. The framework of the proposed tour recommender is outlined in Figure 1which includes four important steps: data collection, location clustering, landmark characterization, and tour recommendation. The third step is to characterize the resulting clusters by grouping them into different categories. This was done by analysing the textual descriptions contained on the hashtags attached to each photo by using a topic modelling algorithm. The topic modeller applied in our work is Latent Dirichlet Allocation, or LDA in short. One of the LDA’s most important characteristics is that it can assign one word to more than one group at the same time. Like in English, one single word can have different meaning depending on the context used. This method has been frequently used in natural language processing in application such as tag recommendations, topic inference, and document summarization. , Fig. 1. The proposed tour recommender framework. LDA is a generative model that represents documents as being generated by a random mixture over latent variables called topics [12]. A topic is defined as a distribution over words. For a given corpus (a collection of documents) of D documents each of length Nd , the generative process for LDA is defined as follows: Fig. 1. The proposed tour recommender framework. 2.5 Tour recommendation Given the value of the input gate activation is, the forget gate activation fs and the candidate state value 𝐶̃𝑠, the new cell state vector Cs can be computed at timestamp s: The last step in our framework is the generation of tours. In this study, a long-short term memory neural network (LSTM) is applied to make the route recommendations. LSTM is a recurrent neural network (RNN) architecture that has good performance and a wide range of applications like text generation and analysis of sequences [13, 14]. This method belongs to the family of machine learning models, more specifically to deep learning. RNN has the peculiarity that its signals can travel in both directions, back and forward. There are many variations of RNN models such as bidirectional RNN, gated recurrent units, recursive neural networks, long-short term memory neural network (LSTM). p 𝐶𝑠= 𝑖𝑠× 𝐶̃𝑠+ 𝑓𝑠× 𝐶𝑠−1 (5) (5) 𝐶𝑠 𝑖𝑠× 𝐶𝑠+ 𝑓𝑠× 𝐶𝑠−1 (5) With the new state of the memory cells, the value of their output gates and, subsequently, their outputs can be computed: 𝐶𝑠 𝑖𝑠 𝐶𝑠+ 𝑓𝑠 𝐶𝑠−1 (5) With the new state of the memory cells, the value of their output gates and, subsequently, their outputs can be computed: p   1 s o s o s o o W x U h b      (6)   1 s s s h o tanh C    (7) (6) (7) (6) (6) (7) A particular LSTM structure called sequence to sequence (seq2seq) model is implemented for event predictions by using touristic landmarks and travelling routes. This model structure is very useful since it offers flexibility at handling the length of both, inputs and outputs. With this particular architecture, it is easier to generate tours under a specified length. g y ( ) Different to traditonal RNN, the LSTM model introduces a new structure called a memory cell. A memory cell, as shown in Figure 2, is composed of four main elements: an input gate, a neuron with a self- recurrent connection (a connection to itself), a forget gate and an output gate. The self-recurrent connection has a weight of 1.0 and ensures that, barring any outside interference, the state of a memory cell can remain constant from one timestamp to another. The gates serve to modulate the interactions between the memory cell itself and its environment. 2.5 Tour recommendation The input gate can allow incoming signal to alter the state of the memory cell or block it. On the other hand, the output gate can allow the state of the memory cell to have an effect on other neurons or prevent it. Finally, the forget gate can modulate the memory cell’s self-recurrent connection, allowing the cell to remember or forget its previous state, as needed. 2.2 Data collection The development of this framework relies on building up a photo dataset to analyse the trajectories followed by the travellers and map the photos taken by the users in order to represent them as Point of Interest (POI). Each POI will constitute an attractive place for tourists such as monuments, museums, parks, and so on. In this study, we will build up the groups of candidates for these POI from social media websites and extract the content from the photo metadata such as geolocation, tags, number of views, timestamp, and encrypted user ID 1. For each topic k, draw a distribution over words ϕk ∼ p ϕ Dirichlet(β) with k ={1,2,...,K} 2. For each document d: Dirichlet(β) with k ={1,2,...,K} 2. For each document d: Dirichlet(β) with k ={1,2,...,K} 2. For each document d: a. Draw a vector of topic proportions θd ∼ Dirichlet(α) b. For each word i i. Draw a topic assignment zd,n ∼ multinomial(θd) with zd,n ∈ {1,2,...,K} i. Draw a topic assignment zd,n ∼ multinomial(θd) with zd,n ∈ {1,2,...,K} ii. Draw a word wd,v ∼ multinomial(ϕk = zd,n ) with wd,v ∈{1,2,...,V} ii. Draw a word wd,v ∼ multinomial(ϕk = zd,n ) with wd,v ∈{1,2,...,V} 2 , 0 (2019) MATEC Web of Conferences 292 100 CSCC 2019 3 https://doi.org/10.1051/matecconf/2019292010033 Note: Only the words w are observed. Second, the forget gate activation fs will be computed at timestamp s: p p   1 s f s f s f f W x U h b      (4) (4) 3 Implementation and experiments 3.1 Flickr data collection and pre-processing First, we built up our dataset by querying photos from Flickr using as a reference the latitude and longitude of the centre of the city of San Francisco, California, under a radius of 5km. The resulting dataset was equivalent to a total of 514,765 pictures containing descriptive tags and geographic coordinates belonging to 15,117 unique users. After we merged the data into one file, we removed the duplicates and analysed the characters on each tag in order to exclude those words containing non-English characters. Forget gate fs Self-recurrent connection Memory cell output hs Output gate os Input gate is Memory cell input xs Fig. 2. The long short-term memory cell. Forget gate fs 3.2 Mean shift clustering Self-recurrent connection With mean shift clustering algorithm, landmarks can be extracted by adjusting the value of the bandwidth until the average distance is short enough. The bandwidth, which work as a windows size, is an argument associated to the convergence rate, density estimation and hence it will influences the resulting number of clusters. For example, a lower bandwidth will generate a higher amount of clusters but it will also slow down the convergence. After using mean shift to analyse the set of latitudes and longitudes from our dataset we got the results shown on Figure 3. Memory cell output hs Memory cell input xs Fig. 2. The long short-term memory cell. Fig. 3. Number of clusters and mean distances by using mean shift clustering algorithm. The equations below describe how a layer of memory cell is updated at every timestamp. xs is the input to the memory cell layer at timestamp s, W and U are weight matrices, b is a bias vector. First, the input gate is will be computed, and the candidate value 𝐶̃𝑠 is for the states of the mem ory cells at timestamp s: ory cells at timestamp s:   1 s i s i s i i W x U h b      (2) (2) 𝐶̃𝑠= tanh(𝑊𝑐𝑥𝑠+ 𝑈𝑐ℎ𝑠−1 + 𝑏𝑓) (3) 𝐶̃𝑠= tanh(𝑊𝑐𝑥𝑠+ 𝑈𝑐ℎ𝑠−1 + 𝑏𝑓) (3) where is a sigmoid activation function and tanh is (3) 𝐶𝑠= tanh(𝑊𝑐𝑥𝑠+ 𝑈𝑐ℎ𝑠−1 + 𝑏𝑓) (3) where  is a sigmoid activation function and tanh is a hyperbolic tangent activation function. Fig. 3. Number of clusters and mean distances by using mean shift clustering algorithm. 𝑓 where  is a sigmoid activation function and tanh is a hyperbolic tangent activation function. where  is a sigmoid activation function and tanh is a hyperbolic tangent activation function. 3 3 , 0 (2019) MATEC Web of Conferences 292 100 CSCC 2019 3 https://doi.org/10.1051/matecconf/2019292010033 Fig. 5. LDA coherence score by number of topics. While a very small bandwidth value might result in too many clusters, a large value might result in very few, merging some of them. Therefore, a short average distance is desired while trying to keep the right amount of clusters. Hence, by using mean shift clustering we decided to choose 0.0150 as the best bandwidth value, generating with this a total of 160 clusters and a mean distance of 3.65 km. 3.2 Mean shift clustering Figure 4 shows how the identified landmarks are well-distributed through entire area of San Francisco city. Fig. 5. LDA coherence score by number of topics. On Figure 5, the amount of topics we tried vary from 4 to 50 by steps of 2. The larger the coherence score the better the results. As it can be noticed, the coherence doesn’t improve much when the model has more than 32 topics (Ct =0.5269). We also compared this results by using LDA with different number of topics such as 30, 24, 20, but the most frequent words among their topics were not as coherent as those gotten from the LDA with 32 topics, then we decided to use 32 topics for the further analysis of this framework. For example, the ten most important words on topic number 30 and their weights are as follow: summer (17.0%), painting (5.8%), green (3.9%), sanfranciscobotanicalgarden (3.0%), potrerohill (2.7%), animal (2.6%), nature (2.2%), sport (2.0%), decoration (1.7%), pink (1.5%). Clearly, topic 30 could be defined as “nature” or “park” since most of these words are related to these terms. Fig. 4. The 160 attractive landmarks by using mean shift clustering. After our LDA model was defined with the right number of topics, the next step consists on assigning every cluster or landmark obtained from our mean shift clustering model to these topics. It is good to remember that by using LDA, one landmark can be assign to one or more topics, which is good since in real life one place could fit into more than one category. In order to decide if a cluster belongs to a topic or not it was necessary to define a threshold parameter. If the probability of a cluster to belong to a specific topic is higher than this threshold, then we can say that it belongs to that topic. After several tries, we found that 0.015 was a threshold value capable to generate good results at distributing the right amount of clusters on each topic. When the threshold was higher than 0.015, very few clusters were assigned on each topics; and when we tried it with a lower threshold we got too many cluster assigned on every topics. Therefore, we choose a threshold value of 0.015 in to continue with the development of our framework. Fig. 4. The 160 attractive landmarks by using mean shift clustering. 3.3 Topic modelling Once potential landmarks have been identified by using clustering algorithms, it is possible to classify those places into different categories (topics) by applying LDA. The LDA method can assign each cluster to one or more topics by analysing the tags contained on every photo and by extracting the similarities among their textual content. LDA has been applied on our photo dataset several times by trying different number of topics in order to find the optimal one. The indicator used to estimate the performance of the results is the semantic coherence value. The semantic coherence has been used on other similar analysis to determine whether the selected number of topics can arrange related words, coherently [15]. The larger the coherence value the more coherent the popular hashtags from a topics will be. This value can be calculated by applying the following formula: 3.4 Tour recommender Next, users need to select one among the 32 topics as their interest so that the generated route will be related to this topic. For example, if the topic we select is about nature, probably the recommended landmarks will be associated to places like parks, beaches, garden, etc. For demonstation purpose, we randomly selected topic 02, which refers to urban related items, as our target. 𝐶𝑡= ∑ ∑ ∑ 𝑙𝑜𝑔 𝐷(𝑡,𝑤𝑖,𝑤𝑗)+1 𝐷(𝑡,𝑤𝑖) 𝑗−1 𝑖=1 𝑚 𝑗=2 \|𝑁\| 𝑡=1 \|𝐾\| (8) (8) \|𝐾\| where Ct represents the coherence value; \|N\| is the amount of topics; m is the value of most frequent word in topic t; D(t, wi, wj) is the number of virtual documents containing wi and wj in topic t; and D(t, wi) represents the amount of virtual documents containing the word wi in the topic t. After we iterated LDA by changing the number of topics, we show the results we got on Figure 5. The next step is to obtain the latitude and longitude of the user’s current location and calculate its nearest centroid. This centroid will represent the starting point of the route recommendation. Once done with this, we trained a traveler behavior modeler to predict the most likely destination to be visited by a new tourist based on 4 , 0 (2019) MATEC Web of Conferences 292 100 CSCC 2019 3 https://doi.org/10.1051/matecconf/2019292010033 the experience of previous travelers. In this modeller, we defined an amount of 125 neurons, a word embedded size of 100, a learning rate of 0.01, a batch size of 100, and 25 epochs. Information and Knowledge Management, 579- 588 (2010). 3. 3. Y. Zheng, and X. Xie, Learning travel recommendations from user-generated GPS traces, Transactions on Intelligent Systems and Technology, 2(1), 2:1-2:29 (2011). Figure 6 shows the four route suggestions when we selected the topic 02 (urban), and a route length of 6 landmarks. The proposed LSTM model needs only less than 1 second to make a recommendation but takes about 3 minutes to finish its training process. Fortunately, the training process will be done just one time by every topic. 4. C. Y. Tsai, and S.H. Chung, A personalized route recommendation service for theme parks using RFID information and tourist behavior, Decision Support Systems 52(2), 514-527 (2012). Fig. 6. Output with LSTM model from four different locations. 5. H. Hsieh, C.T. Li, and S.D. 4. Conclusion 8. 8. Z. Xu, L. Chen, and G. Chen, Topic based context- aware travel recommendation method exploiting geotagged photos, Neurocomputing, 155, 99-107 (2015). After analyzing the geographic coordinates on the photo dataset, we concluded that the mean shift algorithms can generate a suitable result for geospatial clustering. With LDA we successfully grouped related words into topics to later categorize landmarks into topics. Finally, we successfully generate tour recommendations, demonstrating that LSTM model can be used not only for text generation but also to model travelers’ behavior. 9. Y. Sun, H. Fan, M. Bakillah, and A. Zipf, Road- based travel recommendation using geo-tagged images. Computers, Environment and Urban Systems, 53, 110-122 (2015). 10. C.Y. Sun, and A. Lee, Tour reccomendations by mining photo sharing social media, Decision Support Systems, 101, 28-39 (2017). In the future we may consider comparing this framework with the implementation of other modern variation of recurrent neural networks architectures in order to fix the redundancy of landmarks in the generated routes. In a similar way, since it was normal to encounter recommendations where the resulting trajectories went back and forth, we may also consider to combine our framework with heuristic methods with the purpose to replace the recommended routes with more efficient ones by implementing the traveling salesman problem. In this way, tourists will be able to visit more places in the same time, or visit the same number of places in less time. 11. D. J. Crandall, L. Backstrom, D. Huttenlocher, and J. Kleinberg, Mapping the world's photos, Proceedings of the 18th International Conference on World Wide Web, 761-770 (2009). 12. D.M. Blei, A. Y. Ng, M.I. Jordan, Latent dirichlet allocation, Journal of Machine Learning Research, 3(4–5), 993–1022 (2003). 13. N. Tax, I. Verenich, M.L. Rosa, and M. Dumas, Predictive business process monitoring with LSTM neural networks, International Conference on Advanced Information Systems Engineering, 477-492 (2017). ( ) 14. T. Luong, Building Your Own Neural Machine Translation System in TensorFlow. Retrieved on May 25th, 2018 from: https://ai.googleblog.com/2017/07/building-your- own-neural-machine.html (2017). 3.4 Tour recommender Lin, Exploiting large- scale check-in data to recommend time-sensitive routes, Proceedings of the ACM SIGKDD International Workshop on Urban Computing, 55- 62 (2012). 6. K. Jiang, H. Yin, P. Wang, and N. Yu, Learning from contextual information of geo-tagged web photos to rank personalized tourism attractions, Neurocomputing, 119, 17-25 (2013). 7. D. Gavalas, , K. Charalampos, M. Konstantinos, and P. Grammati, A survey on algorithmic approaches for solving tourist trip design problems, Journal of Heuristics, 20(3), 291-328 (2014). Fig. 6. Output with LSTM model from four different locations. 2. T. Kurashima, T. Iwata, G. Irie, and K. Fujimura,Travel route recommendation using geotags in photo sharing sites, Proceedings of the 19th ACM International Conference on References 1. H. Zhang, K. Mohammed, C. David, and L. Gretchen, Mining photo-sharing websites to study ecological phenomena, Proceedings of the 21st International Conference on World Wide Web, 749-758 (2012). 15. D. Mimno, H.M. Wallach, E. Talley, M. Leenders, and A. McCallum, Optimizing semantic coherence in topic models, Proceedings of the Conference on Empirical Methods in Natural Language Processing, 262-272 (2011). 2. T. Kurashima, T. Iwata, G. Irie, and K. Fujimura,Travel route recommendation using geotags in photo sharing sites, Proceedings of the 19th ACM International Conference on 2. T. Kurashima, T. Iwata, G. Irie, and K. Fujimura,Travel route recommendation using geotags in photo sharing sites, Proceedings of the 19th ACM International Conference on 5
https://openalex.org/W4246254076	https://www.researchsquare.com/article/rs-9050/v2.pdf?c=1631846687000	English	null	Ethnomedicine investigation of the medicinal plants and animals in Daur, Inner Mongolia, China	Research Square (Research Square)	2,020	cc-by	10,329	Ethnomedicine investigation of the medicinal plants and animals in Daur, Inner Mongolia, China Xue Li New Inner Mongolia Medical University Ya-Qiong Bi Inner Mongolia Autonomous Region Academy of Traditional Medicin Le-Tai Yi Inner Mongolia Autonomous Region Academy of Traditional Medicin Ying Xia New Inner Mongolia Autonomous Region Academy of Traditional Medicin Lei Zhang Inner Mongolia Medical University Ya-Hong Sun Hailar Bureau of Science and Technology Suo Zhang New Inner Mongolia Medical University Min-Hui Li (  prof_liminhui@yeah.net ) Baotou Medical College https://orcid.org/0000-0002-5366-8464 Research Keywords: Daur, Hulunuir area, Plant medicines, Animal medicines, Eth Posted Date: March 26th, 2020 DOI: https://doi.org/10.21203/rs.2.18430/v2 License:   This work is licensed under a Creative Commons Attribu Xue Li New Inner Mongolia Medical University Ya-Qiong Bi Inner Mongolia Autonomous Region Academy of Traditional Medicine Le-Tai Yi Inner Mongolia Autonomous Region Academy of Traditional Medicine Ying Xia New Inner Mongolia Autonomous Region Academy of Traditional Medicine Lei Zhang Inner Mongolia Medical University Ya-Hong Sun Hailar Bureau of Science and Technology Suo Zhang New Inner Mongolia Medical University Min-Hui Li (  prof_liminhui@yeah.net ) Baotou Medical College https://orcid.org/0000-0002-5366-8464 Research Keywords: Daur, Hulunuir area, Plant medicines, Animal medicines, Ethnomedicine Posted Date: March 26th, 2020 DOI: https://doi.org/10.21203/rs.2.18430/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Xue Li New Inner Mongolia Medical University Ya-Qiong Bi Inner Mongolia Autonomous Region Academy of Traditional Medicine Le-Tai Yi Inner Mongolia Autonomous Region Academy of Traditional Medicine Ying Xia New Inner Mongolia Autonomous Region Academy of Traditional Medicine Lei Zhang Inner Mongolia Medical University Ya-Hong Sun Hailar Bureau of Science and Technology Suo Zhang New Inner Mongolia Medical University Min-Hui Li (  prof_liminhui@yeah.net ) Baotou Medical College https://orcid.org/0000-0002-5366-8464 Ethnomedicine investigation of the medicinal plants and animals in Daur, Inner Mongolia, China Background In the past, many ethnic groups have identified a wide variety of valuable plants and animals that can be used for therapeutic purposes. This empirical knowledge has led to the development of ethnomedicine, i.e., a summary of the experience of different nationalities regarding the treatment of diseases via the use of plants, animals, and minerals in the environment [1–3]. Nowadays, ethnobotany, which includes the study of phytotherapy, is a discipline that can transform popular knowledge into scientific knowledge [4]. The Daur nationality is one of China’s ethnic minorities, and it is one of the “three minors” (Olunchun, Ewenki, and Daur), centrally distributed in Olunchun Autonomous County, Ewenki Autonomous County, and Moridawadaer Autonomous County, respectively, in the Inner Mongolia Autonomous Region. Since the 1970s, research into ethnic minority medicine in China has received increasing attention, especially in the field of medical literature collation, drug resource investigation, development, and utilization. The 2010 Census found that the Daur population in Inner Mongolia was 76255, accounting for 0.31% of the resident population in Inner Mongolia [5]. However, the influence of other ethnic groups has exacerbated the loss of medical knowledge of the ethnic group, which has underestimated the traditional value of the younger generation. Over thousands of years, the Daur people developed medical knowledge with local ethnic characteristics in the struggle against various diseases, and folk medicines that have real curative effects are still used in the Daur region. Records of the use of Daur medicine go back more than 300 years to the Qing Dynasty [6]. Zhang [7] et al. investigated the traditional medicine practices of the Daur ethnic group in Inner Mongolia, especially the traditional authentic medicinal materials and important ethnic medicine resource by consulting the literature records, visiting practitioners, investigating folk traditional knowledge, collecting medicinal fungi and plant specimens, and organizing the original records of the herbarium. The investigation and arrangement according to the geographical distribution of the population provided an objective and scientific basis for the development and utilization of the resource of Daur traditional medicine. Apart from this, Liu [8] et al. researched and investigated 31 ethnic medicines and their uses by the Daur people living in the Hulunbuir League of Inner Mongolia, which reflected the characteristics of Daur ethnic medicine. The Daur people mainly believe in Shamanism and Buddhism. Abstract Background: The traditional medical practices of the Daur region are an important element in Chinese ethnomedicinal knowledge. However, relatively little recording of traditional Daur medicine has been carried out. As Daur medicine is gradually being replaced by traditional Chinese medicine (TCM) and modern medicine, further research is urgently needed. Methods: We collected ethnobotany and ethnozoology data through semi-structured interviews with 114 informants (18 sessions), focus group discussions (6 sessions), and extensive literature reviews. Medicinal samples and specimens were collected during field investigations from June 2015 to October 2018. The diseases and ailments reported as being treated were classified according to the International Classification of Diseases 11th (ICD-11). The expression correlation of medicine classification, medicine, and disease was assessed using Cytoscape 3.6.1 software. Quantitative indices such as informant consensus factor (ICF) and use value (UV) were used to evaluate the importance of ethnomedicinal data. Results: We documented 30 plant species (from 19 families) and 21 animal species (from 11 families). In the past, the majority of plant species were collected from natural habitats, but now they are mainly cultivated. The most widely utilized plants were herbs (21 species), followed by shrubs (4), trees (3), and fungus (2). The most utilized groups of animals were mammals (14 species), followed by birds (5), amphibians (1), and reptiles (1). Medicines were mostly administered orally (43) but were also applied externally (11) or delivered via both routes (9). Informants indicated that medicines were prepared via decoction (21), grinding (11), boiling (10), extraction (8), and burning (7). Medicines were taken as a drink (37), eaten (16), or made into pills and powders (7), but could also be administered as an ointment/wash (6), wrap/dressing (5), in the nose, eyes, and mouth (4), or as a fumigate (2). The traditional Daur medicines investigated in this study treated 76 human diseases or ailments classified under 13 disease categories, based on the ICD-11. The highest ICF (1.00) was reported for neoplasms and visual system diseases and childbirth or the puerperium, followed by injury, poisoning, or other diseases resulting from external causes (0.92), and symptoms, signs or clinical findings, not elsewhere classified (0.82). The most used medicinal species were Cervus elaphus, Cervus nippon, Capreolus, Gallus, Canis lupus familiaris, Betula platyphylla, and Artemisia integrifolia. Conclusions: The current research can make an important contribution to the body of knowledge about the national medicine of the Daur people. Research Keywords: Daur, Hulunuir area, Plant medicines, Animal medicines, Ethnomedicine License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/16 Page 1/16 Abstract This is particularly important because the younger generation is not interested in learning and preserving their traditional medical practices. A substantial body of ethnobotanical and ethnozoological knowledge could lead to the development of new medicines. Therefore, research into Daur ethnic medicines is urgently required. The information documented in the present study will provide a useful basis for future investigations into the ethnopharmacological and traditional knowledge of the Daur region. Background Shamanism is an important part of the traditional culture of the Daur people, and it has profoundly affected all aspects of their social life [9]. However, little attention has been paid to the research and development of modern Daur medicine and even less effort has been made to preserve traditional Daur medicine. In addition, there are few related research reports, and few reports have been written on the ethnomedical resource of the Daur people in China. Plants have always been humanity’s primary source of treatments for disease and injury. Although a high level of cultural and biological diversity has been preserved in the region, research into the utilization of medicinal resource by the Daur people is lacking, research and development of modern Daur medicine attract little attention, and little is done to strengthen the protection of traditional Daur medicine. In addition, the expansion of modern education and medical treatment has further aggravated the loss of knowledge, resulting in the younger generation being unaware of the value, use, and preparation methods of traditional Daur medicine [8]. Page 2/16 Page 2/16 With changes in the lifestyle, natural condition, and social environment of the Daur people, their traditional medical knowledge is disappearing under the influence of mainstream ethnic medicine, such as TCM and Mongolian medicine. The phenomenon of integration is gradually occurring, and unique ethnic characteristics are gradually being lost. It is important to accelerate research into the Daur nation’s traditional knowledge, consolidate the results, and document the inheritance to help with the development of medicines in the future. In this study, we interviewed the few remaining healers in Daur and documented as much ethnic medicinal knowledge as possible with the aim of increasing awareness of the availability and value of ethnic medicines in this region and worldwide. The application of inherited knowledge is a growing field of interest. Research of this type and the comprehensive documentation of traditional medical knowledge are important to increase the chance of discovering new drugs as well as to enhance knowledge about the sustainable use of natural resource. At the same time, it provides a scientific basis for the development and utilization of the resource of the Daur national traditional medicine. With changes in the lifestyle, natural condition, and social environment of the Daur people, their traditional medical knowledge is disappearing under the influence of mainstream ethnic medicine, such as TCM and Mongolian medicine. Background The phenomenon of integration is gradually occurring, and unique ethnic characteristics are gradually being lost. It is important to accelerate research into the Daur nation’s traditional knowledge, consolidate the results, and document the inheritance to help with the development of medicines in the future. In this study, we interviewed the few remaining healers in Daur and documented as much ethnic medicinal knowledge as possible with the aim of increasing awareness of the availability and value of ethnic medicines in this region and worldwide. The application of inherited knowledge is a growing field of interest. Research of this type and the comprehensive documentation of traditional medical knowledge are important to increase the chance of discovering new drugs as well as to enhance knowledge about the sustainable use of natural resource. At the same time, it provides a scientific basis for the development and utilization of the resource of the Daur national traditional medicine. Selection of study area The population of the Daur ethnic group in the Hulunbuir area accounts for more than 90% of the population of this ethnic group in Inner Mongolia. It is mainly distributed in Ewenki county, Arun county, ZhaLanTun county, and Molidawar Daur autonomous county [9], which are in the Greater Khingan Mountains (GKM) and the eastern part of the GKM. Most of the area has a temperate continental monsoon climate, while a small portion lies in the cold temperate zone. Seasonally, the area experiences cold, long winters and cool, short summers. In addition, the region receives an abundance of sun and little precipitation, which occurs mainly between July and August. The area comprises many geographical types, such as the GKM, Hulunbuir Grassland, Inner Mongolia Plateau, and the grasslands at the base of the mountains. The Daur ethnic group, concentrated in the area of Hulunbuir, a district in the northeastern part of Inner Mongolia, is located between longitude 118°48′02″ to 125°13′32″ east and latitude 47°05′40″ to 49°49′18″ north in the northeast part of Inner Mongolia (Fig. 1). The topography of the ethnic minority area is characterized by a temperate climate plus a cold temperate zone within an altitudinal range of 198.0–1706.6 m above sea level, and consists of a plateau, prairie, basin, mountain, and plain. The annual average rainfall and temperatures range from 350.0 mm to 500.0 mm and from -2.4 ℃ to 2.2 ℃, respectively. This area supports a range of minorities that include not only Daur, but also Mongolian, Olunchun, and Ewenki ethnic groups. Therefore, the area has not only a variable topography and highly diversified medicinal flora and fauna, but also multi-ethnic groups and a rich cultural diversity. [10–11]. Social characteristics of study area The ethnomedicinal survey comprised semi-structured interviews with 114 people and focus group discussions with 12 traditional healers. The study informants were divided into different groups according to age: <30, 31–40, 41–50, 51–60, and >60 years old. Most informants were in the 51–60 (50.9%) and 41–50 (20.2%) age groups, i.e., the main users of ethnic medicine and inheritors of related knowledge. There were few young people under 30 (6.1%) and or older people over 60 (7.9%) years of age (Table 1). Reconnaissance survey The fourth national survey of Chinese medical material resource was conducted from June 2015 to October 2018 in Hulunbuir to gain a better understanding of the species, distribution, and reserves of medicinal plants [12]. During this investigation, we carried out a series of traditional knowledge surveys of ethnic medicine, which included Daur medicine. A survey team consisting of botanists, taxonomists, and indigenous people conducted the resource surveys, mainly in summer and autumn, in areas where the medicinal species were extensively found. The team collected specimens of the plants used in Daur medicine, recorded information on the habitats in which they were found, and took photographs for future reference [13,14]. Collect and organize ethnic medicine through the Census of TCM resource, realize the inheritance of traditional medicine, promote the diversity of ethnic culture, improve the theoretical system of innovative ethnic medicine, and welcome the renaissance of ethnic medicine. Additional information on the abundance and distribution of medicinal plants in the study area was also collected. Due to the limited production and preservation facilities for specimens of animal origin, ethnozoological information was obtained only from the interviews with informants, and animal medicinal material samples were not collected. Ethnomedicine data source and collection Two focus group discussions were conducted in the Daur Autonomous Township, during which healers, ethnic medicine gatherers, and local village committees helped facilitate an understanding of the current medical situation of the Daur ethnic minority. Throughout all the investigations, specific information regarding the traditional Daur medicinal materials was recorded. This information included the local name, parts used, disease treated, dosage, and route for administration. The diseases were assigned to different categories according to the International Classification of Diseases 11th (ICD-11), and all the summarized survey information and supplementary information are presented in Tables 2 and 3. Botanical and zoological identification We collected the voucher specimens of the plants for identification by the expert taxonomist with the help of the Flora of Inner Mongolia and Flora of China [16, 17]. Family and scientific names were confirmed with the Angiosperm Phylogeny Website (http://www.mobot.org/MOBOT/research/APweb) and the Plant List database (http://www.theplantlist.org) (Fig.2). Voucher specimens of plants were prepared and deposited at the Baotou Medical College, Inner Mongolia University. Family and scientific names of medicinal animals were determined according to the Fauna of Inner Mongolia and Medicinal Fauna of China [18, 19]. Expression correlation network r medicine and attending disease classifications were analyzed using Cytoscape 3.6.1 software The topological relationships between Daur medicine and attending disease classifications were analyzed using Cytoscape 3.6.1 software (https://cytoscape.org/) to obtain the key factors and to visualize the network [20, 21]. The size of each node in the disease-classification-disease-medicine network was based on the prevalence of the uses. actors and to visualize the network [20, 21]. The size of each node in the disease-classification-disease-medicin e uses. Informant consensus factor Informant consensus factor The informant consensus factor (ICF), was analysis of the general use of plants and animals using the following formula: nt consensus factor (ICF), was analysis of the general use of plants and animals using the following formula: The informant consensus factor (ICF), was analysis of the general use of plants and animals using the follow See formula 1 in the supplemental files. where Nur refers to the number of use reports for a particular disease category, and Nt refers to the number of taxa used for a particular use category by all informants. ICF values are near 0 if medicinal resources are chosen randomly or if there is no exchange of information about their use among informants and close to 1.0 when there is a well-defined selection criterion in the community or if information is exchanged between informants [22, 23]. See formula 2 in the supplemental files. See formula 2 in the supplemental files. where ∑ Ui is the total number of use reports cited by each informant for a given species and N represents the total number of informants. UVs approach zero (0) when there are few reports related to use. Higher UVs indicate that there are many use reports for a plant or animal species, thus implying the importance of the species. Ethnomedicine data source and collection Daur ethnomedicine data were collected from two source: primary source comprising interviews, focus group discussions, and field observations in the study area, and secondary source comprising relevant publications and reviews related to the present study [15]. Semi-structured interviews were conducted with 114 informants. Local therapists automatically become key providers of information (key informants). They are the experts and advisors on traditional indigenous knowledge and its application. Healers can provide basic information on treatment methods, while laypersons described the effects of the treatments on their diseases. The interviews were conducted using a prepared list of questions; however, enough time was allowed for discussions related to any of the questions raised. We then visited experts from the Daur society and key informant healers who had been using Daur medicine for at least 3 years. This group comprised 12 people in total. Our interviewees were chosen through comments and suggestions from religious leaders, elders, administrators, and students, and personal observations by researchers in community groups. The selection of key informants was also based on the quality of the explanations given by specific informants in the interviews. The data collection team paid particular attention to the choice of language in the informant interviews, whether it was Chinese or Mongolian, or the Daur language used by the local people. Prior to the visits, we obtained oral consent from the interviewees that the information provided could be made public. Page 3/16 Page 3/16 Key informants, i.e., those expected to have extensive knowledge of Daur medicine, were selected at the village level with the help of the village committee. Two focus group discussions were conducted in the Daur Autonomous Township, during which healers, ethnic medicine gatherers, and local village committees helped facilitate an understanding of the current medical situation of the Daur ethnic minority. Throughout all the investigations, specific information regarding the traditional Daur medicinal materials was recorded. This information included the local name, parts used, disease treated, dosage, and route for administration. The diseases were assigned to different categories according to the International Classification of Diseases 11th (ICD-11), and all the summarized survey information and supplementary information are presented in Tables 2 and 3. Key informants, i.e., those expected to have extensive knowledge of Daur medicine, were selected at the village level with the help of the village committee. Use value for individual species The use value (UVs), a quantitative measure of the relative importance [24, 25], was calculated for all species in the study area using the following formu The use value (UVs), a quantitative measure of the relative importance [24, 25], was calculated for all species in the study area using the following formula: See formula 2 in the supplemental files. The use value (UVs), a quantitative measure of the relative importance [24, 25], was calculated for all species Diversity of route, preparation, dosage, and usage of ethnomedicines We collected information regarding the Daur ethnomedicine administration route, preparation, dosage, and usage. According to the interviews with the informants, the most popular route was oral (39, 66.1%), followed by external administration only (11, 18.6%), and both oral and external administration (9, 15.2%) (Fig. 4). According to the present study, plant and animal medicines were prepared via decoction (21), grinding (11), boiling (10), extraction (8), and burning (7) (Table 4). Among them, decoction, the most used preparation technique in Chinese medicine, was the method used most often (36.9%). Other preparation methods represent the unique characteristics of traditional Daur medicine. In particular, various medicinal and food ingredient are boiled together and eaten as a meal, which reflects the characteristics of the theory which is medicinal and edible. Most medicines are taken/consumed as a drink (37), which involves decoction, extraction (i.e., solutions and oils), and other methods; eaten (16), made into pills and powder (7), but also used as an ointment/wash (6), wrap/dressing (5), in the nose, eyes, and mouth (4), or as a fumigate (2). (Table 5). Dosages were estimated for most medicines and were dependent on the age of the patient, degree of the illness, diagnosis, and experience of the healers. The ethnic groups adjacent to the Daur group include the Olunchun and Ewenki nationalities. Bi [26] et al. analyzed the ethnic medicine application of the “three minor nationalities” in Inner Mongolia. It appears that Artemisia argyi is used as a medicine by all three nationalities. While the Daur people use it to treat urticaria and rheumatic pain, the Ewenki use it for chronic hepatitis, and the Olunchun use it to treat uterine bleeding and postpartum hemorrhage. The different therapeutic uses of the same medicinal materials in different ethnic groups reflect differences in national characteristics, indicating that they are closely related to the history, culture, and life of each nationality. Table 7 compares Daur medicine with TCM and Mongolian medicine. The known efficacy of the Chinese and Mongolian medicines can help predict the effectiveness of the Daur medicines and identify those that have the potential for the development of new drugs in the future. Diseases treated by diverse medicinal resource Tables 2 and 3 present traditional Daur medicine used to treat 76 human diseases or ailments classified under 13 disease categories, based on the ICD-11. A disease-classification-disease-medicine network of Daur medicine was constructed and visualized with Cytoscape software using the disease classifications and medicinal species targeted (Fig. 5). The availability of the network model structure may improve our understanding of the relationship between plant medicines, animal medicines, diseases, and disease classification. According to statistical information, the classification of ‘symptoms, signs or clinical findings, not elsewhere classified’ was primary, and included 8 diseases and ailments of constipation, cough, diarrhea, dyspepsia, loss of appetite, abnormal sputum, and extravasation of blood. As shown in the constructed network, there were obvious and expressed correlations with 29 medicinal species, such as the plant medicines of R. simsii, P. frutescens, A. integrifolia and the animal medicines of S. scrofa domestica, C. nippon, C. capreolus. Other classifications included ‘Certain infectious or parasitic diseases’ (18 species), ‘Injury, poisoning or certain other consequences of external causes’ (11 species), and other disease classifications. The classification of diseases and ailments clearly indicates a link between Daur medicine and modern medicine; however, traditional medicine still has its inherent characteristics. Thousands of years ago, the Daur people learned to use natural materials (animals, plants, minerals) of their surroundings in the struggle against various diseases. Experience has been passed from generation to generation, gradually forming a body of medical knowledge with local ethnic characteristics, and folk medicines that really have curative effects are still circulating in Daur. These include “Kumula," the Chinese name of which translates as Willowleaf Wormwood, which is derived from A. integrifolia. It has the effect of heat-clearing and detoxifying, and is used to treat hyperlipidemia, excessive drinking, and stomach bleeding [27]. Among the diseases, common day-to-day ailments (cough, constipation, diarrhea, trauma) and endemic diseases (rheumatism, rheumatoid arthritis) were treated with numerous medicines in traditional Daur medicine at Hulunbuir. The Ewenki and Daur people both live in Hulunbuir, Inner Mongolia. Traditional medicines of the two groups focus on different diseases. Ewenki medicine is mainly used for respiratory system diseases, digestive system diseases and skin and subcutaneous tissue diseases; Daur’s drugs are used for certain infectious diseases and parasitic infestations, digestive diseases, injuries, poisoning, trauma, and musculoskeletal and connective tissue diseases. Diversity of Daur ethnobotany In total, 30 medicinal plants were identified, from 19 families (Table 1). Among them, the Compositae was the dominant plant family used (4 species), followed by the Ranunculaceae and Rosaceae (3 species each), and the Gramineae, Betulaceae, and Cucurbitaceae (2 species each). There were 13 other commonly used families including Tricholomataceae, Auriculariales, Typhaceae, Salicaceae, Moraceae, Polygonaceae, Euphorbiaceae, Araliaceae, Umbelliferae, Ericaceae, Labiatae, Solanaceae, and Caprifoliaceae (1 species each). While it has been observed that, these days, many medicinal plants are cultivated, in our study most plant medicines were collected from the natural environment. Herbs (21 species, 70.0%) were the most commonly used plant type, followed by shrubs (4 species, 13.3%), trees (3 species, 10.0%), and fungus (2 species, 6.7%) (Fig. 3a). Several parts of different plants are used to deal with various diseases. According to the interviews with the informants, the most commonly used parts are the seeds (8); followed by the roots and fruits (6, each); whole plant (5); branches (3); barks, rhizomes, flowers, and fruiting bodies (2, each), and root nodules and leaves (1, each) (Fig. 3b). Daur medicine differs from TCM in which the most used plant parts are the roots. These differences may be influenced by factors involving the ease of collection and application. Diversity of Daur ethnozoology Page 4/16 Page 4/16 In the present study, 20 animal species from 11 families were documented from the study site (Table 2). The most widely used family was Cervidae (4 species), followed by Phasianidae, Canidae, and Bovidae (3 species each). Other families that were commonly used were Ranidae, Trionychidae, Picidae, Corvidae, Erinaceidae, Mustelidae, and Suidae (1 species, each). Mammals were the dominant animal group, accounting for 14 species (66.6%), followed by birds (5, 23.8%), and amphibians and reptiles (1, 4.8%) (Fig. 3c). Animal-based drugs mainly came from organs (12), such as the kidney, liver, gallbladder, heart, gizzard, uterus, and viscera, involving numerous applications. Animal-based drugs were also prepared from tissues (11), including meat, hamstrings, grease, hair, and blood; from bones and the whole body (3 each); secretions and antlers (2 each); and carapaces (1) (Fig. 3d). Conclusions The Hulunbuir area has a rich biodiversity and traditional culture in which numerous important ethnomedicines have been preserved. In Daur, the traditional plant and animal medicines that are used for ethnomedicinal purposes are obtained close to the place of residence of users. During the 3 years of our ethnobotany and ethnozoology investigation, we identified 30 species of medicinal plants, belonging to 19 families, and 21 species of medicinal animals, belonging to 11 families. Analyses of the collected data revealed that herbs were the dominant plant medicine, and seeds and roots were the parts most often used in ethnobiology. Mammals dominated the list of medicinal animals, and several kinds of organs and tissues were used in ethnozoology. Most medicines were administered orally. The dominant preparation technique was decoction, and medicines were most frequently taken/consumed as a drink. A comparative analysis of the medicines of the Daur nationality, TCM, and Mongolian medicine reflects the uniqueness and national characteristics of the medicines of the Daur nationality. Apart from a handful of experienced and knowledgeable traditional therapists, the route and dosage of plant-based medicine is usually based on haphazard application. Even a qualified therapist is not perfect. The implication of this situation is that improper use of medicinal plants may have severe short-term and long-term effects on the health of patients and may sometimes even endanger life. In drug research and development, some plants with higher medicinal value are candidate plants for further phytochemical analysis. Comprehensive documentation of traditional Daur medicines is critical to promote the discovery of new source of drugs and the sustainable use of natural resource. One of the main characteristics of traditional medicinal knowledge is its dynamism. As revealed by the analysis of the data collected from both field investigations and interviews, the use of plant and animal medicine based on traditional knowledge is gradually declining in the study region with the increased use of modern medication in recent generations. It is therefore essential to build awareness among community members of the importance of protecting traditional knowledge and medicinal plants before they disappear and to take urgent measures to protect threatened plant species. This will enable people to continue to use traditional practices, which have proven to be safe and effective. Informant consensus factor ICF is used to identify plants of particular intercultural relevance. With the ICF, we evaluated how homogenous the information was.Thirteen disease categories were identified. The ICF was calculated for each disease category, and the range was from 0.29 to 1.00 (Table 6). The highest ICF (1.00) was Page 5/16 Page 5/16 reported for the neoplasms and visual system diseases and childbirth or the puerperium all for 1 species and 1 use reports, followed by injury, poisoning or certain other medical problems arising from external causes (0.92) with 2 species and 13 use reports, symptoms, signs or clinical findings, not elsewhere classified (0.82) with 18 species and 95 use reports. The highest ICF for the neoplasms and visual system diseases and childbirth or the puerperium and injury, poisoning or certain other medical problems arising from external causes can be probably related to the low number of cases and trauma and poisoning are mainly treated by taking agaric and Artemisia integrifolia medicines. reported for the neoplasms and visual system diseases and childbirth or the puerperium all for 1 species and 1 use reports, followed by injury, poisoning or certain other medical problems arising from external causes (0.92) with 2 species and 13 use reports, symptoms, signs or clinical findings, not elsewhere classified (0.82) with 18 species and 95 use reports. The highest ICF for the neoplasms and visual system diseases and childbirth or the puerperium and injury, poisoning or certain other medical problems arising from external causes can be probably related to the low number of cases and trauma and poisoning are mainly treated by taking agaric and Artemisia integrifolia medicines. Use value The most used medicines were C. elaphus and C. nippon with UVs of 2.70 followed by C. capreolus, G. gallus, C.lupus familiaris, and B. platyphylla with UVs of 1.82, 1.81, 1.33, 1.07, respectively. Many parts of these species are used as medicine and they are readily available or are typical species of the area. Specifically, A. integrifolia, with an UV of 1.00, is a well-known local species that is used for both food and medicine. In the published literature, A. integrifolia has reportedly been used to treat various diseases including hypertension, hyperlipidemia, diabetes, and hepatitis, besides its use in traditional Daur medicine [28]. Species that are used to treat three diseases and more (39%) were dominant, followed by species used to treat single diseases (37%), and plants or animals used to treat two diseases (24%). With respect to animal parts, the Daur tribe uses bile (for example, from bear, pig, and sheep) the most. The next most commonly used animal-derived medicines are made from bone, followed by those made from meat, and those that use the whole body of the animal. The most widely used families for animal-based drugs are Corvidae (crows) (4 species), followed by Anatidae (ducks), Canidae (canines), and Bovidae (bovines) (3 species each). Animal-based medicines are mainly composed of kidney, liver, gallbladder, heart, sac, uterus, and other organs and are widely administered. With respect to the animals used, mammals dominate, accounting for 14 species (66.6%), followed by birds (5 species, 23.8%) and amphibians (1 species, 4.8%). Conclusions There are many types of medicinal fungi, animals, and plants in the Daur region, most of which are folk medicines and have significant effect, but data on phytochemistry, pharmacology, and quality control is lacking. The systematic research into medicinal fungi, animals, and plants provides an objective and scientific basis for folk applications of the ethnomedicines of the Daur group. Ethics approval and consent to participate Ethics approval and consent participate Permission to conduct the ethnobotanical study was obtained from the district administration and village office in the study area. The purpose of the study was explained to all participants, and they agreed to provide information. Consent for publication Consent for publication Our manuscript does not contains any individual's person data. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Page 6/16 Authors' information 1Inner Mongolia Medical University, Hohhot (010020), China 1Inner Mongolia Medical University, Hohhot (010020), China 2Inner Mongolia Institute of Traditional Chinese Medicine, Hohhot (010020), China. 3Jilin Agricultural University, Changchun (130000), China. 3Jilin Agricultural University, Changchun (130000), China. Acknowledgements The authors would like to acknowledge the traditional healers and other local people who shared their traditional ethnomedicine knowledge unselfishly. And thanks for the help of the people of villagers committee at Daur main distribution areas. Furthermore, we would like to extend our gratitude to the botanists, taxonomists and indigenes peoples which help resource surveys during 3 years. Funding This work was supported by Capacity Building of Sustainable Utilization of valuable traditional Chinese Medicine Resources (grant no. 2060302), 2018 Chinese medicine public health service subsidy special “the fourth survey on Chinese materia medica resource” (grant no. Finance Society [2018] 43), National Natural Science Foundation of China (grant no. 81760776). Authors' contributions XL and YQB: participated in research survey, data evaluation and wrote the manuscript as the major contributor of the study. MHL: supervised the project and designed the study. LZ: analyzed data and provided technical support. YHS: designed the research survey and collected data. SZ and MHL: contributed to the structure, discussion contents of the study, and helped with species identification. All authors read and approved the final manuscript. A k l d The authors declare that they have no competing interests. The authors declare that they have no competing interests. References 1. 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Tables Table. 1 Socio-demographic characteristics of the informants Table. 1 Socio-demographic characteristics of the informants Table. 1 Socio-demographic characteristics of the informants Basic information Number of informants Percentage (%) Basic information Number of informants Percentage (%) Sex Male 68 59.6 Female 46 40.4 Age ≤30 7 6.1 31-40 17 14.9 41-50 23 20.2 51-60 58 50.9 ＞60 9 7.9 Table. 2 List of plant species for medicinal purposes in Daur, Inner Mongolia, China Table. 2 List of plant species for medicinal purposes in Daur, Inner Mongolia, China The 13 diseases according to ICD-11 are classified as follows:(A)Certain infectious or parasitic diseases, (B)Neoplasms, (C)Diseases of the nervous system, (D)Diseases of the visual system, (E)Diseases of the circulatory system, (F)Diseases of the respiratory system, (G)Diseases of the digestive system, (H)Diseases of the skin, (I)Diseases of the musculoskeletal system or connective tissue, (J)Diseases of the genitourinary system, (K)Pregnancy, childbirth or the puerperium, (L)Symptoms, signs or clinical findings, not elsewhere classified, (M)Injury, poisoning or certain other consequences of external causes. O O l E E l Or: Oral, Ex: External Or: Oral, Ex: External Page 8/16 Family Scientific name Local name Parts used Disease treated Classification of diseases （ICD-11） Taxonomic Route, dose and usage UV Voucher Specimens Tricholomataceae T. mongolicum S. Imai Qi gan mog Fruiting body Measles A Fungus Or:50g cut and used in decoction 0.04 20170703001 Auriculariales A. auricula (L. ex Hook.) Underw Bakerte Fruiting body Cervical cancer, hypertension, constipation, vaginal cancer, trauma B, E, L, M Fungus Or: 5-30g used in food or burned to carbon powder 0.94 20170628021 Typhaceae Typha orientalis Presl. Laesu Whole grass Rheumatoid arthritis, rheumatism I Herb Ex: fumigated with fresh grass for 1 hour 0.06 20170703021 Poaceae Setaria italica (L.) Beauv. var. italica. Narem baei tos Fruit Several kinds of ringworm and scabies A Herb Ex: anointed the affected area with oil 0.36 20170802002 Avena sativa L. Kua lin po Seed Loss of appetite, constipation L Herb Or: 30-60g used in decoction 0.99 20170608013 Salicaceae Populus davidiana Dode Holordaan koltus Bark Dental disease G Tree Ex: decocted for gargling 0.10 20170703020 Betulaceae C. heterophylla Fisch Ququu Seed Diarrhea, loss of appetite, cough L Tree Or: 30-60g used in decoction or grinded to powder 0.77 20170802003 Betula platyphylla Suk. Tables Qaalbaan koltus Bark Bacillary dysentery, diarrhea, gastric ulcer A, G, L Tree Or: 5g burned to carbon powder and taken with water 1.07 20170703019 Qaalbaan tos Branch Open wound of lower limb M Ex: burned defined amount branch into oil and wiped at the affected area Cannabaceae Cannabis sativa L. - Seed Smallpox A Herb Ex: grinded seeds mixed with Raphanus sativus leaves and applied the exuding latex to the affected area 0.23 20170703024 Polygonaceae Fagopyrum esculentum Moench Haul Seed Cholera, dyspepsia A, L Herb Or: boiled seed mixed with hot cow’s milk 0.99 20170608012 Ranunculaceae P. chinensis (Bunge) Regel - Root,flower Rheumatism I Herb Or: used in decoction or extracted in wine Ex: grinded fresh herbs for applying to affected area 0.09 20170412014 A. kusnezoffii Reichb. - Root Rheumatism I Herb Or: 3- 6g used in decoction or made for pills and powder (If used in soup should be prepared 0.03 20170703011 C. dahurica L. A Page 9/16 Page 9/16 herbs applied to affected area Rosaceae R. davurica Pall. Jaami gada Root nodule Rheumatoid arthritis I Shrub Or: 7 root nodules used in decoction Ex: used in decoction to wash the affected area 0.14 20170802102 Potentilla flagellaris Willd. ex Schlecht. Suadelil eus Whole grass Gaseous abdominal distention, urethritis G,J Herb Or: drank it like tea or used in decoction 0.15 20170602101 S. sorbifolia (L.) A. Br. Caakunku Branch Costal chondritis, trauma I, M Shrub Or:9- 15g used in decoction Ex: used in decoction to wash the affected area 0.82 20170703034 Euphorbiaceae E. fischeriana Steud. Temee mek Root Tuberculosis, tuberculosis of bones, several kinds of scabies A Herb Or: 50g dried root decocted mixed with 100g jujube， and eat 2 jujube twice a day 0.38 20170613024 Araliaceae Panax ginseng C. A. Mey. Aorgiden Root All kinds of disease - Herb - 0.61 20170703018 Umbelliferae C. virosa L. Juma qin Rhizome Venomous snakes or insects bite, extravasation of blood, osteomyelitis, gout, rheumatism I, L, M Herb Ex: decocted or grinded rhizome appling to the affected area, mixed with egg white or Cicuta virosa to use together 0.04 20170703023 Ericaceae R. simsii Planch. Nadeg yilga Flower or fruit Tracheitis, pneumonia, irregular periods, amenorrhe, uterine bleeding, rheumatism, trauma A, F, J, I, M Shrub Or: 5g flowers used in decoction or extracted in wine, 2- 3g fruits grinded to powder 0.62 20170710021 Labiatae Perilla frutescens (L.) Britt. Tables Balei tos Seed Abnormal sputum, cough, constipation L Herb Or: drank 5- 10 ml or boiled seeds mixed with G. gallus domesticus 0.93 20170906011 bal Seed and Fruit Hepatitis, cough, constipation G, L Or: 6- 10g used in decoction or made for pills and powder， 15g eat as raw S. baicalensis Georgi - Root Abdominal pain, diarrhea, dysentery, cough A, G, L Herb Or: drank it like tea 0.39 20170812007 Solanaceae Nicotiana tabacum L. danga larq Leaves Carbuncle, furuncle, dermatitis, eczema, trauma A, H, M Herb Or: 6- 9g used in decoction Ex: grinded and wrapped around the affected area 0.61 20170703016 Caprifoliaceae S. williamsii Hance Caawui mood Branch Trauma, costal chondritis M, I Shrub Or: 25g used in decoction Ex: 25g used in decoction to wash the ff d 0.27 20170703017 Rosaceae Asteraceae X. sibiricum Patrin ex Widder Zhang guozi Fruit Rheumatism I Herb Or: used in decoction 0.52 20170703013 Artemisia argyi Levl. et Vant. Qigaan suaig Whole grass Urticaria, rheumatism H, I Herb Or: used in decoction Ex: used in decoction to fumigate affected area 0.84 20170608011 F. sibiricum (L.) Kitam. Taoli suaig Whole grass Pneumonia F Herb Or: 3g whole grass decocted mixed with 30g flowers and leaves of R. dauricum, and taken for twice a day 0.27 20170703009 A. integrifolia L. Kumile Whole grass Dysentery, diarrhea, laryngeal carcinoma A, B, L Herb Or: defined amount bud boiled mixed with Phaseolus vulgaris or drank as tea 1.00 20170608008 Table. 3 List of animal species for medicinal purposes in Daur, Inner Mongolia, China Table. 3 List of animal species for medicinal purposes in Daur, Inner Mongolia, China Table. Tables 3 List of animal species for medicinal purposes in Daur, Inner Mongolia, China Page 11/16 Family Scientific name Local name Parts used Disease treated Classification of diseases （ICD-11） Taxonomic Route, dose and usage UV Ranidae Rana chensinensis David Meleg Whole body Rheumatic heart disease E Amphibian Or: cut 5 whole animals and boiled with 50g pork for once a day 0.42 Trionychidae Trionyx sinensis Wiegmann Kaberin miaal Tissue (meat) Urgency of urination J Reptile Or: boiled meat or taken soup 0.19 - Carapace Urgency of urination J Or: burned to carbon and taken with water Phasianidae Gallus domesticus - Tissue (meat) Rheumatism I Bird Or: boiled meat with pork together 0.84 Coturnix japonica Temminck et Schlegel Bedeni xirbes Tissue (hamstring) Injury of fascia or tendon M Bird Or: grinded hamstring and applied at the site of wound 0.10 G. gallus Kakera chulqi Organ (gallbladder) Cough, abnormal sputum L Bird Or: extracted bile and mixed with sugar to drinking 1.81 - Organ ( viscera) Trauma M Ex: applied the viscera organ to affect area with hot compress Kakera toturig Organ (gizzard) Traumatic hemorrhag, dyspepsia M, L Or: grinded to powder Ex: applied the gizzard to affect area with hot compress kakraa tos Tissue (grease) Rheumatic heart disease ， coronary heart disease L, E Or: 15ml taken for twice a day Picidae Dendrocopos major Linnaeus Tontroookil Whole body Cramp or spasm C Bird Or: burned to carbon and taken with water 0.17 Corvidae Corvus macrorhynchos Vagler Gawu miag Tissue (meat) Diseases of stomach, hepatic cirrhosis G Bird Or: boiled meat or taken soup 0.84 Erinaceidae Erinaceus amurensis Schrenk Jierie Whole body Urgency of urination J Mammal Or: lightly burnt whole body to eat meat and drink water soaked with skin 0.13 Canidae C. lupus Linnaeus Guska tos Tissue (grease) Tuberculosis A Mammal Or: 10ml take for 3 times a day 0.01 Guske ios Bone Rheumatism, rheumatoid arthritis I Or: bone extracted in vinegar, and 10ml taken for twice a day - Tissue (hair) Allergic contact dermatitis H Ex: anointed at the affected area C. Tables lupus familiaris Linnaeus Nowuei tos Tissue (grease) Tuberculosis A Mammal Or: 10ml taken for twice a day 1.33 Nowuei ios Bone Rheumatism, rheumatoid arthritis I Or: boiled water to drink Vulpes vulpes Linnaeus Hunuwuei jurwu Organ (heart) Palpitation, rheumatic heart disease E Mammal Or: mixed with Cinnabaris, Ambrum and Angelica sinensis to eat together 0.18 Mustelidae Meles meles Linnaeus Hewur tos Tissue (grease) Tuberculosis, burn A, M Mammal Or: 10ml taken for three times a day Ex: wrapped around the affected area 0.51 Suidae Sus scrofa domestica Brisson gag chulqi Organ (gallbladder) Constipation, cough ， asthma, carbuncle, dermatophytosis of scalp, furunculosis, eczema, trauma A, H, F, L, M Mammal Ex: dressed at the affected area until the bile dry 0.87 Suidae for pills and powder Ex: applied the blood of antler to affected area - Organ (uterus) Irregular menstruation, infertility, uterine bleeding J Or: 15-25g made for pills and powder, or boiled the fresh uterus C. nippon Temminck Bawoi jurwu qow Tissue (blood) Palpitation E Mammal Or: a little quantity for once a day 2.70 Pentu Antlers Cough, frostbite L, M Or: cut into pieces, and used in decoction or extracted in wine. 1-3g grinded to powder, and made for pills and powder Ex: applied the blood of antler to affected area - Organ (uterus) Irregular menstruation, infertility, uterine bleeding J Or: 15-25g made for pills and powder, or boiled the fresh uterus Moschus moschiferus Linnaeus - Secretion Epilepsy, contraception, stroke, carbuncle, pyogenic abscess of the skin, trauma A, C, E, K, L Mammal Or: 0.06-0.1g made for pills and powder Ex: inhaled drugs from the nose in moderation 0.10 Moschus sifanicus Przewalski - Secretion Epilepsy, contraception, stroke, carbuncle, pyogenic abscess of the skin, trauma A, C, E, K, L Mammal Or: 0.06-0.1g made for pills and powder Ex: inhaled drugs from the nose in moderation 0.10 C. Tables capreolus Linnaeus Juri bast Organ (kidney) Cough L Mammal Or: Eaten with salt 1.82 Juri beleg Organ (liver) Night blindness, astigmatism, glaucoma D Or: Eaten with salt Juri ios Bone Rheumatism I Or: bone extract in vinegar, and 10ml taken for twice a day Bovidae Ovis aries Linnaeus honi chulqi Organ (gallbladder) Constipation, cough, asthma, carbuncle, dermatophytosis of scalp, furunculosis, eczema, trauma A, F, H, L, M Mammal Ex: dressed at the affected area until the bile dry 0.62 Capra hircus Linnaeus Honi chulqi Organ (gallbladder) Constipation, cough, asthma, carbuncle, dermatophytosis of scalp, furunculosis, eczema, trauma A, F, H, L, M Mammal Ex: dressed at the affected area until the bile dry 0.64 Bostaurus domesticus Gmelin Wuker chulq Organ (gallbladder) Tracheitis, icterus , infantile convulsions, constipation, haemorrhoids, carbuncle A, C, G, F, L Mammal Or: 0.3-0.9g made for pills or powder Ex: anointed at the affected area or dropped in eyes 0.30 - Organ (uterus) Irregular menstruation, infertility, uterine bleeding J C. nippon Temminck Bawoi jurwu qow Tissue (blood) Palpitation E Pentu Antlers Cough, frostbite L, M - Organ (uterus) C. nippon Temminck Bawoi jurwu qow Tissue (blood) Pentu Antlers Moschus sifanicus Przewalski C. capreolus Linnaeus Juri bast Organ (kidney) Cough L Mammal Or: Eaten with salt 1.82 Juri beleg Organ (liver) Night blindness, astigmatism, glaucoma D Or: Eaten with salt Juri ios Bone Rheumatism I Or: bone extract in vinegar, and 10ml taken for twice a day Bovidae Ovis aries Linnaeus honi chulqi Organ (gallbladder) Constipation, cough, asthma, carbuncle, dermatophytosis of scalp, furunculosis, eczema, trauma A, F, H, L, M Mammal Ex: dressed at the affected area until the bile dry 0.62 Capra hircus Linnaeus Honi chulqi Organ (gallbladder) Constipation, cough, asthma, carbuncle, dermatophytosis of scalp, furunculosis, eczema, trauma A, F, H, L, M Mammal Ex: dressed at the affected area until the bile dry 0.64 Bostaurus domesticus Gmelin Wuker chulq Organ (gallbladder) Tracheitis, icterus , infantile convulsions, constipation, haemorrhoids, carbuncle A, C, G, F, L Mammal Or: 0.3-0.9g made for pills or powder Ex: anointed at the affected area or dropped in eyes 0.30 Page 13/16 Table. 4 Methods of preparation of plant and animal parts used in Daur ethnomedicine No. Type of preparation Number of medicines Percentage (%) 1 Decoction 21 36.9 2 Grinding 11 19.3 3 Boiling 10 17.5 4 Extraction 8 14.1 5 Burning 7 12.2 Table. Tables 4 Methods of preparation of plant and animal parts used in Daur ethnomedicine No. Type of preparation Number of medicines Percentage (%) 1 Decoction 21 36.9 2 Grinding 11 19.3 3 Boiling 10 17.5 4 Extraction 8 14.1 5 Burning 7 12.2 4 Methods of preparation of plant and animal parts used in Daur ethnomedicine able. 5 Methods of usage of plant and animal parts used in Daur ethnomedicine s of usage of plant and animal parts used in Daur ethnomedicine No. Type of usage Number of medicines Percentage (%) 1 Drink/take 37 48.1 2 Eaten 16 20.7 3 Made for pills and powder 7 9.1 4 Anointment/wash 6 7.8 5 Wrap/dressing 5 6.5 6 Used at nose, eyes and mouth touth 4 5.2 7 Fumigate 2 2.6 Table 6 Informant consensus factor for ailment categories Disease categories Number of use reports (Nur) Number of taxa (Nt) Informant consensus factor (ICF) Certain infectious or parasitic diseases 69 23 0.68 Neoplasms 1 1 1.00 Diseases of the nervous system 5 3 0.50 Diseases of the visual system 1 1 1.00 Diseases of the circulatory system 23 8 0.68 Diseases of the respiratory system 9 5 0.50 Diseases of the digestive system 46 6 0.89 Diseases of the skin 23 6 0.77 Diseases of the musculoskeletal system or connective tissue 32 13 0.29 Diseases of the genitourinary system 24 6 0.78 Pregnancy, childbirth or the puerperium 1 1 1.00 Symptoms, signs or clinical findings, not elsewhere classified 95 18 0.82 Injury, poisoning or certain other consequences of external causes 13 2 0.92 Table 7 Comparison of the efficacy of different ethnic characteristics Scientific name Daur Traditional Chinese medicine Mongolian medicine Artemisia integrifolia Linn. Hypertension, stomach bleeding Detoxification[29] Scabies Allium macrostemon Bunge. Pneumonia Insecticide[30] Irregular menstruation, Trauma bl Pycnonotus sinensis Detoxification Cooling blood[31] Fractures and tendons Sorbaria sorbifolia (L.) A. Br Bruises, chondritis Activating blood removing stasis[32] Cold Perilla frutescens (L.) Britt. Hepatitis Constipation[33] Excessive phlegm Table 6 Informant consensus factor for ailment categories Number of use reports (Nur) Number of taxa (Nt) Informant consensus factor (ICF) Table 7 Comparison of the efficacy of different ethnic characteristics Scientific name Daur Traditional Chinese medicine Mongolian medicine Artemisia integrifolia Linn. Hypertension, stomach bleeding Detoxification[29] Scabies Allium macrostemon Bunge. Pneumonia Insecticide[30] Irregular menstruation, Trauma bleeding Pycnonotus sinensis Detoxification Cooling blood[31] Fractures and tendons Sorbaria sorbifolia (L.) A. Figure 1 Map of Daur main distribution in Hulunbuir, Inner Mongolia, China. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Map of Daur main distribution in Hulunbuir, Inner Mongolia, China. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Tables Br Bruises, chondritis Activating blood removing stasis[32] Cold Perilla frutescens (L.) Britt. Hepatitis Constipation[33] Excessive phlegm Table 7 Comparison of the efficacy of different ethnic characteristics Scientific name Daur Traditional Chinese medicine Mongolian medicine Artemisia integrifolia Linn. Hypertension, stomach bleeding Detoxification[29] Scabies Allium macrostemon Bunge. Pneumonia Insecticide[30] Irregular menstruation, Trauma bleeding Pycnonotus sinensis Detoxification Cooling blood[31] Fractures and tendons Sorbaria sorbifolia (L.) A. Br Bruises, chondritis Activating blood removing stasis[32] Cold Perilla frutescens (L.) Britt. Hepatitis Constipation[33] Excessive phlegm Figures Page 14/16 Page 14/16 Page 14/16 Figure 1 Map of Daur main distribution in Hulunbuir, Inner Mongolia, China. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Figure 2 Parts of plants used in Daur medicine at study area （a. Panax ginseng C. A. Mey., b. Scutellaria baicalensis Georgi, c. Sorbaria sorbifolia (L.) A. Br., d. Euphorbia fischeriana Steud., e. Rosa davurica Pall., f. Cicuta virosa L., g. Cucumis sativus L., h. Rhododendron dauricum L., i. Auricularia auricula (L. ex Hook.) Underw, j. Corylus heterophylla Fisch., k. Cimicifuga dahurica (Turcz.) Maxim., l. Xanthium sibiricum Patrin ex Widder, m. Sambucus williamsii Hance, n. Pulsatilla chinensis (Bunge) Regel, o. Filifolium sibiricum (L.) Kitam., p. Aconitum kusnezoffii Reichb.） Page 15/16 Figure 3 Figure 3 Page 15/16 Graphical representation showing plant and animal species of taxonomic and various used parts (a. plant species of taxonomic; b. plants according to various used parts; c. animal species of taxonomic; d. plants according to various used parts) Graphical representation showing plant and animal species of taxonomic and various used parts (a. plant species of taxonomic; b. plants according to various used parts; c. animal species of taxonomic; d. plants according to various used parts) Figure 4 Figure 4 Figure 4 Rount of diversity Daur medicines Rount of diversity Daur medicines Figure 5 Network visualization of the relationship between disease and Daur medicines (The orange nodes represent plant species, the blue nodes represent anim species, the nodes represent different diseases, and the green nodes represent disease classification. The edges represent interactions.The network was built and visualized with Cytoscape. Figure 5 Network visualization of the relationship between disease and Daur medicines (The orange nodes represent plant species, the blue nodes represent animal species, the nodes represent different diseases, and the green nodes represent disease classification. The edges represent interactions.The network was built and visualized with Cytoscape. Network visualization of the relationship between disease and Daur medicines (The orange nodes represent plant species, the blue nodes represent animal species, the nodes represent different diseases, and the green nodes represent disease classification. The edges represent interactions.The network was built and visualized with Cytoscape. Page 16/16 Page 16/16
W2511619189.txt	https://opendata.uni-halle.de//bitstream/1981185920/57053/318/179977059.pdf	de		Metaphysische Anfangsgründe der Naturwissenschaft (1786)	J.B. Metzler eBooks	2,010	public-domain	37,941	Ex legato dr. AUGusTI sSCcHoL? professoris Halensis d. d. ei desponsa MARGARETE MINDNER Vratislaviensis 1922 Metaphyſiſche Anfangsgrunde der Naturwiſſenſchaft vou 8 Jmmanmnel Kant. Dritte Auflage. Leipzig, bey Johanu KLrredrich Sartkinonh. 1800. Vorrede. Wen das Wort Natur blos in formaler Bedeu— tung genommen wird, da es das erſte innere Princip alles deſſen bedeutet, was zum Daſeyn eines Dinges gehort ſo kann es ſo vielerley Naturwiſſen— ſchaften geben, als es ſpecifiſch verſchiedene Dinge giebt, deren jedes ſein eigenthumliches inneres Princip der zu ſeinem Daſeyn gehorigen Beſtimmungen enthalten muß. Sonſt wird aber-auch Natur in materieller Bedeu— tung genommen, nicht als eine Beſchaffenheit, ſondern als der Jnbegriff aller Dinge, ſo fern ſie Gegeuiſtau— de unſerer Sinne, mithin auch der Erfahrung ſeyn konnen, worunter alſo das Ganze aller Erſcheinungen, d. i. die Sinnenwelt, mit Ausſchließung aller nicht ſinnlichen Objecte, verſtanden wird. Die Natur, in dieſer Bedeutung des Worts genommen, hat nun, nach a 2 der 4) Weſen iſt das erſte innere Prineip alles deſſen, was Jir Moglichkeit eines Dinges gehort. Daber kann man den geometriſchen Figuren, (da in ihrem Begriße nichts, was ein Daſeyn ausdruckte, gedacht wird) nur ein Weſen, nicht aber eine Natur beylegen. 2 1v —S— der Hauptverſchiedenheit unſerer Sinne, zwey Haupt.“ theile, deren der eine die Gegenſtande außerer, der an— dere den Gegenſtand des inneren Sinnes enthalt, mit— hin iſt von ihr eine zwiefache Naturlehre, die Kör— perlehre und Seelenlehre maoglich, wovon die erſte die ausgedehnte, die zweyte die denkende Natur, in Erwagung zieht. Eine jede Lehre, wenn ſie ein Syſtem, d. i. ein nach Principien geordnetes Ganze der Erkenntniß ſeyn ſoll, heißt Wiſſenſchaft, und, da jene Principien entwe— der Grundſate der empiriſchen oder der rationalen Verknupfung der Erkenntniſſe in einem Ganzen ſeyn konnen, ſo wurde auch die Naturwiſſenſchaft, ſie mag nun Korperlehre oder Seelenlehre ſeyn, in hiſtoriſche oder rationale Naturwiſſenſchaft eingetheilt werden muſſen, wenn nur nicht das Wort Natur (weil dieſes eine Ableitung des Mannigfaltigen zum Daſeyn der Dinge gehorigen aus ihrem inneren Princip bezeichnet) eine Erkenntniß durch Vernunft von ihrem Zuſammen— hange nothwendig machte, wofern ſie den Namen von Naturwiſſenſchaft verdienen ſoll. Daher wird die Na— turlehre beſſer in hiſtoriſche Raturlehre, welche nichts als ſyſtematiſch geordnete Faecta der Naturdinge enthalt (und wiederum aus Naturbeſchreibung, als einem Claſſenſyſtem derſelben nach Aehnlichkeiten, und Natur geſchichte, als einer ſyſtematiſchen Darſtellung derſel— ben νv ben in verſchiedenen Zeiten und Oertern, beſtehen wur- de), und Naturwiſſenſchaft eingetheilt werden kon— nen. Die Naturwiſſenſchaft wurde nun wiederum ent— weder eigentlich, oder uneigentlich ſogenannte Na— turwiſſenſchaft ſeyn, wovon die erſtere ihren Gegenſtand ganzlich nach Principien a priori; die zweyte nach Er fahrungsgeſetzen behandelt. Eigentliche Wiſſenſchaft kann nur diejenige ge— nannt werden, deren Gewißheit apodictiſch iſt; Erkennt— niß, die blos empiriſche Gewißheit enthalten kann, iſt ein nur uneigentlich ſogenanntes Wiſſen. Dasjenige Ganze der Erkenntniß, was ſyſtematiſch iſt, kann ſchon darum Wiſſenſchaft heißen, und, wenn die Verknu— pfung der Erkenntniß in dieſem Syſtem ein Zuſammen hang von Grunden und Folgen iſt, ſo gar rationale Wiſſenſchaft. Wenn aber dieſe Grunde oder Princi— pien in ihr, wie z. B. in der Chemie, doch zuletzt blos empiriſch find, und die Geſetze, aus denen die gegebenen Facta durch die Vernunft erklart werden, blos Erfah rungsgeſetze ſind, ſo fuhren ſie kein Bewußtſeyn ihrer Nothwendigkeit bey ſich (ſind nicht apodictiſch-gewiß) und alsdenn verdient das Ganze in ſtrengem Sinne nicht den Namen einer Wiſſenſchaft, und Chymie ſollte daher eher ſyſtematiſche Kunſt, als Wiſſenſchaft heißen. Eine rationale Naturlehre verdient alſo den Na— men einer Naturwiſſenſchaft nur alsdenn, wenn die Na— a3 tur vr turgeſetze, die in ihr zum Grunde liegen, a priori er— kannt werden, und nicht bloße Erfahrungsgeſetze ſind. Man nenut eine Raturerkenntniß von der erſteren Art rein; die ven der zweyten Aet aber wird angewandte Wernunnfterkenntniß genannt. Da das Wort Natur ſchon den Begriff von Geſetzen bey ſich fuhrt, dieſer aber den Begriff der Nothwendigkeit aller Beſtimmungen eines Dinges, die zu ſeinem Daſeyn gehoren, bey ſich fuhrt, ſo ſieht man leicht, warum Naturwiſſenſchaft die Rechtmaßigkeit dieſer Benennung nur von einem reinen Theil derſetben, der namlich die Principien a priori aller ubrigen Naturerklarungen enthalt, ableiten muſſe und nur Kraft dieſes reinen Theils eigentliche Wiſſenſchaft ſey, imgleichen daß, nach Foderungen der Vernunft, jede Naturlehre zuletzt auf Naturwiſſenſchaft hinausgehen und darin ſich endigen muſſe, weil jene Nothwendigkeit der Geſetze dem Bezgriffe der Natur unzertrennlich anhäugt und daher durchaus eingeſehen ſeyn will; daher die voll— ſtandigſte Erklarung gewiſſer Erſcheinungen aus chymi ſchen Principien noch immer eine Unzufriedenheit zu— rucklaßt, weil man von dieſen, als zufalligen Geſetzen, die blos Erfahrung gelehrt hat, keine Grunde a priori anfuhren kann. Alle eigentliche Naturwiſſenſchaft bedarf alſo einen reinen Theil, auf dem ſich die apodietiſche Gewißheit, die die Vernunft in ihr ſucht, grunden konne, und weil die vir dieſer, ſeinen Principien nach, in Vergleichung mit de— nen, die nur empiriſch ſind, ganz ungleichartig iſt, ſo iſt es zugleich von der großten Zutraglichkeit, ja, der Natur der Suche nach, von unerlaßlicher Pflicht in Anſehung der Methode, jenen. Theil abgeſondert, und von dem an— dern ganz unbemeilgt, ſo viel moglich in ſeiner ganzen Vollſtändigkeit vorzutragen, damit man genau beſiim— men konne, was die Verüunft fur ſich zu leiſten vermag, und wo ihr Vermogen anhebt der Beyhulfe der Erfah— rungsprincipien nothig zu haben. Reine Vernunfter— kenntniß aus bloßen Begriffen heißt reine Phileſophie, oder Metaphyſik; dagegen wird die, welche nur auf der Conſtruction der Begriffe, vermittelſt Darſtellung des Gegenſtandes in einer Anſchauung a priori, ihre Erkenntniß grundet, Mathematik genannt. Eigentlich ſo zu nennende Naturwiſſenſchaft ſetzt zuerſt Metaphyſik der Natur voraus; denn Geſetze, d. i. Principien der Nothwendigkeit deſſen, was zum Da— ſeyn eines Dinges gehort, beſchaftigen ſich mit einem Begriffe, der ſich nicht conſtruiren laßt, weil das Da— ſeyn in keiner Anſchauung a priori dargfſtellt werden kann. Daher ſetzt eigentliche Naturwiſſenſchaft Meta— phyſik der Natur voraus. Dieſe muß nun zwar jeder— zeit lauter Prineipien, die nicht empiriſch ſind, enthalten (denn darum fuhrt ſie eben den Namen einer Metaphy— ſik), aber ſie kann doch entweder ſegar ohne Beziehung a auf viri S auf irgend ein beſtimmtes Erfahrungsobject, mithin un beſtimmt in Anſehung der Natur dieſes oder jenen Din— ges der Sinnenwelt, von den Geſcetzen, die den Begriff einer Natur uberhaupt moglich machen, handeln, und alsdenn iſt es der transſcendentaäle Theil der Meta— phyſik der Natur: oder ſie beſchaftigt ſich mit einer be— ſonderen Natur dieſer oder jener Art Dinge, von denen ein empiriſcher Begriff gegeben iſt, doch ſo, daß außer dem, was in dieſem Begriffe liegt, kein anderes empiri— ſches Princip zur Erkenntniß derſelben gebraucht. wird, (z. B. ſie legt den empiriſchen Begriff einer Materie, oder eines denkenden Weſens, zum Grunde, und ſucht den Umfang der Erkenntniſt, deren die Vernunft uber dieſe Gegenſtande a priori fahig iſt) und da muß eine ſolche Wiſſenſchaft noch immer eine Metaphyſck der Na— tur, namlich der korperlichen oder denkenden Natur, hei Pen, aber es iſt alsdenn keine allgemeine, ſondern be-— ſondere metaphyſeſche Naturwiſſenſchaft, (Phyſik und Yſychologie) in. der jene transſcendentalen Principien auf die zwey Gattungen der Gegenſtande unſerer Sinne angewandt werden. Jch behaupte aber, daß in jeder beſonderen Natur— lehre nur ſo viel eigentliche Wiſſenſchaft angetroffen werden konne, als darin Mathematik anzutreffen iſt. Denn nach dem Vorhergehenden erfodert eigentliche Wiſſenſchaft, vornamlich der Natur, einen reinen Theil, der 1X der dem empiriſchen zum Grunde liegt, und der auf Er— kenntniß der Naturdinge a priori beruht. Nun heißt etwas a priori erkennen, es aus ſeiner bloßen Moglich— keit erkennen. Die Moglichkeit beſtimmter Naturdinge kann aber nicht aus ihren bloßen Begriffen erkannt wer— den; denn aus dieſen kann zwar die Moglichkeit des Gedanken, (daß er ſich ſelbſt nicht widerſpreche) aber nicht des Objects, als Naturdinges erkannt werdeu, welches außer dem Gedanken (als exiſtirend) gegeben werden kann. Alſo wird, um die Moglichkeit beſtimm— ter Naturdinge, mithin um dieſe a priori zu erkennen, noch erfodert, daß die dem Begriffe correſpondirende Anſchauung a priori gegeben werde, d. i. daß der Be— griff conſtruirt werde. Nun iſt die Vernunfterkenntniß durch Conſtruction der Begriffe mathematiſch. Alſo mag zwar eine reine Philoſophie der Natur uberhaupt, d.i. diejenige, die nur das, was den Begriff einer Na— tur im Allgemeinen ausmacht, unterſucht, auch ohne Mathematik moglich ſeyn, aber eine reine Naturlehre uber beſtimmte Naturdinge (Körperlehre und Seelen— lehre) iſt nur vermittelſt der Mathematik moglich, und, da in jeder Naturlehre nur ſo viel eigentliche Wiſſen— ſchaſt angetroffen wird, als ſich darin Erkenntniß a priori befindet, ſo wird Naturlehre nur ſo viel eigent. liche Wiſſenſchaft enthalten, als Mathematik in ihr angewandt werden kann. a5 So x —S So lange alſo noch fur die chymiſchen Wirkungen der Materien auf einander kein Begriff ausgefunden wird, der ſich conſtruiren läßt, d. i. kein Geſetz der Annaherung oder Entfernung der Theile angeben laßt, nach welchem etwa in Proportion ihrer Dichtigkeiten u. d.g. ihre Be— wegungen ſammt ihren Folgen ſich im Raume a priori anſchaulich meichen und darſtellen laſſen, (eine Foderung, die ſchwerlich jemals erfullt werden wird) ſo kann Chy mie nichts mehr als ſyſtematiſche Kunſt, oder Experi— mentallehre, niemals aber eigentliche Wiſſenſchaft wer— den, weil die Principien derſelben blos empiriſch ſind und keine Darſtellung a priori in der Anſchauung erlauben, folglich die Grundſaßze chymiſcher Erſcheinungen ihrer Moglichkeit nach nicht im mindeſten begreiflich machen, weil ſie der Anwendung der Mathematik unfahig ſind. Noch weiter aber, als ſelbſt Chymie, muß empiri— ſche Seelenlehre jederzeit von dem Range einer eigent. lich ſo zu nennenden Naturwiſſenſchaft entfernt bleiben, erſtlich weil Mathematik auf die Phanomene des inneren ESinnes und ihre Geſetze nicht anwendbar iſt, man muß te denn allein das Geſetz der Stetigkeit in dem Abfluſſe der inneren Veranderungen deſſelben in Anſchlag brin— gen wollen, welches aber eine Erweiterung der Erkennt— niß ſeyn wurde, die ſich zu der, welche die Mathematik der Korperlehre verſchafft, ohngefahr ſo verhalten wurde, wie die Lehre von den Eigenſchaften der geraden Linie, zur gan xt ganzen Geometrie. Denn die reine innere Anſchauung, in welcher die Seelen-Erſcheinungen conſtruirt werden ſollen, iſt die Zeit, die nur eine Dimenſion hat. Aber auch nicht einmal als ſyſtematiſche Zergliederungskunſt, oder Experimentallehre, kann ſie der Chymie jemals nahe kom men, weil ſich in ihr das Mannigfaltige der inneren Beobachtung nur durch bloße Gedankentheilung von einander abſondern, nicht aber abgeſondert aufbehalten und beliebig wiederum verknupfen, noch weniger aber ein anderes denkendes Subject ſich unſeren Verſuchen der Ab— ſicht angemeſſen von uns unterwerfen laßt, und ſelbſt die Beobachtung an ſich ſchon den Zuſtand des beobachteten Gegenſtandes alterirt und verſtellt. Sie kann daher nie— mals etwas mehr als eine hiſtoriſche, und, als ſolche, ſo viel moglich ſyſtematiſche Naturlehre des inneren Sin— nes, d. i. eine Naturbeſchreibung der Seele, aber nicht Seelenwiſſenſchaft, ja nicht einmal pſychologiſche Experi— mentallehre werden; welches denn auch die Urſache iſt, weswegen wir uns zum Titel dieſes Werks, welches eigentlich die Grundſatze der Korperlehre enthalt, dem gewohnlichen Gebrauche gemaß des allgemeinen Namens der Naturwiſſenſchaft bedient haben, weil ihr dieſe Be— nennung im eigentlichen Sinne allein zukommt und alſo hiedurch keine Zweydeutigkeit veranlaßt wird. Damit aber die Anwendung der Mathematik auf die Korperlehre, die durch ſie allein Naturwiſſenſchaft wer xit werden kann, moglich werde, ſo muſſen Principien der Conſtruction der Begriffe, welche zur Moglichkeit der Macerie uberhaupt gehoren, vorangeſchickt werden; mit— hin wird eine vollſtandige Zergliederung des Begriffs von einer Materie uberhaupt zum Grunde gelegt werden muſ— ſen, welches ein Geſchafte der reinen Philoſophie iſt, die zu dieſer Abſicht ſich keiner beſonderen Erfahrungen, ſon— dern nur deſſen, was ſie im abgeſonderten (ob zwar an ſich empiriſchen) Begriffe ſelbſt antrifft, in Beziehung auf die reinen Anſchauungen im Raume, und der Zeit (nach Geſetzen, welche ſchon dem Begriffe der Natur uberhaupt weſentlich anhangen) bedient, mithin eine wirkliche Metaphyſtik der korperlichen Natur iſt. Alle Naturphiloſophen, welche in ihrem Geſchafte mathematiſch verfahren wollten, haben ſich daher jeder— zeit (ob ſchon ſich ſelbſt unbewußt) metaphyſiſcher Prin— ripien bedient und bedienen muſſen, wenn ſie ſich gleich ſonſt wider allen Anſpruch der Metaphyſik auf ihre Wiſ— ſenſchaft feyerlich verwahrten. Ohne Zweifel verſtanden ſie unter der letzteren den Wahn, ſich Moglichkeiten nach Belieben auszudenken und mit Begriffen zu ſpielen, die ſich in der Anſchauung vielleicht gar nicht darſtellen laſ ſen, und keine andere Beglaubigung ihrer objectiven Realitat haben, als daß ſie blos mit ſich ſelbſt nicht im Widerſpruche ſtehen. jAlle wahre Metaphyſtk iſt aus dem Weſen des Denkungsvermogens ſelbſt genommen, und XxIIt und keinesweges darum erdichtet, weil ſie nicht von der Erfahrung entlehnt iſt, ſondern enthalt die reinen Hand— lungen des Denkens, mithin Begriffe und Grundſatze a priori, welche das Mannigfaltige einpiriſcher Vorſtel— lungen allererſt in die geſetzmaßige Verbindung bringt, dadurch es empiriſches Erkenntniß, d.i. Erfah— rung, werden kann. So konnten alſo jene mathemati— ſchen Phyſiker metaphyſiſcher Principien gar nicht entbeh— ren, und unter dieſen auch nicht ſolcher, welche den Be— griff ihres eigentlichen Gegenſtandes, nanilich der Mate— rie, a priori zur Anwendung auf außere Erſahrung tauglich machen, als des Begriffs der Bewegung, der Erfullung des Raums, der Tragheit, u. ſ. w. Daruber aber blos empiriſche Grundſatze gelten zu laſſen; hiel— ten ſie mit Recht der apodictiſchen Gewißheit, die ſie ihren Naturgeſetzen geben wollten, gar nicht gemaß, daher ſie ſolche lieber poſtulirten, ohne nach ihren Quellen a priori zu forſchen. Es iſt aber von der großten Wichtigkeit zum Vor— theil der Wiſſenſchaften ungleichartige Principien von einander zu ſcheiden, jede in ein beſonderes Syſtem zu bringen, damit ſie eine Wiſſenſchaft ihrer eigenen Art ausmachen, um dadurch die Ungewißheit zu verhuten, die aus der Vermengung entſpringt, da man nicht wohl unterſcheiden kann, welcher von beyden theils die Schran— ken, theils auch die Verirrungen, die ſich im Gebrauche der xiv derſelben zutragen mochten, beyzumeſſen ſeyn durften. Um deswillen habe ich fur nothig gehalten, von dem reinen Theile ber Naturwiſſenſchaft (phyſica generalis), wo metaphyſiſche und mathematiſche Conſtructionen durch einander zu laufen pflegen, die erſtere, und mit ihnen zu— gleich die Principien der, Conſtruction dieſer Begriffe, alſo der Moglichkeit einer mathematiſchen Naturlehre ſelbſt, in einem Syſtem darzuſtellen. Dieſe Abſonde— rung hat, außer dem ſchon erwahnten Nutzen, den ſie ſchafft, noch einen beſonderen Reiz, den die Einheit der Erkenntniß bey ſich fuhrt, wenn man verhutet, daß die Grenzen der Wiſſenſchaften nicht in einander laufen, ſondern ihre gehorig abgerheilten Felder einnehmen. Es kann noch zu einem zweyten Anpreiſungsgrunde dieſes Verfahrens dienen: daß in Allem, was Metaphy ſik heißt, die abſolute Vollſtandigkeit der Wiſſenſchaf ten gehofft werden kann, dergleichen man ſich in keiner anderen Art von Erkenntniſſen verſprechen darf, mithin eben ſo, wie in der Metaphyſik der Natur uberhaupt, alſo auch hier die Vollſtandigkeit der Metaphyſik der korperlichen Natur zuverſichtlich erwartet werden kann; wovon die Urſache iſt, daß in der Metaphyſik der Ge— genſtand nur, wie er blos nach den allgemeinen Geſetzen des Denkens, in andern Wiſſenſchaften aber, wie er nach datis der Anſchauung (der reinen ſowohl, als empiri— ſchen) vorgeſtellt werden muß, betrachtet wird, da denn. jene XxXv jene, weil der Gegenſtand in ihr jederzeit mit alien noth— wendigen Geſetzen des Denkens verglichen werden muß, eine beſtimmte Zahl von Erkenntniſſen geben muß, die ſich vollig erſchopfen laßt, dieſe aber, weil ſie eine unend— liche Mannigfaltigkeit von Anſchauungen (reinen oder empiriſchen), mithin Objecte des Denkens darbieten; niemals zur abſoluten Vollſtandigkeit gelangen, ſondern ins Unendliche erweitert werden konnen; wie reine Ma— thematik und empiriſche Naturlehre. Auch glaube ich dieſe metaphyſiſche Korperlehre ſo weit, als ſie ſich immer nur erſtreckt, vollſtandig erſchopft, dadurch aber doch eben kein großes Werk zu Stande gebracht zu haben. Das Schema aber zur Vollſtandigkeit eines meta— phyſiſchen Syſtems, es ſey der Natur uberhaupt, oder der korperlichen Natur insbeſondere, iſt die Tafel der Categorien“). Denn mehr giebt es nicht reine Verſtan des 2) Nicht wider dieſe Tafel der reinen Verſtandesbegriffe, ſon: dern die daraus gezogenen Schluſſe auf die Grenzbeſtim mung des ganzen reinen Vernunftvermogens, mithin auch aller Metaphyſit, ſinde ich in der Allgem. Litt. Zeit. Nt. 295. in der Recenſion der lnſtitutiones Logicae et Metaph. des Herrn Prof. Ulrich Zweifel, in welchen der tiefforſchende Recenſent mit ſeinem nicht minder prufenden Verfaſſer ubereinzukommen ſich erklaet, und zwar Zweifel, die, weil ſie gerade das Hauptfundament meines in der Critik aufge— ſtallten Syſtems treffen ſollen, Urſache waren, daß dieſes in Anſehung ſeines Hauptzieles noch lange nicht diejenige apo— dictiſche Ueberzeugung bey ſich fuhre, welche zur Abnothit gung XVI desbegriffe, die die Natur der Dinge betreffen konnen. Unter die vier Claſſen derſelben, die der Große, der Qua gung einer uneingeſchrankten Annahme erfoderlich iſt; dier ſes Hauptfundament ſey meine, theiis dort, thetls in den Prolegomenen, vorgetragene Deduction der reinen Vexr— ſtandesbegriffe, die aber in dem Thetrle der Cririk, welcher gerade der helleſte ſeyn mußte, am meiſten dunkel ware, oder wohl gar ſich im Cirkel herumdrehete ee. Jch richte meine VBeantwortung dieſer Einwurfe nur auf den Hauptt punct derſetben, daß namlich, ohue eine ganz klare und ge nugthurude Deduction der Categorien, das Syſtem der Critik der reinen Vernunft in ſeinem Fundamente wanke. Dagegen behaupte ich, daß fur denjenigen, der meine Satze von der Sinnlichkeit aller unſerer Anſchauung und der Zu— langlichkeit der Tafel der Categorien, als von den logiſchen Functionen in Urtheilen uberhaupt entlehnter Beſtimmun— gen unſeres Bewußtſeyns, unterſchreibt, (wie dieſes denn der Recenſent thut) das Soſtem der Critik apodictiſche Get wißheit bey ſich fuhren müſſe, weil dieſes auf dem Satze erbauet iſt: daß der ganze ſpeculative Gebrauch unſerer Vernunft niemals weiter, als auf Gegenſtande moglicher Erfahrung, reiche. Denn, wenn bewieſen werden kann: daß die Categorien, deren ſich die Vernunft in allem ihrem Erkenntniß bedienen muß, gar leinen anderen Gebrauch, als blos in Beziehung auf Gegenſtande der Erfahrung hat ben konnen (dadurch daß ſie in dieſer blos die Form des Denkens moglich machen), ſo iſt die Beantwortung der Frage, wie ſie ſolehe moglich machen, zwar wichtig genug, um dieſe Deduction, wo moglich, zu vollenden, aber in Bet ziehung auf den Hauptzweck des Syſtems, naämlich die Grenzbeſtimmung der reinen Vernunft, keinesweges noth— wendig, ſondern blos verdienſtlich. Denn in dieſer Abſicht iſt die Deduction ſchon alsdenu weit genug gefuhrt, wenn ſie XVYII Qualitut, der Relation und endlich der Modalitat, muſſen ſich auch alle Beſtimmungen des allgemeinen Be— griffs ſie zeigt, daß gedachte Categorien nichts anders, als bloße GVormen der Urtheile ſind, ſo fern ſie auf Anſchauungen (oie bey uns immer nur ſinnlich ſind) angewandt werden, dadurch aber allererſt Objecte bekommen und Erkenniniſſe weiden; weil dieſes ſchon hinreicht, das ganze Syſtem der eigentli—t chen Critik darauf mit volliger Sicherheit zu grunden. So ſteht Newtons Syſtem der allgemeinen Gravitaten feſt, ob es gleich die Schwierigkeit beyh ſich fuhrt, daß maun nicht err klaren kann, wie Anziehung in die Ferne moglich ſey; aber Schwierigkeiten ſind urcht Zweifel. Daß nun jenes Hauptfundament auch ohne voliſtandige Deductten ber Ca— tesgorien feſt ſtehe, beweiſe ich aus dein Zugeſtandenen alſo: 1. Zugeſtanden: daß die Tafel der Categorien alle reme Vert ſtandesbegtiffe vollſtandig enihalte und eben ſeo alle formale Verſtandeshandiaugen in Uetheilen, von welchen ſie abget leitet und auch in nichts unterſchieden ſind, als daß durch den Verſtandesbegriff ein Object in Anſehung einer oder der andern Function der Uztheile als beſtimmt gedacht wird; (z. B. ſo wird in dem categoriſchen Urtheile, der Stein iſt hart, der Stein fur Subject und hart als Pradicat ge—t braucht, ſo doth, daß es dem-Verſtande unbenommen bleibt, die iogiſche Function dieſer Begriffe umzutauſchen und zu ſagen: eiaiges Harte iſt zin Stein; dagegen, wenn ich es mir im Objecte als beſtimmt vorſtelle, daß der Stein in jeder moglichen Beſtimmung eines Gegepyſtandes, nicht des bloßen Begriffs, nur' als Subjeet, die Harte aber nur als Pradicat gedacht werden muſſe, dieſelben logiſchen Functio— nen nun veine Verſtandesbegriffe von Objeecten, namlich als Subſtanz vnd Aecidens, werden); 2. zugeſtanden: daß der Verſtand durch ſeine Natur ſynther tiſche Grundſatze a priori bey ſich fuhre, durch die er alle 2 b— Gegens XVIII griffs einer Materie uberhaupt, mithin auch alles, was a priori von ihr gedacht, was in der mathematiſchen Con Gegenſtande, die ihm gegeben-werden mogen, jenen Cate— gorien unterwirft, mithin es auch Anſchauungen a priori geben muffe, welche die jJur Anwendung jener reinen Ver— ſtandesbegriffe erforderlichen Bedingungen enthalten, weil ohne Auſchauung kein Object, in Anſehung deſſen die lo— giſche Function als Categorie beſtimmt werden konne, mit— hin auch keine Eckenntniß irgend eines Gegenſtandes, und alſo auch ohne reine Anſchauung kein Grundſatz, der ſit à priori in dieſer Abſicht beſtimmte, Statt findet; z. zugeſtanden: daß dieſe reinen Anſchauungen niemals etwas anders, als bloße Formen der Erſcheinungen außeren oder des inneren Sinnes (Raum und Zeit), folglich nur allein der Gegenſtande moglicher Erfahrungen ſeyn konnen: So folgt: daß aller Gebrauch der reinen Vernunft niemals worauf anders, als auf Gegenſtande der Erfahrung gehen konne, und, weil in Grundſatzen a priori nichts Empiri— ſches die Bedingung ſeyn kann, ſie nichts weiter als Prin— cipien der Moglichkeit der Erfahrung uberhaupt ſeyn konnen. Dieſes allein iſt das wahre und hinlaugliche Fundament der Grenzbeſtimmung der reinen Vernunſt, aber nicht die Aufloſung der Aufgabe: wie nun Erfaht rung vermittelſt jener Categorien und nur allein durch die— ſelbe moglich ſey. Die letztere Aufgabe, obgleich auch vhne ſie das Gebaude feſt ſteht, hat indeſſen große Wich— tigkeit, und, wie ich es jetzt einſehe, eben ſo große Leichtigkeit, da ſie beynahe durch einen einzigen Schluß aus der genau beſtimmten Definition eines Urtheils uberhaupt (einer Handlung, durch die gegebenen Vorſtellungen zuerſt Erkenntniſſe eines Objects werden) verrichtet werden kann. Die Dunkelheit, die in dieſem Theile der Deduction mei— nen vorigen Verhandlungen anhangt, und die ich nicht in Xx Conſtruction dargeſtellt, oder in der Erfahrung, als be— ſtimmter Gegenſtand derſelben, gegeben werden mag, brin— in Abrede ziehe, iſt dem gewohnlichen Schickſale des Verſtandes im Nachſforſchen beyzumeſſen, dem der kurze— ſte Weg gemeiniglich nicht der erſte iſt, den er gewahr wird. Daher ich Lie nachſte Gelegenheit ergreifen wer— de, diefen Mangel (welcher auch nur die Art der Dar— ſtellung, nicht den dort ſchon richtig augegebenen Erkla— rungsgrund, betrifft) zu erganzen, ohne daß der ſcharft ſinnige Recenſent in die ihm gewiß felbſt unangenehm fal—t lende Rothwendigkeit verſetzt werden darf, wegen der be—r fremdlichen Einſtinmung der Erſcheinungen zueden Ver— ſtandesgeſetzen, ob dieſe gleich von jenen ganz verſchisdene Quellen haben, zu einer praäſtabilirten Harmonie ſeine Zu— flucht zu nehmen; einem Rettungsmittel, welches wert ſchlinmer ware, als das Uebel, dawider es helfen ſoll, und das dagegen doch wirklich nichts helfen kamm. Denn auf dieſe komme doch jene objective Nothwendiakeit nicht heraus, welche die reinen Verſtandesbegriffe (und die Grundſatze ihrer Anwendung auf Erſcheinungen) characte: riſirt, z. B. in dem Begriffe der Urſache in Verknupfung mit der Wirkung, ſondern alles bleibt blos ſubjectiv— nothwendige, objectiv aber blos zufallige Zuſammenſtel- lung, gerade wie es Hume will, wenn er ſie bloße Tau— ſchung aus Gewohnheit nennt. Auch kann kein Syſtem in der Welt dieſe Nothwendigkeit wo anders herleiten, als aus den a priori zum Grunde liegenden Principien der Moglichkeit des Denkens ſelbſt, wodurch allein die Erkenntniß der Objecte, deren Erſcheinung uns gegeben iſt, d. i. Erfahrung, moglich wird, und geſetzt, die Art, wie Erfahrung dadurch allererſt moglich werde, konnte niemals hinreichend erklart werden, ſo bleibt es doch un widerſprechlich gewiß, daß ſie blos durch jene Begriffe b 2 moglich, xx SSJ— bringen laſſen. Mehr iſt hier nicht zu thun, zu entdecken oder hinzuzuſetzen, ſondern allenfalls, wo in der Deut— lichkeit oder Grundlichkeit gefehlt ſeyn mochte, es beſ— ſer zu muchen. Der Begriff der Materie mußte daher durch alle vier genannte Functionen der Verſtandesbegriffe (in vier Hauptſtucken) durchgefuhrt werden, in deren jedem eine neue Beſtimmung deſſelben hinzukam. DVie Grumndbe— ſtunmung eines Erwas, das ein Gegenſtand außerer Sinue ſeyn ſoll, mußte Bewegung ſeyn; denn dadurch allein konnen dieſe Sinne afficirt werden. Auf dieſe fuhrt auch der Verſtand alle ubrige Pradicate der Ma— terie, die zu ihrer Natur gehoren, zuruck, und ſo iſt die Naturwiſſenſchaft durchgängig eine entweder reine oder angewandte Bewegungslehre. Die metaphyſiſchen Anfangsgrunde der Naturwiſſenſchaft ſind alſo unter vier Hauptſtucke zu bringen, deren erſtes die Bewe— gung als ein reines Quantum, nach ſeiner Zuſammen— ſetzung, ohne alle Qualitat des Beweglichen, betrachtet, und Phorono mie genannt werden kann, das zwey— te ſie als zur Qualitat der Materie gehorig, unter dem Namen einer urſprunglich bewegenden Kraft, in Erwa— gung moglich, und jene Begriffe umgekehrt auch in keiner an: deren Beziehung, als auf Gegenſtande der Erfahrung, einer Bedkutung und irgend etines Gebrauchs fahig ſind. p xxi gung zieht, und daher Dy na mik heißt, das dritte die Materie mit dieſer Qualitat durch ihre eigene Be— wegung gegen einander. in Relation betrachtet, und unter dem Namen Mechanik vorkommt, das vier— te aber ihre Bewegung oder Ruhe blos in Beziehung auf die Vorſtellungsart, oder Modalitat, mithin als Erſcheinung außerer Sinne, beſtimmt, und Pha— nomeno logie genannt wird. Aber außer jener inneren Nothwendigkeit, die me— taphyſiſchen Anfangsgrunde der Korperlehre nicht allein von der Phyſik, welche empiriſche Principien braucht, ſondern ſelbſt von den rationalen Pramiſſen derſelben, die den Gebrauch der Mathematik in ihr betreffen, ab zuſondern, iſt noch ein außerer, zwar nur zufalliger, aber gleichwohl wichtiger Grund da, ihre ausfuhrliche Bearbeitung von dem allgemeinen Syſtem der Meta— phyſik abzutrennen, und ſie als ein beſonderes Ganze ſy— ſtematiſch darzuſtellen. Denn, wenn es erlaubt iſt, die Grenzen einer Wiſſenſchaft nicht blos nach der Beſchaf fenheit des Objects und der ſpecifiſchen Erkenntnißart deſſelben, fondern auch nach dem Zwecke, den man mit J der Wiſſenſchaft ſelbſt zum anderweitigen Gebrauche vor Augen hat, zu zeichnen, und findet, daß Metaphyſtk ſo viel Kopfe bisher nicht darum beſchaftigt hat und ſie ferner beſchaftigen wird, um Naturkenntniſſe dadurch iu erweitern, (welches viel leichter und ſicherer durch b 3 Beobach— XXII Beobachtung, Erperiment und Anwendung der Mathe— matik auf außere Erſcheinungen geſchieht, ſondern um zur Erkenntniß deſſen, was ganzlich uber alle Grenzen der Erfahrung hinausliegt, von Gott, Freyheit und Un— ſterblichkeit zu gelangen; ſo gewinnt man in Beforde— rung dieſer Abſicht, wenn man ſie von einem zwar aus ihrer Wurzel ſproſſenden, aber doch ihrem regelmaßigen Wuchſe nur hinderlichen, Sproßlinge befreyet, dieſen beſonders pflanzt, ohne dennoch deſſen Abſtammung aus jener zu verkennen und ſein volliges Gewachs aus dem Syſtem der allgemeinen Metaphyſik wegzulaſſen. Dieſes thut der Vollſtändigkeit der letzteren keinen Ab— bruch und erleichtert doch den gleichformigen Gang die— ſer Wiſſenſchaft zu ihrem Zwecke, wenn man in allen Fallen, wo man der allgemeinen Korperlehre bedarf, ſich nur auf das abgeſonderte Syſtem derſelben berufen darf, ohne jenes großere mit dieſem anzuſchwellen. Es iſt auch in der That ſehr merkwurdig, (kann aber hierr nicht ausfuhrlich vor Augen gelegt werden) daß die all— gemeine Metaphyſik in allen Fallen, wo ſie Beyſpiele (Anſchauungen) bedarf, um ihren reinen Verſtandesbe— griffen Bedeutung zu verſchaffen, dieſe jederzeit aus der allgemeinen Korperlehre, mithin von der Form und den Principien der außeren Anſchauung hernehmen muſſe, und, wenn dieſe nicht vollendet darliegen, unter lauter finnleeren Begriffen unſtat und ſchwankend herumtappe. Daher XXIII Daher die bekannten Streitigkeiten, wenigſtens die Dunkelheit in den Fragen: uber die Moglichkeit eines Widerſtreits der Realitaten, die der intenſiven Gro— ße, u. a. m., bey welchen der Verſtand nur durch Bey— ſpiele aus der korperlichen Natur belehrt wird, welches die Bedingungen ſind, unter denen jene Begriffe allein objective Realitat, d. i. Bedeutung und Wahrheit ha— ben konnen. Und ſo thut eine abgeſonderte Metaphyſik der korperlichen Natur der allgemeinen vortrefliche und unentbehrliche Dienſte, indem ſie Beyſpiele (Falle in Concreto) herbeyſchafft, die Begriffe und Lehrſatze der letzteren (eigentlich der Transſcendentalphiloſophie) zu realiſiren, d. i. einer bloßen Gedankenform Sinn und Bedeutung unterzulegen. Jch habe in dieſer Abhandlung die mathematiſche Methode, wenn gleich nicht mit aller Strenge befolgt, (wozu mehr Zeit erfoderlich geweſen ware, als ich dar— auf zu verwenden hatte) dennoch nachgeahmt, nicht, um ihr durch ein Geprange von Grundlichkeit beſſeren Eingang zu verſchaffen, ſondern weil ich glaube, daß ein ſolches Syſtem deren wohl fahig ſey und dieſe Voll. kommenheit auch mit der Zeit von geſchickterer Hand wohl erlangen konne, wenn, durch dieſen Entwurf ver— anlaßt, mathematiſche Naturforſcher es nicht unwichtig finden ſollten, den metaphyſiſchen Theil, deſſen ſie ohnedem nicht entubrigt ſeyn konnen, in ihrer allgemei. J nen xxtv —SJ nen Phyſtk als einen beſonderen Grundtheil zu behan— deln und mit der mathematiſchen Bewegungslehre in Vereinigung zu bringen. Newton ſagt in der Vorrede zu ſeinen mathem. Grundlehren der Nat. Wiſſ., (nachdem er angemerkt. hatte, daß die Geemetrie von den mechaniſchen Hand griffen, die ſie poſtulirt, nur zweyer bedurfe, namlich eink gerade Linie und einen Cirkel zu beſchreiben). Die Geometrie iſt ſtolz Warauf, daß ſie mit ſo weni— gem, was ſie anderwarts hernimmt, ſo viel zu. leiſten vermag“). Veon der Metaphyſik konnte man dagegen ſagen: ſie ſteht beſturzt, daß ſie mit ſo vie— lem, als ihr die reine Mathematik darbietet, doch nur ſo wenig ausrichten kann. Jndeſſen iſt doch dieſes Wenige etwas, das ſelbſt die Mathematik in ihrer Anwendung auf Naturwiſſenſchaft unumganglich braucht, die ſich alſo, da ſie hier von der Metaphyſik nothwendig borgen muß, auch nicht ſchamen darf, ſich mit ihr in Gemeinſchaft ſehen zu laſſen. Gloriatur Geometria, quod rtam paueis prineipiis aliunde petitis tam multa praeſtet. Newton Princ. Phil. Nat. Math. Pruefut. Erſtes Hauptſtuck. Metaphyſiſche Anfangsgrunde der Phoronomie— Erklarung 1. Bylaterie iſt das im Raume. 24 Der Raum, derBewegliche ſelbſt beweglich iſt, heißt der materielle, oder auch der relative Raum; der, in welchem alle Bewegung zuletzt gedacht werden muß, (der mithin ſeibſt ſchlechterdings unbeweglich iſt) heißt der reine, oder auch abſo— lute Raum. Anmerkung 1. Da in der Phoronomte von nichts als Bewegung ge— redet werden ſoll, ſo wird dem Subjzekt derſelben, namlich der Materie, hier keine andere Eigenſchaft beygelegt, als die Beweglichkeit. Sie ſelbſt kann aiſo ſo lange auch fur einen Punct gelten, und man abſtrahirt in der Phoronomie von aller innern Beſchaffenheit, mirhin auch der Große des Beweglichen, und hat es nur mit der Bewegung und dem, was in dieſer als Große betrachtet werden kann, (Geſchwin digkeit und Richtung) zu ihun. Wenn gleichwohl der Ausdruck eines Korpers hier bisweilen gebraucht werden A ſo llte, ſollte, ſo geſchieht es nur, um die Anwendung der Principien der Phoronomie auf die noch folgenden beſtimmteren Begriffe der Materie gewiſſermaaßen zu anticipiren, damit der Vor— trag weniger abſtract und faßlicher ſey. Anmerkung 2. Wenn ich den Begriff der Materie nicht durch ein Pra— dicat, was ihr ſelbſt als Objeet zukommt, ſondern nur durch das VBerhaltniß zum Erkenntnitzvermogen, in welchem mir die Vorſtellung allererſt gegeben werden kann, erkiaren ſoll, ſo iſt Materie ein jeder Gegenſtand außerer Sinne, und dieſes ware die blos metaphyſiſche Erklarung derſelhen. Der Rarm aber ware blos die Form aller außeren ſinnlichen An— ſchauung, (ob eben dieſelbe auch dem außeren Object, das wir Materie nennen, an ſich ſelbſt zukomme, oder nur in der Beſchaffenheit unſeres Sinnes bleibe, davon iſt hier gar nicht die Frage). Die Materie ware im Gegenfatz der Form das, was in der außeren Anſchauung ein Gegenſtand der Empfindung iſt, folglich das Eigentlich-empiriſche der ſinnlichen und außeren Anſchauung, weil es gar nicht a priori gegeben werden kann. Jn aller Erfahrung muß et— was empfunden werden, und das iſt das Reale der ſinn lichen Anſchauurig, folglich muß auch der Raum, in wel— chem wir uber die Bewegungen Erfahrung anſtellen ſollen, empfindbar, d. i. durch das, was empfunden werden kann, bezeichnet ſeyn, und dieſer, als der Jnbegriff aller Gegen— ſtande der Erfahrung und ſelbſt ein Object derſelben, heißt der ewnpiriſche Raum. Dieſer aber, als materiell, iſt ſelbſt beweglich. Ein beweglicher Raum aber, wenn ſeine Bevwoegung ſoll wahrgenommen werden konnen, ſetzt wiederum einen anderen erweitertern materiellen Raum voraus, in welchem er beweglich iſt, dieſer eben ſowohl einen andern, und ſo forthin ins Unendliche. Alſo iſt alle Bewegung, die ein Gegenſtand der Er— fahrung iſt, blos relativ, der Raum, in dem ſie wahrge— nommen wird, iſt ein relativer Raum, der ſelbſt wiederum, und J3 Nund vielleicht in entgegengeſetzter Richtung, in einem erwei— terten Raume bewegt, mithin auch die in Beziehung auf den erſtern bewegte Matetie in Verhaltniß auf den zweyten Raum ruhig genaunt werden kann, uno dieſe Abanderuigen des Begriffs der Bewegungen gehen mit der Veranderung des relativen Raums ſo ins Unendliche fort. Einen abſo— luten Raum, d. i. einen ſolchen, der, weil er nicht mate— riell iſt; auch kein Gegenſtand der Erfahrung ſehn kanu, als fur ſich gegeben annehmen, heiat etwas, das weder an ſich, noch in ſeinen Folgen (der Bewegung im abſoluten Raum) wahrgenommen werden kann, um der Mdoalichkeit der Erſahrung willen annehmen, die docy jederzeit ohne ihn angeſte t werden muß. Der abſolute Raum iſt auſo au ſich nichts und gar lein Object, ſondern bedeutet nur einen zeden andern relatipen. Faum, den ich mir außer dem gegebenen jederzeit denken lann, und den ich nur uber jeden gegebenen ins Unendliche hinausrücke, als einen ſolchen, der dieſen einſchlietßt und in welchem ich den erſteren als bewegt autieh— men kann. Weil ich den erweiterten, obgleich immer noch materiellen, Raum nur in Gedanken habe und mir von der Materie, die ihn bezeichnet, nichts betannt iſt, ſo ab— ſtrahtre ich von dieſer, und er wird daher wie ein reiner, nicht empiriſcher und abſoluter Raum vorgeſtellt, mit dem ich jeden emptriſchen vergleichen und dieſen in ihm als be— weglich vorſtellen lann, der alſo jederzeit ars unbeweglich gilt. Jhn zum wirklichen Dinge zu ma hen, heißt die lo— giſche Allgemeinbeit irgend eines Rauns, mit dem ich jeden empirtichen als darin eingeſchloſſen vergleichen kann, in eine phyſiſche Allgemeinheit des wirklichen Unfanges verwechſeln, und die Vernunft in ihrer Jdee misverſtehen. Schueßitch merke ich noch an: daß, da die Beweg— lichkeit eines Gegenſtandes im Raum a priori und ohne Belehrung durch Erfahrung nicht erkannt werden kann, ſie von mir eben darum in der Critik der r. V. auch nicht unter die reinen Verſtandesbeariffe gezahlt werden konnte, und daß dieſer Begriff, als empiriſch, nur in einer Naturwiſſen— A2 ſchaft, 4 ſchaft, als angewandter Metaphyſik, welche ſich mit einem durch Erfahrung gegebenen Begriffe, obwohl uach Princi— pien a priori, beſchaftigt, Platz finden konne. Erklarung 2. Bewegung eines Dinges iſt die Verande— rung der außeren Verhaltniſſe deſſelben zu einem gegebenen Raum. Anmerkung 1. Vorher habe ich dem Begriffe der Materie ſchon den Begriff der Bewequng zum Grunde gelegt. Denn, da ich denſelben ſelbſt unabhangig vom Begriffe der Ausdehnung beſtimmen wollte, und die Materie alſo auch in einem Puncte betrachten konnte, ſo durfte ich einraumen, daß man ſich daſelbſt der gemeinen Erklarung der Bewegung als Weranderung des Orts bedienete. Jetzt, da der Be— griff einer Materie allgemein, mithin auch auf bewegte Korper paſſend, erklart werden ſoll, ſo reicht jene Defini— tivn nicht zu. Denn der Ort eines jeden Korpers iſt ein Punct. Wenn man die Weite des Mondes von der Erde beſtimmen will, ſo will man die Entfernung ihrer Oerter wiſſen, und zu dieſem Ende mißt man nicht von einem be— liebigen Puncte der Oberflache, oder des Jnwendigen der Erde, zu jedem beliebigen Punete des Mondes, ſondern nimmt die kurzeſte Linie vom Mittelpunete des einen zum Mittelpuncte des andern, mithin iſt von jedem dieſer Kor— per nur ein Punct, der ſeinen Ort ausmacht. Nun kaun ſich ein Korper bewegan, ohne ſeinen Ort zu verandern, wie die Erde, indem ſie ſich um ihre Achſe dreht. Aber ihr Verhältniß zum äußeren Raume verandert ſich hiebey doch; denn ſie kehrt z. B. in 24 Stunden dem Monde ihre verſchiedenen Seiten zu, woraus denn auch allerley wandel— bare Wirkungen auf der Erde erfolgen. Nur von einem beweglichen, d. i. phyſiſchen, Puncte kann man ſagen: Bewe —S 5 Bewegung ſey jederzeit Veranderung des Ort. Man konnte wider dieſe Erklarung erinnern: daß die innere Be— wegung, z. B. einer Gahrung, nicht in ihr mit eingeſchloſſen ſey: aber das Ding, was man bereegt nennt, muß ſo fern als Einheit betrachtet werden. Die Materie, als z. B. ein Faß Bier, iſt bewegt, bedeutet alſo etwas anderes, als das Bier im Faſſe iſt in Bewegung. Die Bewegung eines Dinges iſt mit der Bewegung in dieſem Dinge nicht einerley, von der erſteren aber iſt hier nur die Rede. Die— ſes Begriffs Anwendung aber auf den zweyten Fall iſt nachher leicht. Anmerkung 2. Die Bewchrungen konnen drehend (ohne Veranderung des Orts) oder fortſchreitend, dieſe aber entweder den Raum erweiternd, oder auf einen gegebenen Raum einge— ſchrankte Bewegungen ſeyn. Von der erſteren Art ſind die geradlinigten, oder auch krummlinigten, in ſich nicht zuruck— kehrenden Bewegungen. Die von der zweyten ſind die in ſich zuruckkehrenden. Die letztern ſind wiederum entweder circulirende oder oſcillirende, d. i. Kreis-, oder ſchwan— kende Bewegungen. Die erſtern legen eben denſelben Raum immer in derſelben Richtung, die zweyten immer wechſels— weiſe in entgegengeſetzter Richtung zuruck, wie ſchwankende Penduln. Zu beyden gehort noch Bebung (mortus tre— mulus), welche nicht eine fortſchreitende Bewegung eines Korpers, dennoch aber eine reciprocirende Bewegung einer Materie iſt, die dabey ihre Stelle im Ganzen nickt veran— dert, wie die Zitterungen einer geſchlagenen Glocke, oder die Bebungen einer durch den Schall in Bewegung geſetzten Luft. Jch thue dieſer verſchiedenen Arten der Bewegung blos darum in einer Phoronomie Erwahnung, weil man bey allen, die nicht fortſchreitend ſind, ſich des Worts Ge ſchwindigkeit gemeiniglich in anderer Bedeutung bedient, als bey den fortſchreitenden, wie die folgende Anmer— kung zeigt. Az An Anmerkung 3. Jn jeder Bewegung ſind Richtung und Geſchwindig— keit die beyden Momente der Erwagung. derſelben, wenn man von allen anderen Eigenſchafren des Beweglichen ab— ſtrahirt. Jch ſetze hier die gewohnliche Deſinttion beyder voraus; allein die der Richtung bedarf unoch verſchtedener Etutchrankungen. Ein im Kreiſe bewegter Korper vetan— dert ſeine Richtung coutunnrlich, ſo, daß er bis zu ſetner Rucktehr zum Punecte, voie em er auvging, alle in einer Flache nur mogliche Richtungen eingeſchlagen iſt, und doch ſugt man: er bewege ſich immer in derſelben Rechtung, z. B. der Planet von Abend gegen Morgen. Allein, was iſt hier die Seite, nach der die Bewe— gung gerichtet iſt? eine Frage, die mit der einqe Verwandt— ſchaft hat, worauf beruhet der innere Unterſchieb der Schne— cken; die ſonſt ahnlich und ſo gar gleich, aber davon eine Species rechts, die andere links gewunden iſt; oder des Windens der Schwerdtbohnen und des Sopfens, deren die erſtere wie ein Pfropfenzieher, oder, wie die Seeleute es ausdrucken wurden, wider die Sonne, der andere mit der Sonne um ihre Stange laufen? ein Begriff, der ſich zwar conſtruiren, aber, als Begriff, fur ſich durch allge— meine Merkmale und in der discurſiven Erkenntnißart gar nicht deutlich machen laßt, und der in den Dingen ſelbſt (z. B. an denen ſeltenen Menſchen, bey denen die Leichener— offnung alle Theile nach der phyſiologiſchen Regel mit an— dern Menſchen einſtimmig, nur alle Eingeweide links oder rechts, wider die gewohnliche Ordnung verſetzt fand) keinen erdenklichen Unterſchied in den innern Folgen geben kann und demnach ein wahrhafter mathematiſcher und zwar inuerer Unterſchied iſt, womit der, von dem Uhnterſchiede zweyer ſonſt in allen Stucken gleichen, der Richtung nach aber ver ſchiedenen Kreisbewegungen, obgleich nicht vollig einerley, dennoch aber zuſammenhangend iſt. Jch habe anderswo gezeigt, daß, da ſich dieſer Unterſchied zwar in der Au— ſchauung ſchauung geben, aber gar nicht auf deutliche Begriffe brin— gen, mithin nicht verſtandlich ertlaren (dari, non intelligi) laßt, er einen guten beſtätigenden Beweisgrund zu dem Satze abgebe: daß der Raum uberhaupt nicht zu den Eigen— ſchaften oder Verhaltnifſfen der Dinge an ſich ſelbſt, die ſich nothwendig auf objectire Begriffe mußten bringen laſſen, ſondern blos zu der ſubjectiven Form unſerer ſinnlichen An— ſchauung von Dingen oder Verhaltniſſen, die uns, nach dem, was ſie an ſich ſeyn mogen, vollig unbekannt bleiben, gehore. Doch dies iſt eine Abſchweifung von unſerem jetzi— gen Geſchafte, in welchem wir den Raum ganz nothwendig als Eigenſchaft der Dinar, die wir in Betrachtung ziehen, namlich korperlicher Weſen, behandeln mufſen, weil dieſe ſelbſt nur Erſcheinungen außerer Sinne ſind und nur als ſolche hier erklart zu werden bedurfen. Was den Be— griff der Geſchwindigkeit betrifft, ſo bekommt dieſer Aus— druck im Gebrauche auch bisweilen eine abweichende Beden: tung. Wir ſagen: die Erde dreht ſich geſchwinder um ihre Achſe als die Sonne, weil ſie es in kürzerer Zeit thut; obgleich die Bewegung der letzteren viel geſchwinder iſt. Der Blutumlauf eines kleinen Vogels iſt viel geſchwinder, alsſder eines Menſchen, obgleich ſeine ſtrmende Bewegung im erſteren ohne Zweifel weniger Geſchwindigkeit hat, und ſo auch bey den Bebungen elaſtiſcher Materien. Die Kurze der Zeit der Wiederkehr, es ſey der circulirenden oder oſtcil— lirenden Bewegung, macht den Grund dieſes Gebrauchs aus, an welchem, wenn ſonſt nur die Misdeutung vermie— den wird, man auch nicht unrecht thut. Denn dieſe bloße Vergroßerung der Eile in der Wiederkehr, ohne Vergroße— rung der raumlichen Geſchwindigkeit, hat ihre eigenen und ſehr erheblichen Wirkungen in der Natur, worauf in dem Cirkellauf der Safte der Thiere vielleicht noch nicht genug Rückſicht genommen worden. Jn der Phorondmie brauchen wir das Wort Geſchwindigkeit blos in raumlicher Bedeu— 8 tung C— T Aa4 Erkla— Erklärung z. Ruhe iſt die beharrliche Gegenwart (praeſentia perdurabilis) an demſelben Orte; beharr— lich aber iſt das, was eine Zeit hindurch exiſtirt, d. i. dauret. Aanmerkung. n Ein Korper, der in Bewegung iſt, iſt in jedem Puncte der Linte, die er durchlauft, einen Argenblick. Es fragt ſich nun, oh er darin rutze, oder ſich bemege. Ohne Zweifel wird man das letztere ſagen; denn er iſt in dieſem Puucte nur ſo fern, als er ſich bewegt, genenwartig. Man nehme aber die Bewegvong deſſelben ſo an: A aO, o0oB —o. daß der Korper mir gleichtdrmiger Geſchwindigkeit die Linie AB vorwarts und ructwarts von Benach A zurücilege, ſo daß, weil der Augenblick, da er in B iſt, beyden Bewe— gungen gemein tſt, ote Bewegung von Annach Bin Sec., die von hunach A aber auch in Sec., beyde zuſammen aber in einer ganzen Secunde zuruckgelegt worden, ſo daß auch nicht der kleinſte Theil der Zeit auf die Gegenwart des Korpers in B aufgewandt worden: ſo wird, ohne den min— deſten Zuwachs dieſer Bewegungen, die letztere, die in der Richtung BA geſchahe, in die nach der Richtung Ba, wel— ches mit AB in einer geraden Linie liegt, verwandelt werden fonnen, wo denn der Korper, indem er in B iſt, darin nicht als ruhig, ſondern als bewegt angeſehen werden muß. Er mußte daher auch in der erſteren in ſich ſelbſt wiederkeh— renden Bewegung in dem Puncte B als bewegt angeſehen werden, welches aber unmoglich iſt; weil, nach dem, was angenommen worden, es nur ein Augenblick iſt, der zur Bewegung AL und zugleich zur gleichen Bewegung BA ge— hort, die der vorigen entgegengeſetzt und mit ihr in einem und demſelben Augenblicke verbunden iſt, volligen Mangel der Bewegung, folglich, wenn dieſer den Begriff der Ruhe aus 2 ausmachte, auch in der gleichformiger Bewegung An Ruhe des Korpers in jedem Puncte, z. B. in B, beweiſen munte, welches der obigen Behauptung widerſpricht. Man ſtelle ſich dagegen die Linie AB als uber den Punct A aufgerichtet vor, ſo, daß ein Korper von An nach B ſteigend, nachdem er durch die Schwere im Puncte B feine Bewegung verloh— ren hat, von B enach Aneben ſv wiederum zuruckfalle; ſo fräge ich, ob der Korper in Bels bewegt, oder als ruhig angeſehen werden konne. Olne Zweifel wird man ſagen, als ruhig: weil ihm alle vocherige Bewegung genomnien worden, nachdem er dieſen Punct erreicht hat, und hernach eine gleichmaßige Bewegung zuruck allererſt folgen ſoll, folglich noch nicht da iſt; der Mangel aber der Bewegung, wird man hinzufetzen, iſt Ruhe. Aber in dem erſteren Falle einer angenommeuen gleickformigen Bewegung konnte die Bewegung EA auch nicht anders eintreten, als dadurch, daß vorher die Beweqgung Ab aufgehort hatte und die von Bnach A noch nicht war, folglich, daß in Bein Mangel aller Bewegung, und, aach der gewohnlichen Ertlarung, Ruhe mußte angenommen werden, aber man durfte ſte doch nicht aunehmen, weil, bey einer gegebenen Geſchwindig— keit, kein Korper in einan Puncte ſeiner gleichformigen Be— wegung als ruhend gedacht werden muß. Worauf beruht denn im zweyten Fale die Anmaßung des Begriffs der Ruhe, da doch dieſes Steigen und Fallen gleichfalls nur durch einen Attgeublie von einander getrennt wird. Der Grund davon liegt darin, daß die letztere Bewegung nicht als gleichformig mit gegebener Geſchwindigkeit gedacht wird, ſondern zuerſt als gleichformig verzogert und hernach als gleichformig beſchleanigt, ſo doch, daß die Geſchwindigkeit im Puncte B nicht ganzlich, ſondern nur bis zu einem Grad, der kleiner iſt, als zede nur anzugebende Geſchwindigkeit, mit welcher, wenn, anſtatt zuruckzufallen, die Linie ſeines Falles BA in die Richtung Ba geſtellet, mithin der Korper immer noch als ſteigend betrachtet wurde, er, als mit einent bloßen Moment der Geſchwindigkeit, Gder Widerſtand der A5 Schwe 10 ptec Schwere wird alsdenn bey Seite geſetzt), in jeder noch ſo großen anzugebenden Zett gleichſdrmig doch nur einen Raum, der kieiner iſt, a.s jeder anzugebende Roum, zurucklegen, mirhin ſeinen Ort (für irgeno eine mogliche Erfahrung) in alle Ewigkeit gar nicht verandern wurde. Folglich murd er in den Zuſtand einer dauecaden Gegenwart an demſelben Orte, d. i. der Ruhe, verſitzt, ob ſie gleich wegen der con— tinuſrlichen Einwirkung der Schwere, d. i. der Veranderung dieſes Zuſtandes, ſo fort aufzehoben wird. Jn einem be harrlichen Zuſtande ſeyn ind darin beharren (wenn nichts anderes ihn verruckt) ſind zwey verſchiedene Begriffe, deren einer dem anderen keinen Abbruch thut. Alſo kann die Ruhe nicht durch den Maungel der Bewegung, der ſich, als So, gar nicht conſtruiren laßt, ſondern muß durch die beharrliche Gegenwart an deniſelben Orte ertlart wer— den, da denn dieſer Begriff auch durch die Vorſtellung einer Bewegung mit unendlich kletier Geſchwindigkeit, eine endüiche Zeit hindurch conſtruirt, mithin zu nachheriger Anwendung der Mathematik auf Naturwiſſenſchaft benutzt werden kann. Erklarung 4. Den Begriff einer zuſammengeſetzten Bewegung conſtruiren— heißt eine Bewe— ung, ſo fern ſie aus zweyen oder mehreren ge gebenen in einem Beweglichen vereinigt ent— ſpringt, a priori in der Anſchauung darſtellen. Anmerkung. Zur Conſtruction der Begriffe wird erfodert: daß die Bedingung ihrer Darſtellung nicht von der Erſahrung ent— lehnt ſey, alſo auch nicht gewiſſe Krafte vorausſetze, deren Exiſtenz nur von der Erfahrung abgeleitet werden kann, oder uberhaupt, daß die Bedingung der Conſtruction nicht ſelbſt ein Begriff ſeyn muſſe, der gar nicht a priori in der An 11 Anſchauung gegeben werden kann, wie z. B. der von Urſa— che und Wirkung, Handlung und Widerſtand rc. Hier iſt nun vorzuglich zu bemerken: daß Phorononue durchaus zuerſt Conſtruction der Bewegungen uberhaupt als Großen, und, da ſie die Materie blos als Etwass Bewegliches, mithin an welchem gar auf keine Große derſelben Ruckſicht genom men wird, zum Gegenſtande hät, dieſe Bewegungen allein als Großen, ſo wohl ihrer Geſchwindigkeit als Richtung nach, und zwar ihrer Zufammenſetzung nach a priori zu be— ſtimmen habe. Denn ſo viel muß ganzlich a priori und zwar anſchauend zumn Behuf der angewandten Mathemattk ausgemacht werden. Denn die Regeln der Verknupfung der Bewegungen durch phyſiſche Urſachen, d. i. Krafte, laſ— ſen ſich, ehe die Grundſatze ihrer Zuſammenſetzung uberhaupt vorher rein mathematiſch zum Grunde gelegt worden, nie— mals grundlich vortragen. Grundſatz 1. Eine jede Bewegung, als Gegenſtand einer moglichen Erfahrung, kann nach Belteben, als Bewegung des Korpers in einem ruhigen Rau— me, oder als Ruhe des Korpers und dagegen Bewegung des. Raumes in entgegengeſetzter Richtung mit gleicher Geſchwindigkeit angeſe— hen werden. Anmerkung. Von der Bewegung eines Korpers eine Erfahrung zu machen, dazu wird erfodert: daß nicht allein der Korper, ſondern auch der Raum, darin er ſich bewegt, Gegenſtande der außern Erfahrung, mithin materiell ſeyn. Eine abſo— lute Bewegung alſo, d. i. in Beztehung auf einen nicht ma— teriellen Raum, jſt gar keiner Erfahrung fahig und fur uns alſo nichts (wenn man gleich einraumen wollte, der abſolute Raum ſey an ſich etwas). Aber auch in aller relativen Be we 12 S wegung kann der Raum ſelbſt, weil er als materiell ange— .nommen wird, wiederum als ruhig oder bewegt vorgeſtellt Swerden. Das erſtere geſchteht, wenn mir uber den Raum, in Beziehung auf welchen ich einen Korper als bewegt an— ſehe, kein mehr erweiterter und ihn einſchließender gegeben iſt, (wie wenn ich in der Cajute eines Schiffs eine Kugel auf dem Tiſche bewegt ſehe); das zweyte, wenn mir uber dieſen Raum hinaus noch ein anderer Raum, der ihn ein— ſchließt, (wie im genanntea Falle das Ufer des Fluſſes) ge— geben iſt, da ich denn in Anſehung des letzteren den nach— ſten Raum (die Caqute) als bewegt und den Korper ſelbſt alt afalls als ruhig anſehen kann. Da es nun ſchlechter— dings unmoglich iſt, von einem empiriſch gegebenen Raume, wie erwrutert er auch ſey, auszumachen, ob er nicht in An— ſehung eines in einem noch großeren Umfange ihn einſchlie— ßenden Raumes ſelbſt wiederum bewegt ſey, oder nicht, ſo muß es aller Erfahrung und jeder Folge aus der Erfahrung vollig einerley ſeyn, ob ich einen Korper als bewegt, oder ihn als ruhig, den Raum aber in entgegengeſetzter Richtung mit gleicher Geſchwindigkeit bewegt anſehen will. Noch mehr: da der abſolute Raum fur alle mogliche Erfahrung nichts iſt, ſo ſind auch die Begriffe einerley, ob ich ſage: ein Korper bewegt ſich in Anſehung dieſes gegebenen Rau— mes in dieſer Richtung mit dieſer Geſchwindigkeit, oder ob ich ihn mir als ruhig denken, und dem Raum alles dieſes, aber in entgegengeſetzter Richtung, beylegen will. Denn ein jeder Begriff iſt mit demjenigen, von deſſen Unterſchiede vom erſteren gar kein Beyſpiel moglich iſt, vollig einerley und nur in Beziehung auf die Verknupfung, die wir ihm im Verſtande geben wollen, verſchieden. Auch ſind wir gar nicht im Stande, in irgend einer Erfahrung einen feſten Punct anzugeben, in Beziehung auf welchen, was Bewegung und Ruhe abſolut heißen ſollte, beſtimmt wurde; denn alles, was uns auf die Art gegeben wird, iſt materiell, alſo auch beweglich, und (da wir im Raume keine außerſte Grenze moglicher Erfahrung kennen,) viel 13 vielleicht auch wirklich bewegt, ohne daß wir dieſe Bewe— gung woran wahrrehmen toönnen. Von dieſer Lieres gung eines Korpero im empiriſchen Raume kann ich nun einen Theil der gegebenen Geſchwindigkeit dem Korper, den andern dem Raume, abet in entgegengeſetzter Richtung, ge— ben, und die ganze mogliche Erfahrung in Antehung der Folgen dieſer zwey verbundenen Bewegungen iſt vollig iner— ley mit derjenigen, da ich den Korper mit der ganzen Ge— ſchwindigkeit allein bewegt, oder ihn als ruhig und den Raum mit derſelben Geſchwindigkeit in entgegengeſetzter Richtung bewegt denle. Jch nehme hier aber alle Be— wegungen als gerarlinigt an. Denn, wes die krumm— linigte betrifft, da es nicht in allen Studlen einerley iſt, ob ich den Korper (z. B. die Erde in ihrer taglichen Um— drehung) als bewegt, und den umgebenden Raum (den be— ſtirnten Himmel) als ruhig, oder dieſen als bewegt und jenen als ruhig anzuſehen befugt bin, davon wird in der Folge beſvnders gehandelt werden. Jn der Phoronomie alſo, wo ich die Bewegung eines, Korpers nur mit dem vcaume, (auf deſſen. RFuhe vder Bewegung jener gar keinen Einfluß hat,) in Verpaltniß betrachte, iſt es an ſich ganz unbeſtimmt und beliebig, ob unb wie viel ich Geſchwindig— keit dem einen oder dem andern von der gegebenen Bewegung beylegen will; kunftig in der Mechanik, da ein bewegter Korper in wirkſamer Beziehung auf andere Korper im Rau— me ſeiner Bewegung betrachtet werden ſoll, wird dieſes nicht mehr ſo vollig einerley ſeyn, wie es an ſeinem Orte gezeigt werden ſoll. Erklarung z5. Die Zuſammenſetzung der Bewegung iſt die Vorſtellung der Bewegung eines Puncts als einerley mit zweyen oder mehreren Bewe— gungen deſſelben zuſammen verbunden. Anmer— 14 Anmerkung. Jn der Phoronomie, da ich die. Materie durch keine andere Eigenſchaft als ihre Beweglichkeit kenne, mithin ſie ſeibſt nur als einen Punct betrachten darf, kann die Bewe— gung nur als Beſchreibung eines Raun.es betrachtet werden, doch ſo, daß ich nicht blos, wie in der Geome— trie, auf den NRaum, der beſchrieben wird, ſondern auch auf die Zeit darin, mithin auf die Gelchwindigkelt, Womit ein Punct den Raum beſchreibt, Acht habe, Phoronomie iſt alſo die reine Groſenlehre Matheſis) der Bewrgungen. Der beſtimmte Begriff von einer Große iſt der Begriff der Erzeugung der Vorſtellung eines Gegenſtandes dur.h die Zu ſammenſetzung des Gleichartigen. Da nun der Bewegung nichts gleichartig iſt, als wiederum Bewegung, ſo iſt die Phoronomie eine Lehre der Zuſammenſetzung der Bewegun— gen eben deſſelben Puncts nach ihrer Richtung und Ge— ſchwindigkert, d. i. die Vorſtellung einer einzigen Bewegung, als einer felchen, die zwey und ſo mehrere Bewegungen zu— gleich in ſich enthalt, oder zweyer Bewegungen eben deſſel— ben Puncts zugleich, ſo ferne ſie zuſamn.en Eine ausma— chen, d. i. mit dieſer einerley ſind, und nicht etwa ſo fern ſie die letztere, als Urſachen ihre Wirkung, hervorvringen. Um die Bewegung zu finden, die aus der Zuſammenſetzung von mehreten, ſo viel man will, entſpringt, darf man nur, wie bey aller Großenerzeugung, zuerſt diejenige ſuchen, die unter gegebenen Bedingungen aus zweyen zuſammengeſetzt iſt; datauf dieſe mit einer dritten verbunden u. ſ. w. Folglich laßt die Lehre der Zuſammenſetzung aller Bewegun— gen ſich auf die von zweyen zuruckfuhren. Zwey Bewegun— gen aber eines und deſſelben Punets, die zugleich an demſel— ben angetroffen werden, konnen auf zwiefache Weiſe unter— ſchieden ſeyn, und als ſolche auf dreyfache Art an ihm verbunden werden. Erſtlich geſchehen ſie entweder in einer und derſelben Linie, oder in verſchiedenen Linien zu— oleich; die letztere ſind Bewegungen, die einen Winkel ein— ſchlie— S 15 ſchließen. Die, ſo in einer und dexſelben Linie geſchehen, ſind nun der Richtung nach entweder einander eulgegen geſetzt, oder halten einerien ?ichetrng. Da alle dieſe Bewegungen als zugleich geſcucers letiuchtet werden, ſo ergiebt ſich aus dem Verhaltnik der Lintien, d. t. der be— ſchriebenen Raume der Bewegung, in gleicher Zert, ſo fort auch das Verhältniß der Geſchwundigkeit. Alſo ſind der Falle drey. 1) Da zwey Beweguncgeen ſee mogen von gleichen oder ungleichen Geſchwindtaleiten ſeyn) in einem Korper in derſelben Richtung verbunden, eine daraus zu— ſammengeſetzte Bewegung auemachen ſollen. 2, Da zwey Bewegungen deſſelben Puncts (von gleicher oder ungtet— cher Geſchwindigkeit) in entgegengeſetzter Richtung verbun— den durch ihre Zuſammenſetzung eine dritte Bewegung in der— ſelben Linie ausmachen ſollen. 3) Da zwey Bewegrngen eines Puncts, mit gleichen oder ungleichen Geſchwindigkei— ten, aber in verſchiedenen Linten, die einen Winlel ein— ſchließen, als zuſammengeſetzt betrachtet werden. Lehrſatz 1. Die Zuſammenſetzung zweyer Bewegungen eines und deſſelben Puncts kann nur dadurch ge— dacht werden, daß die eine derſelben im abſoluten Raume, ſtat? der anderen aber eine mit der glei— chen Geſchwindigkeit in entgegengeſetzter Rich— tung geſchehende Bewegung des relativen Raunis, als mit derſelben einerley, vorgeſtellt wird. Beweis. Erſter Fall. Da zwey Bewegungen in eben derſelben Linie und Richtung einem und demſelben Punete zugleich zukommen. Es 16 —S Es ſollen in einer Geſchwindigkeit der Bewegung zwey Geſchwindigkeiten AB und ab als enthalten vor geſtellt werden. Man nehme dieſe Geſchwindigkeiten fur diesmal als gleich an, ſo daß ABab iſt, ſo ſa— ge ich, ſie konnen in einem und demſelben Raum, (dem abſoluten oder dem relativen) an demſelben Puncte nicht zugleich vorgeſtellt werden. Denn, weil die Li— nien Ab und ab, welche die Geſchwiudigkeiten bezeich— nen, eigentlich die Raume ſind, welche ſte in gleichen Zeiten durchlaufen, ſo wurde die Zuſammenſetzung die— ſer Rauume Aß und ab —BC, mithin die Linie AC, als die Summe der Raume, die Summe beyder Ge— ſchwindigkeiten ausdrucken muſſen. Aber die Theile AB und B0 ſtellen, jede fur ſich, nicht die Geſchwindigkeit Sab vor; denn ſie werden nicht in gleicher Zeit wie ab zuruckgelegt. Alſo ſtellt auch die doppelte Linie AC, die in derſelben Zeit zuruckgelegt wird, wie die Linie ab, nicht die zwiefache Geſchwindigkeit der letztern vor, welches doch verlangt wurde. Allſo laßt ſich die Zuſammenſetzung zweyer Geſchwindigkeiten in einer Richtung in demſelben Raume nicht anſchaulich darſtellen. Dagegen, wenn der Korper Anmit der Geſchwin— digkeit AB im abſoluten Raume als bewegt vorgeſtellt wird, und ich gebe uberdem dem relativen Raume eine Geſchwindigkeit ab AB in entgegengeſehzter Rich— tung ba —Ch, ſo iſt dieſes eben daſſelbe, als ob ich die letztere Geſchwindigkeit dem Korper in der Rich— tung Ab ertheilt hätte (GGrundſatz 1.). Der Korper bewegt ſich aber alsdenn in derſelben Zeit durch die Summe der Linien Ab und BCæaab,in welcher er die die Linie abAB allein wurde zuruckgelegt haben, und ſeine Geſchwindigkeit iſt doch als die Summe der zweyen gleichen Geſchwindigkeiten AB und ab vorge— ſtellt, welches das iſt, was verlangt wurde. Zweyter Fall. Da zweh Bewegungen itt gerade entgegengeſetzten Richtungen an einem und demſelben Puncte ſollen verbunden werden. D J7c Es ſey AB die eine dieſer Bewegungen und AC die andere in entgegengeſetzter Richtung, deren Geſchwin— digkeit wir hier der erſten gleich annehmen wollen: ſo wurde der Gedanke ſelbſt, zwey ſolche Bewegungen in einem und demſelben Rauine an eben demſelben Puncte als zugleich vorzuſtellen, mithin der Fall einer ſolchen Zuſammenſetzung der Bewegungen ſelbſt unmoglich ſeyn, welches der Vorausſetzung zuwider iſt. Dagegen denket euch die Bewegung Aß im abſo— luten Raume, ſtatt der Bewegung AC aber in dem— ſelben abſoluten Raume, die entgegengeſetzte CA des relativen Raumes mit eben derſelben Geſchwind gtat, die (nach Grundſatz 1.) der Bewegung AC vollig gleich gilt und alſo ganzlich an die Stelle derſelben ge— ſetzt werden kann: ſo laſſen ſich zwey gerade entgegen— geſetzte und gleiche Bewegungen deſſelben Puncts zu gleicher Zeit gar wohl darſtellen. Weil nun der rela— tive Raum mit derſelben Geſchwindigkeit CA ABb in derſelben Richtung mit dem Puncte A bewegt iſt, ſo verandert dieſer Punct, oder der in ihm befindliche Bo Kor 18 Korper, in Anſehung des relativen Raumes Ort nicht, d. i. ein Korper, der nach zwey ein gerade entgegengeſetzten Richtungen mit gleicher ſchwindigkeit bewegt wird, ruhet, oder, allgemein gedruckt: ſeine Bewegung iſt der Differenz der ſchwindigkeiten in der Richtung der großeren (welches ſich aus dem Bewieſenen leicht folgern le Dritter Fall. Da zwey Bewegungen deſſelben Puncts, nach Richtungen, die Winkel einſchließen, verbunden vergeſtellt wer A. M d f Fn C c a r a er o r o r Ê Die zwey gegebenen Bewegungen ſind Al AC, deren Geſchwindigkeit und Richtungen durchk Uinien, der Winkel aber, den die letzteren einſchl durch BAC ausgedruckt wird, (er mag, wie hier rechter, aber auch ein jeder beliebiger ſchiefer ſeyn). Wenn nun dieſe zwey Bewegungen z1i in den Richtungen Ab und AC und zwar in und demſelben Raume geſchehen ſollen: ſo wurl doch nicht in dieſen beyden Linien AB und AC zi geſchehen konnen, ſondern nur in Linien, die 19 parallel laufen. Es wurde alſo angenommen werden mufſen: daß eine dieſer Bewegungen in der anderen eine Veranderung (namlich die Avbringung von der gegebenen Bahn) wirkte, wenn gleich biyee ſeits Riche tuugen dieſelben olieben. Dacfes iſt aber der Voraus— ſetzung des Lehrſatzes zuwider, wel.he unter dem Wor— te Zuſammenqezung andeutet: daß beyde gegebene Be— wegungen in einer dritren enthalten, mithin mit die— ſer einerley ſeyn, und nicht, daß, indem eine, die an— dere vera.tert, ſie eine dritte hervorkrivaen. Dagegen nehme man die Bewegung AC als im abſoluten Raume vor ſich gehend an, anſtatt der Be— wegung AB aber die Bewegung des relativen Raumes in entgegengeſetzter Richtung. Die Linie AC eyn in drey gleiche Theile Ak, EF. FC gerheilt. Wahrend daß nun der Korper Anim abſoluten Raume die Liuuie All durchlauft, durchlauft der relative Raum, und mit ihm der Punct E, den Raum LKe—MAz wah— rend daß der Korper die zwey Theile zuſanmen A durchlauft, beſchreibt der relative Raum, und mit ihm der Punct F, die Linie Ul NA: wahrend daß der Korper endlich die ganze Linie AC durchlauft, ſo be— ſchreibt der Raum, und mit ihm der Punct C, die i— nie CoBA; welches alles eben daſſelbe iſt, als ob der Korper A in dieſen drey Zeittheilen die Linien Em, Fn, und CDAM. AN. AB und in der ganzen Zeit, darin er AC durchlauft, die Linie CDAs durch laufen hatte. Alſo iſt er im letzten Augenblicke im Puncte D und in dieſer ganzen Zeit nach und nach in allen Puneten der Diagonallinie AD, welche alſo ſo— wohl die Richtung als Geſchwindigkeit der zuſammen— geſetzten Bewegung ausdruckt. B 2 Anmer— 20 Anmerkung 1. Die geometriſche Conſtruction erfodert, daß eine Große mit der andern, oder zwey Großen in der Zuſammen ſehung mit einer dritten einerley ſeyn, nicht daß ſte als Urſachen die dritte hervorbringen, welches die mechaniſche Conſtruction ſeyn. wurde. Die vollige Aehnlichkeit und Gleichheit, ſo fern ſie nur in der Anſchauung erkannt wer— den lann, iſt die Congruenz. Alle geometrifche Con— ſtruction der volligen Jdentitat beruht auf Congruenz. Dirſe Congruenz zweyer zuſammenverbundenen Bewegungen mit einer dritten (als dem motu compoſito ſelbſt) kann nun niemals Statt haben, wenn jene beyden in einem und dem— ſelben Raume, z. B. dem relativen, vorgeſtellt werden. Daher ſind alle Verſuche, obigen Lehrſatz in ſeinen dreh Fallen zu beweiſen, immer nur mechaniſche Aufloſungen ge— weſen, da man namlich bewegende Urſachen durch die eine gegebene Bewegung, mit einer andern verbunden, eine dritte hervorbringen ließ, nicht aber Beweiſe, daß jene mit die— ſer einerley ſind, und ſich, als ſolche, in der reinen An ſchauung a priori darſtellen laſſen. Anmerkung 2. Wenn z. B. eine Geſchwindigkeit AB doppelt genannt wird: ſo kann darunter nichts anders verſtanden werden, als daß ſie aus zwey einfachen und gleichen Ab und EC (ſiehe Fig. 1.) beſtehe. Erklart man aber eine doppelte Ge— ſchwindigkeit dadurch, daß man ſagt, ſie ſey eine Bzwe gung, dadurch in derſelben Zeit ein doppelt ſo großer Raum zuruckgelegt wird, ſo wird hier etwas angenommen, was ſich nicht von ſelbſt verſteht, namlich: daß ſich zwey glei che Geſchwindigkeiten eben ſo verbinden Jaſſen, als zwey gleiche Raäume, und es iſt nicht fur ſich klar, daß eine ge gebene Geſchwindigkeit aus kleinern und eine Schnelligkeit aus Langſamkeiten eben ſo beſtehe, wie ein Raum aus lei neren; denn die Theile der Geſchwindigkeit ſind nicht außer halb 21 halb einander, wie die Theile des Raumes, und wenn jene als Große betrachtet werden ſoll, ſo muß der Begriff ihrer Große, da ſie intenſiv iſt, auf andere Art conſtruirt wer den, als der in der extenſiven Große des Raumes. Dieſe Conſtructton iſt aber auf keine andere Art moglich, als durch die mittelbare Zuſammenſetzung zweyher gleichen Bewegun— gen, deren eine die. des Korpers, die andere des relativen Raumes in entgegengeſetzter Richtung, aber eben darum mit einer ihr gleichen Bewegung des Korpers in der vorigen Richtung vollig einerlen iſt. Denn in derſelben Richtung laſſen ſich zwey gleiche Geſchwindigkeiten in einem Korper gar nicht zuſammenſetzen, als nur durch außere bewegende Urſachen, z. B. ein Schiff, welches den Korpet mit einer dieſer Geſchwindigkeiten tragt, indeſſen daß eine andere mit dem Schiffe uubeweglich verbundene bewegende Kraft dem Korper die zwehte, der vorigen gleiche, Geſchwiudigkeit ein druckt; wobey doch immer vorausgeſetzt werden muß: daß der Kotper ſich mit der erſten Geſchwindigkeit in freher Be wegung erhalte, indem die zweyte hinzukommt; welches ein YNaturgeſetz bewegender Krafte iſt, wovon gar nicht die Rede ſejn kann, wenn die Frage lediglich. iſt, wie der Begriff. der Geſchwindigkeit als eine Große conſtruiret werde. So viel von der Hinzuthuung der Geſchwindigkeiten zu einan— der. Wenn aber von der Abziehung einer von der anderen die Rede iſt, ſo laßt ſich zwar dieſe letztere leicht denken, wenn einmal die Moglichkeit einer Geſchwindigkeit als Gro— ße durch Hinzuthunng eingeraumt worden, aber jener Be— griff laßt ſich nicht ſo leicht eonſtruiren. Denn zu dem Ende muſfen zwey entgegengeſetzte Bewegungen in einem Korper verbunden werden; aber wie ſoll dieſes geſchehen? Unmittelbar, d. i. in Anſehung eben deſſelben ruhenden Rau— mes iſt es unmoglich, ſich zwey gleiche Bewegungen in ent— gegengeſetzter Richtung au demſetben Korper zu denken; aber die Vorſtellung der Unmoglichkeit dieſer beyden Bewegungen in einem Korper iſt nicht der Begriff von der Ruhe deſſel ben, ſondern der Unmogüchkeit der Conſtruction dieſer 3 Zu 22 Zuſammenſetzung entgegengeletzter Bewegungen, die doch im Lehrſatz alv moglich angenuuimen wird. Dieſe Conſtruction iſt aber nicht anders urda ich, als durch die Verbindung der Bewegung der Korpers mit der Bewequtig dt Roums, wie gewieſen wocden. Eadlich, wais die nuſanmenſetzung zwener Bewegungen, deren Richting einen Wintel ein— ſchließt, betrifft, ſe latzt ſr ſich an dem Korper, in Bezie— hung auf einen und denſelben Raum, gleichfalis nicht dent ken, wenn mun nicht gar eine derſelben durch außere con— tinutriich einfließende Kraft (z. E. ein den Korper forttrat gendes Fahrzeug) gewirkt, die andern als ſich ſelbſt hiebey underandert erbaltend, annimmt, oder uberhaupt, man muß bewegende Krafte und Erzeugung einer dritten Bewegung aus zwey vereinigten Kraften zum Grunde legen, welches zwar die mechaniſche Ausführung defſen, was ein Begriff enthalt, aber nicht die mathematiſche Conſtruction der— ſelben iſt, die nur anſchaulich machen ſoll, was das Objeet (als Quantum) ſey; unicht, wie es durch Ratur oder Kunſt, vermittelſt gewiſſer Werkzeuge und Krafte hervorgebracht werden konne. Die Zuſammenſetzung der Bewegungen, um ihr Verhaltniß zu andern als Große zu beſtimmen, m iß nach den Regeln der Congruenz geſchehen, welches in allen dreyen Fallen nur vermittelſt der Bewegung des Raums, die mit einer der zwey gegebenen Bewegungen congruirt, und dadurch beyde mit der zuſammengeſetzten congruiren, mog— lich iſt. Anmerkung 3. Phoronomie, unicht als reine Bewegungslehre, ſon— dern blos als reine Großenlehre der Bewegung, in wel— cher die Materie nach keiner Eigenſchaft mehr als der blo— ßen Beweglichkeit gedacht wird, enthalt alſo nichts mehr, als blos dieſen einzigen, durch die angefuhrten deey Falle gefuhrten Lehrſatz von der Zuſammenſetzung der Beweaung und zwar von der Moglichkeit der geradlinigten Bewe gung allein, nicht der krummlinigten. Denn, weil in die 23 dieſer die Bewegung continuirlich (der Richtung nach) ver— andert wird, ſo muß eine ürfche diefer Veranderung, wel che nun nicht der bloße Raum ſeyn tann, herbeygezogen werden. Daß man, aber gewoöhnlich unter der Benennung der zuſammeungeſetzten Bewegung nur den emuigen Fall, da die Richtungen derſelben einen Winkel einſchlteßen, ver— ſtand, dadurch ward zwar wohl eben nicht der Phyſik, wohl aber dem Princip der Etutheilung einer reinen philoſophi— ſchen Wiſſenſchaft uberhaupt einiger Abbruch gethan. Denn was die erſtere betrifft, ſo laſſen ſich alle im obigen Lehrſatze behandelte drey Falle im dritten allein hinreichend darſtel— len. Denn, wenu der Winkel, den die zwey gegebenen Bewegungen einſchließen, als unendlich klein gedacht wird, ſo enthalt er den erſten; wird er aber als von einer einzigen geraden Linie nur unendlich wenig unterſchieden vorgeſtellt, ſo enthalt er den zwehten Fall; ſo daß ſich freylich in dem bekannten Lehrfatze der zuſammengeſetzten Bewegung alle drey von uns genanute Falle, als in einer allgemeinen For— mel, geben laſſen. Man konnte aber auf dieſe Art nicht wohl die Großenlehre der Bewegung nach ihren Theilen a priori einfehen leren, welches. in. maucher Abſicht auch ſeinen Nutzen hat. Hat jemand Luſt, die gedachten drey Theile des all— gemeinen phoronomiſchen Lehrſatzes an das Schema der Eintheilung aller reinen Verſtandesbegriffe, namentlich hier der des Begriffs der Große zu halten, ſo wird er bemerken: daß, da der Begriff einer Große jederzeit den der Zuſammenſetzung des Gleichartigen enthält, die Lehre der Zuſammenſetzunge der Bewegungen zugleich die reine Großenlehre derſelben ſeh, und zwar nach allen drey Momenten, die der Raum an die Hand giebt, der Ein— heit der Linie und Richtung, der Vielheit der Richtun— gen in einer und derſelben Linie, endlich der Aliheit der Richtungen ſowohl, als der Linien, nach denen die Bewegung geſchehen mag, welches die Beſtimmung al— B 4 ler 24 —S ler mööglichen Bewegung als eines Quantum enthalt, wiewohl die Quantitat derſelben (an einem beweglichen Puncte) blos in der Geſchwindigkeit beſteht. Dieſe Bemerkung hat nur in der TCranſcendentalphiloſophie Nihren Rutzen. Zweytes Hauptſtuck der Metaphyſiſchen Anfangsgrunde der Dynamiek. Erklarung 1. Materie iſt das Bewegliche, ſo fern es einen Raum erfullt. Einen Raum erfullen, heißt allem Beweglichen widerſtehen, das durch ſeine Bewegung in einen gewiſſen Raum einzudrin— gen beſtrebt iſt. Ein Raum, der nicht erfullt iſt, iſt ein leerrer Raum. Anmerkung. Dieſes iſt nun die dynamiſche Erklarung des Begriffs der Materie. Sie ſetzt die phoronomiſche voraus, aber thut eine Eigenſchaft hinzu, die ſich als Urſache auf eine Wirkung bezieht, namlich das Vermogen, einer Bewegung innerhalb eines gewiſſen Raumes zu widerſtehen, wovon in der vorhergehenden Wiſſenſchaft gar nicht die Rede ſeyn mußte, ſelbſt nicht, wenn man es mit Bewegungen eines und deſſelben Punctes in eutgegengeſetzten Richtungen zu thun 25 thun hatte. Dieſe Erfullung des Rauins hult einen gewif— ſen Jaum von dem Eindringen irgend eines anderen Beweq— lichen freh, wenn ſeine Bewegurg auf irgend einen Ort in dieſem Raume hingerichtet iſt. Worauf nun der nach allen Seiten gerichtete Widerſtand der Materie beruhe und was er ſey, muß noch unterſucht werten. So viel ſieht man aber ſchon aus der obigen Erklarung: daß die Materie hier nicht ſo betrachtet wird, wie ſr widerſteht, wenn ſie aus ihrem Orte getrieben uw alſo ſelbſt bewegt werden ſoll, (dieſer Fall wird kunftig, als mechaniſcher Widerſtand, noch in Erwaguna kommen) ſondern wenn blos der Raum ihrer eigenen Ausdehnung berringert werden ſoll. Man bedient ſich des Worts: einen Raum einnehmen, d. i. in allen Puncten deſſelben unmittelbar gegenwartig ſeyn, um die Ausdehnung eiues Dinges im Raume dadurch zu be— zeichnen. Weil aber in dieſem Begriffe nicht beſtimmt iſt, welche Wirkung, oder ob gar uberall eine Wirkung aus die— ſer Gegenwart entſpringe, ob andern zu widerſtehen, die hineinzudringen beſtrebt ſeyn, oder ob es blos einen Raum ohne Materie bedeute, ſo fern er ein Jubegriff mehrerer Raumr iſt, wie man von jeder geometriſchen Figur ſagen kann, ſie nimmt einen Raum ein (ſie iſt ausgedehut), oder ob wohl gar ini Raume etwas ſey, was ein anderes Be— wegliche nothigt, tiefer in denſelben einzudringen (andere anzieht), weil, fage ich, durch den Begriſf des Einnehmens eines Raumes dieſes alles unbeſtimmt iſt, ſo iſt: einen Raum erfullen, eine nahere Beſtimmung des Begriffs: einen Raum einnehmien. kLehrſatz 1. Die Naterie erfullt einen Raum, nicht durch ihre bloße Exiſtenz, ſondern durch eine beſondere bewegende Kraft. B 5 Be— 26 SS Beweis. 1 Das Eindringen in einen Raum (im Anfangs— augenblicke heiß,t ſolches die Beſtrebung einzudringen) iſt eeae Bewegung. Der Widerſtand, gegen Bewe— gunq iſt die Urſache der Verminderung, oder auch Veranderung derjelben in Ruhe. Nun kann mit kei— ner Bewegung etwas verbunden werden, was ſie ver— mindert eder aufhebt, als eine andere Bewegung eben deſſelben Beweglichen in emgegengeſetzter Richtung (Phoron. Lehrſ.). Alſo iſt der Widerſtand, den eine Materie in dem Raum, den ſie erfullt, allem Ein— dringen anderer leiſtet, eine Urſache der Bewegung der letzteren in entgegengeſetzter Richtunug. Die Ur— ſache einer Bewegung heißt aber bewegende Kraft. Alſo erfullet die Materie ihren Raum durch bewegen— de Kraft, und nicht durch ihre bloße Exiſtenz. Anmerkung. Lambert und andere nannten die Eigenſchaft der Ma— terie, da ſie einen Raum erfullt, die Soliditat, (ein ziemlich vieldeutiger Ausdruck) und wollen, man muſſe ſie an jedem Dinge, was exiſtirt, (Subſtanz) annehmen, we— nigſtens in der außeren Sinuenwelt. Nach thren Begrif— fen wußte die Anweſenheit von etwas Reellem im Raume dieſen Widerſtand ſchon durch ſeinen Begriff, mithin nach dem Satze des Widerſpruchs, bey ſich fuhren, und es ma— chen, daß nichts anderes in dem Raume der Anweſenheit eines ſolchen Dinges zugleich ſeyn konne. Allein der Satz des Widerſpruchs treibt keine Materie zuruck, welche an— ruckt; um in einen Raum einzudringen, in welchem eine andere anzutrefſen iſt. Rur alsdann, wenn ich dem, was eiuen Raum einnimint, eine Kraft beylege, alles außere Bewegliche, welches ſich annahert, zuruckzutreiben, ver— ſtehe pν 27 ſtehe ich, wie es einen Widerſpruch enthalte, daß in den Raum, den ein Ding einntnimt, noch ein anderes von derſelben Art eindringe. Hier hat der Mathematiker etwas als ein erſtes Datum der Conſtruction des Begriffs einer Materie, welches ſich ſelbſt nicht weiter conſtrutren laſſe, angenommen. Nun kann—.er zwar von jedem beltebigen Dato ſeine Conſtruction eines Begriffs anfangen, ohne ſich dar— auf einzulaſſen, dieſes Datum auch wiederum zu erilaren; darum aber iſt er doch nicht befugt, jenes fur etwas aller mathematiſchen Conſtruction ganz Unfahiges zu erklaten, um dadurch das Zurugkgehen zu den erſten Principien in der Naturwiſſenſchaft zu hemmene« Erklarung 2. Anziehungskraft iſt diejenige bewegende Kraft, wodurch eine Materie die Urſache der Annaherung anderer zu ihr ſeyn kann (oder, wel— ches einerley iſt, dadurch ſie der Entfernung an— derer von ihr widerſteht). Zuruckſtoßungskraft iſt diejenige, wo— durch eine Materie Urſache ſeyn kann, andere von ſich zu entfernen (oder, welches einerley iſt, wodurch ſie der Annaherung anderer zu ihr wi— derſteht). Die letzteren werden wir auch zuwei— len treibende, ſo wie die erſteren ziehende Krafte nennen. Zuſatz. Es laſſen ſich nur dieſe zwey bewegende Krafte der Materie denken. Denn alle Bewegung, die eine Materie einer anderen eindrucken kann, da in dieſer Ruckſicht jede derſelben nur wie ein Punct betrachtet wird, muß jederzeit als in der geraden Linie zwiſchen zweyen ST 28 zweyen Puncten ertheilt angeſehen werden. Jn dieſer geraden Linie aber ſind nur zweyerley Bewegungen moglich: die eine, dadurch ſich jene Puncte ven einan— der entfernen, die zweyte, dadurch ſie ſich einander nahern. Die Kraft aber, die die Urſache der erſte— ren Bewegung iſt, heißt Zuruckſtoßungs-, und die der zweyten, Anziehungskraft. Alſo konnen nur dieſe zwey Arten von Kraften, als ſolche, worauf alle Bewegungskrafte in der materlellen Natur zuruckge— fuhrt werden muſſen, gedacht werden. Lehrſatz 2. Die Naterie ekfullet ihre Raume durch re— pulſive Krafte aller ihrer Theile, d. i. durch eine ihr eigene Ausdehnungskraft, die einen beſtimm— ten Grad hat, uber den kleinere oder großere ins Unendliche konnen gedacht werden. Beweis. Die Materie erfullet einen Raum nur durch be— wegende Kraft (Lehrſ. 2.) und zwar eine ſolche, die dem Eindringen anderer, d. i. der Annaherung wider ſteht. Nun iſt dieſe eine zuruckſtoßende Kraft. (Er— klarung 2.) Alſo erfullet die Materie ihren Raum nur durch zuruckſtoßende Krafte, und zwar aller ihrer Thei— le, weil ſonſt ein Theil ihres Raums (wider die Vor— ausſetzung) nicht erfullet, ſondern nur eingeſchloſſen ſeyn wurde. Die Kraft aber eines Ausgedehnten vermoge der Zuruckſtoßung aller ſeiner Theile iſt eine Ausdehnungskraft (expanſive). Aliſo erfullet die Materie ihren Raum nur durch eine ihr eigene Aus— deh J 29 dehnungskraft; welches das Erſte war. Ueber jede gegebene Kraft muß eine großere gedacht werden kon— nen, denn die, uber welche keine großere moglich iſt, wurde eine ſolche ſeyn, wodurch in einer endlichen Zeit ein unendlicher Raum zuruckgelegt werden wurde, (wel— ches unmoglich iſtz). Es muß ferner unter jeder gege— benen bewegenden Kraft eine kleinere gedacht werden konnen, (denn die kleinſte wurde die ſeyn, durch deren unendliche Hinzuthuung zu ſich ſelbſt eine jede gegebene Zeit hindurch keine endliche Geſchwindigkeit erzeugt werden konnte, welches aber den Mangel aller bewe— genden Kraft bedeutet). Alſo muß unter einem jeden gegebenen Grad einer bewegenden Kraft immer noch ein kleinerer gegeben werden konnen, welches das Zwey te iſt. Mithin hat die Ausdehnungskraft, womit jede Materie ihren Raum erfullt, ihren Grad, der niemals der großte oder kleinſte iſt, ſondern uber den ins Unendliche ſowohl großere als kleinere konnen ge— funden werden. Zuſatz 1. Die eppanſive Kraft einer Materie nennt man auch Elaſticitat. Da nun jene der Grund iſt, wor— auf die Erfullung des Raumes, als eine weſentliche Eigenſchaft aller Materie, beruht, ſo muß dieſe Ela— ſticitat urſprunglich heißen; weil ſie von keiner aude— ren Eigenſchaft der Materie abgeleitet werden kann. Alle Materie iſt demnach urſprunglich elaſtiſch. Zuſatz 2. Weil uber jede ausdehnende Kraft eine großere bewegende Kraft gefunden werden kann, dieſe aber auch J 30 auch jener entgegenwirken kann, wodurch ſie alsdenn den Naum der letz'eren verengen wurde, den dieſe za erweitern trachtet, in welchem Falle die erſtere eine zuſammendruckende Kraft heißen wurde; ſo muß auch fur jede Materie eine zuſammendruckeade Kraft gefun— den werden konnen, die ſie von einem jeden Raum, den ſie erfullt, in einen engeren Raum zu treiben vermag. Erklarung 3. Eine Materie durchdringt in ihrer Bewe— gung ein: andere, wenn ſie vurch Zuſammen— druckung den dcaum ihrer Ausdehnung vollig aufhebt. Anmerkung. Wenn in einem mit Luft angefullten Stiefel einer Luftpumpe der Kolben dem Boden immer naher getrieben wird, ſo wird die Luftmaterie zuſammengedruckt. Konnte nun dieſe Zuſammendruckung ſo weit getrieben werden, daß der Kolben den Boden vollig beruührte, (ohne daß das Min— deſte von Luft entwiſcht ware) ſo wurde die Luftmaterie durchdrungen ſeyn; denn die Materien, zwiſchen denen ſie iſt, laſſen leinen Raum fur ſie ubrig, und ſie waäre alſo zwiſchen dem Kolben und Boden anzutreffen, ohne doch einen Raum einzunehmen. Dieſe Durchdringlichkeit der Materie durch außere zuſammendruckende Krafte, wenn je— mand eine ſolche aunehmen oder auch nur denken wollte, wurde die mechaniſche heißen konnen. Jch habe Urſache, durch eine ſolche Einſchrankung dieſe Durchdringlichkeit der Materie von einer andern zu unterſcheiden, deren Be— griff vielleicht eben ſo unmoglich, als der erſtere iſt, von der ich aber doch kunftig etwas anzumerken Anlaß haben mochte. Lehr— 31 Lehrſatz z. Die Naterie kann ins Unendliche zuſam— mengedruckt, aber niemals von euier wate— rie, wie groß auch die druckende Kraft derſerben ſey, durchorungen werden. Beweis. Eine urſprunaliche Kraft, womit eine Materie ſich uber einen gegebenen Raum, den ſie einnimmt, allerwarts auszudehnen trachtet, muß, in einen klei— neren Raum eingeſchleſſen, großer, und, in einen unendlich kleinen Raum. zuſammengepreßt, unendlich ſeyn. Nun kann fur gegebene ausdehnende Keaft der Materie eine großere zuſemmendruckende gefunden wer— den, die dieſe in einen engeren Raum zwingt, und ſo ins Unendliche; welches das Erſte war. Zum Durch— dringen der Materie aber wurde eine Zuſammentrei— bung derſelben in einen unendlich kleinen Raum, mit— hin eine unendlich zuſammendruckende Kraft erfodert, welche unmoglich iſt. Alſo kann eine Materie durch Zuſammendruckung von keiner anderen durchdrungen werden; welches das Zweyte iſt. Anmerkung. IJch habe in dieſem Beweiſe gleich zu Anfange ange— nommen, daß eine ausdehnende Kraft, je mehr ſie in die Enge getrieben worden, deſto ſtarker entgegenwirken muſſe. Dieſes wurde nun zwar nicht ſo fur jede Art elaſtiſcher Krafte, die nur abgeleitet ſind, gelten; aber bey der Ma— terie, ſo fern ihr als Materie uberhaupt, die einen Raum erfullt, weſentliche Elaſticitat zutommt, laßt ſich dieſes poſtuliren. Denn expanſive Kraft aus allen Puncten nach allen 32 S allen Seiten hin ausgeubt, macht ſogar den Begriff derſel— ben aus. Eben daſſelbe Quantum aber von ausſpannenden Kraften in einen engeren Raum gebracht, muß in jedem Puncte deſſelben ſo viel ſtarker zurücktreiben, ſo viel umge— kehrt der Raum kleiner iſt, in welchem ein gewiſſes Quan tum von Kraft ſeine Wirkſamkeit verbreitet. Erklarung 4. Die Undurchdringlichkeit der Materie, die auf dem Widerſtande beruht, der mit den Graden der Zuſammendruckung proportionirlich wachſt, nenne ich die relative; diejenige aber, welche auf der Vorausſetzung beruht, daß die Raterie, als ſolche, gar keiner Zuſan mendru— ckung fahig ſey, heißt die abſolute Undurch— dringlichteit. Die Erfullung des Raumes mit abſoluter Undurchdringlichkeit kann die ma thematiſche, die mit blos relativer, die dyna miſche Erfullung des Raums heißen. Anmerkung 1. Nach dem blos mathematiſchen Begriffe der Undurchdringlichkeit (der keine bewegende Kraft als urſprunglich der Materie eigen vorausſetzt) iſt keine Materie einer Zuſam— mendruckung fahig, als ſo fern ſie leere Raume in ſich ent halt; mithin die Materie als Materie widerſteht allem Ein— dringen ſchlechterdings und mit abſoluter Rothwendigkeit. Nach unſerer Erorterung dieſer Eigenſchaft aber beruht die Undurchdringlichkeit auf einem phyſiſchen Grunde; denn die ausdehnende Kraft macht ſie ſelbſt, als ein Ausgedehntes, das ſeinen Raum erfullt, allererſt moglich. Da aber dieſe Kraft einen Grad hat, welcher uberwaltigt, mithin der Raum der Ausdehnung vertingert, d. i. in denſelhen bis auf ein gewiſſes Maaß von einer gegebenen zuſammen drucken: t 83 druckenden Kraft eingedrungen werden kann, doch ſo, daß die ganzliche Durchdtingeng, weil ſie eine unendliche zu— ſammendruckende Kraft erfodern wurde, unmoglich iſt: ſo muß die Erfullung des Raums aur au reiative Un— durchdringlichkeit angeſehen werden. Anmerkung 2. Die abſolute Undurchdringlichkett iſt in der That nichts mehr, oder weniger, als qualitas orculta. Dean man fragt, was die Urſache ſey, daß Materien einander in ihrer Bewegung nicht durchdringen konnen, und betommt die Antwort: weil ſie undurchdringlich ſind. Die Berufung auf zurucktreibende Kraft iſt von dieſem Vorwurfe frey. Denn ob dieſe aleich ihrer Moglichleit nach auch nicht wei— ter erklart werden kann, mithin als Grundkraft geiten muſt, ſo giebt ſie doch einen Begriff von einer wirkenden ürſache und ihren Geſetzen, nach welchen die Wirkung, namm— lich der Widerſtand in dem erfulleten Raum, ihren Graden nach geſchatzt werden kann. Erklarung 5. Materielle Subſtanz iſt dasjenige im Raume, was fur ſich, d. i. abgeſondert von al— lem anderen, was außer ihm im Raume eriſtirt, beweglich iſt. Die Bewegung eines Theils der Naterie, dadurch ſie aufhort ein Theil zu ſeyn, iſt die Treunung. Die Trennung der gTheile einer Materie iſt die phyſiſche Theilung. Anmerkung. Der Begriff einer. Subſtanz bedeutet das letzte Sub— ject der Exiſtenz, d. i. dasjenige, was ſelbſt nicht wiederu n blos als Pradicat zur Exiſtenz eines anderen gehort. Nun iſt Materie das Subject alles deſſen, wav im Raume zur C Exr 34 e r Exiſtenz der Dinge gezahlt werden mag; denn außer ihr wurde ſonſt kein Subject gedacht werden konnen, als der Raum ſelbſt; welcher aber ein Begriff iſt, der noch gar nichts Exiſtirendes, ſondern blos die nothwendigen Bedin— gungen der außeren Relation moglicher Gegenſtande außerer Sinne enthalt. Alſo iſt Materie, als dass Bewegliche im Raume, die Subſtanz in demſelben. Aber eben ſo werden auch alle Theile derſelben, ſo fern man von ihnen nur ſagen kann, daß ſie ſelbſt Subjecte und nicht blos Pradicate von anderen Materten ſeyn, Subſtanzen, mithin ſelbſt wie— derum Materie heißen muſſen. Sie ſind aber ſelbſt Sub— jecte, wonn ſie fur ſich beweglich und alſo auch außer der Verbindung mit anderen Nebentheilen etwas im Raume exi— ſtirendes ſind. Alſo iſt die eigene Beweglichkeit der Ma— terie, oder irgend eines Theils derſelben, zugleich ein Be weis dafur, daß dieſes Bewegliche, und ein jeder bewegli— cher Theil deſſelben, Subſtanz ſey. Lehrſatz 4. Die Naterie iſt ins Unendliche theilbar, und zwar in Theile, deren jeder wiederum Ma— terie iſt. Beweis. Die Materie iſt undurchdringlich, und zwar durch ihre urſprungliche Ausdehnungskraft (Lehrſ. 3.), dieſe aber iſt nur die Folge der repulſiven Krafte eines jeden Puncts in einem von Materie erfullten Raum. Nun iſt der Raum, den die Materie erfullet, ins Unendliche mathematiſch theilbar, d. i. ſeine Theile konnen ins Unendliche unterſchieden, obgleich nicht be— wegt, folglich auch nicht getrennt werden, (nach Be— weiſen der Geometrie). Jn einem mit Materie erful— leten Raume aber enthalt jeder Theil deſſelben repul— ſive J 35 ſive Kraft, allen ubrigen nach allen Seiten entgegen— zuwirken, mithin ſie zuruckzutreiben und von ihnen eben ſo wohl zuruckgetrieben, d. i. zur Entfernung von denſelben bewegt zu werden. Mitcthin iſt cin jeder Theil eines durch Materie erfullten Raums fur ſich ſelbſt beweglich, folglich trennbar von den ubrigen als inaterielle Subſtanz durch phyſiſche Theilung. So weit ſich alſo die mathematiſche Theilbarkeit des Rau— mes, den eine Materie erfullt, erſtreckt, ſo weit er— ſtreckt ſich auch die mogliche phyſiſche Theilung der Subſtanz, die ihn erfullt. Die mathematiſche Theil— barkeit aber geht ins Unendliche, ſolglich auch die phy ſiſche, d. i. alle Materie iſt ins Unendliche theilbar, und zwar in Theile, deren jeder ſelbſt wiederum ma— terielle Subſtanz iſt. Anmerkung 1. Durch den Beweis der unendlichen Theilbarkeit des Raunms iſt die-der Materie lange noch nicht bewieſen, wenn nicht vorher.dargethan worden: daß in jedem Thetle des Raumes materieclle Subſtanz ſey, d. i. fur ſich bewegliche Theile anzutreffen ſind. Denn, wollte ein Mongdiſt an— nehmen, die Materie beſtande aus phyſiſchen Punteten, dee ren ein jeder zwar (eben darum) keine beweglirhe Theile habe, aber dennoch durch bloße repulſive Kraft einen Raum erful— lete; fo wurde er geſtehen konnen, daß zwar dieſer Raum, aber nicht die Subſtanz, die in ihm wirkt, mithin zwar die Sphare der Wirkſamkeit der letzteren, aber nicht das wirkende bewegliche Subject ſelbſt durch die Theilung des Rauins zugleich getheilt werde. Alſo wurde er die Mate— rie aus phyſiſch untheilbaren Theilen zuſammenſetzen, und ſie doch auf dynamiſche Art einen Raum einnehmen laſſen. Durch den obigen Beweis aber iſt dem Monadiſten dieſe Ausflucht ganzlich benommen. Denn daraus iſt klar: C2 daff 36 SS daß in eineen erfulleten Raume kein Punct ſeyn konne, der nicht ſelbſt nach allen Seiten Zuruckſtoßung ausubete, ſo wie er zuruckgeſtoßen wind, mithin als ein außer jedem an— deren zuruckſtoßenden Puncte befindliches gegenwirkendes Subject an ſich ſelbſt beweglich ware, und daß die Hypo theſe eines Puncts, der durch bloße treibende Kraft, und nicht vermittelſt anderer gleichfalls zuruckſtoßenden Krafte, einen Raum erfullete, ganzlich unmoglich ſeh. Um dieſes und dadurch auch den Beweis des vorhergehenden Lehrſatzes anſchaulich zu machen C.. OOO O nehme man an, A ſey der Ort einer Monas im Raume, ab ſey der Durchmeſſer der Sphare ihrer repulſiven Kraft, mithin aa der Halbmeſſer derſelben, ſo iſt zwiſchen a, wo dem Eindringen einer äußeren Monade in den Raum, den jene Sphare einnimmt, widerſtanden wird, und dem Mit— telpuncte derſelben A, ein Punct e anzugeben moglich (laut der unendlichen Theilbarkeit des Raumes). Wenn nun A demjenigen, was in a einzudringen trachtet, widerſteht, ſo muß auch e den beyden Puncten A und a widerſtehen. Denn ware dieſes nicht, ſo wurden fie ſich einander ungehindert nahern, folglich A und a im Puncte c zuſammentreffen, d. i. der Raum wurde durchdrungen werden. Alſo muß in c et— was ſeyn, was dem Eindringen von Anund a widerſteht und alſo die Monas A zurucktreibt, ſo wie es auch von ihr zu— ruckgetrieben wird. Da nun Zurucktreiben ein Bewegen iſt, ſo iſt e etwas Bewegliches, im Raum mithin Materie, und der Raum zwiſchen A und a konnte nicht durch die Sphare der Wirkſamkeit einer einzigen Monade angefullt ſeyn, alſo auch nicht der Raum zwiſchen e und A, und fo ins Unendliche. Wenn Mathematiker die repulſiven Krafte der Theile elaſtiſcher Materien, bey großerer oder kleinerer Zuſam- men S 37 mendruckung derſelben, als nach einer gewiſſen Proportion ihrer Entfernungen von einander abnehmend oder zunehmend ſich vorſtellen, z. B. daß de kleinſten Theile der Luft ſich in ungekehrtem Verbaltniß ihrer Entfernungen von einander zurücktreiben, weil die Elaſticttat derſelben in umgekehrtem Verhaltnitß der Raume ſteht, darin ſie zuſammengedruckt werden: ſo verfehlt man ganzlich ihren Sinn und misdeutet ihre Sprache, wenn man das, was zum Verfahren der Conſtruction eines Begriffs nothnendig gehort, dem Be— griffe im Object ſelbſt beylegt. Denn nach jenem kann eine jede Beruhrung als eine unendlich kleine Entfernung vorge— ſtellt werden; welches in ſolchen Fallen auch nothwendig ge— ſchehen muß, wo ein großer oder kleiner Raum durch eben dieſelbe Quantitat der Materie, d. i. einerley Quantum repulſiver Krafte, als ganz erfullt vorgeſtellt werden ſoll. Bey einem ins Unendliche Theilbaren darf darum dennoch keine wirkliche Eutfernung der Theile, die bey aller Erwei— terung des Raums des Ganzen immer ein Continuum aus: machen, angenommen werden, obgleich die WMoglichkeit dieſer Erweiterung nur unter der Jdee einer unendlich klei— nen Entfernung anſchaulich gemacht werden kann. Aymerkung 2. Die Mathematik kann zwar in ihrem inneren Gebrau— che in Anſehung der Chicane einer verfehlten Metaphyſik ganz gleichgültig ſeyn, und im ſicheren Beſitz ihrer eviden— ten Behauptungen von der unendlichen Theilbarkeit des Raumes beharren, was fur Einwurſe auch eine an blo— ßen Begriffen klaubende Vernunfteley dagegen auf die Bahn bringen mag; allein in der Anwendung ihrer GSatze, die vom Raume gelten, auf Subſtant, die ſie erfullt, muß ſie ſich doch auf Prufung nach bloßen Begriffen, mithin auf Metaphyſik einlaſſen. Obiger Lehrſatz iſt ſchon ein Beweis davon. Denn es folgt nicht nothweudig, daß Materie ins Unendliche phyſiſch theilbar ſey, wenn ſie es gleich in ma— Cz thema thematiſcher Abſicht iſt, wenn gleich ein jeder Theil des Raums wiederunein Raum iſt, und alſo immer Theile außerhalb einander in ſich faßt, woferne nicht bewieſen werden kann, daß in jedem aller moglichen Theile dieſes erfuleten Raumes auch Subſtanz ſey, die folglich auch, abgeſondert von allen ubrigen, als fur ſich beweglich exi— ſtire. Alſo fehlete doch bisher dem mathematiſchen Be— weiſe noch etwas, ohne welches er auf die Naturwiſſen— ſchaft keine ſichere Anwendung haben konnte, und dieſem Mangel iſt in obſtehendem Lehrſatz abgeholfen worben. Was nun aber die ubrigen Angriffe der Metaphyſik auf den nun— mehro phyſiſchen Lehrſatz der unendlichen Theilbarkeit der Materie betrifft, ſo muß ſie der Mathemattker ganzlich dem Philoſophen uberlaſſen, der ohnedem durch dieſe Ein— wurfe ſich ſelbſt in ein Labyrinth begiebt, woraus es ihm ſchwer wird, auch in denen ihn unmittelbar angehenden Fragen, herauszufinden, und alſo mit ſich ſelbſt genug zu thun hat, ohne daß der Mathematiker ſich in dieſes Ge— ſchafte durfte einflechten laſfſen. Wenn namlich die Mate— rie ins Unendliche theilbar iſt, ſo (ſchließt der dogmatiſche Metaphvſiker) beſteht ſie aus einer uneudlichen Menge von Theilen; denn ein Ganzes muß doch alle die Theile zum voraus insgeſammt ſchon in ſich enthalten, in die es getheilt werden kann. Der letztere Satz iſt auch von einem jeden Ganzen, als Dinge an ſich ſelbſt, ungezweifelt ge— wiß, mithin, da man doch nicht einraumen kann, die Ma— terde, ja gar ſelbſt nicht einmal der Raum, beſtehe aus unendlich viel Theilen, weil es ein Widerſpruch iſt, eine unendliche Menge, deren Begriff es ſchon mit ſich fuhrt, daß ſie niemals vollendet vorgeſtellt werden konne, ſich als ganz vollendet zu denken) ſo muſſe man ſich zu einem ent— ſchließen, entweder dem Geometer zum Trotz zu ſagen: der Raum iſt nicht ins Unendliche theilbar, oder dem Metaphyſiker zur Aergerniß: der Raum iſt keine Eigen ſchaft eines Dinges au ſich ſelbſt, und alſo die Materie kein Ding an ſich ſſelbſt, ſondern bloße Erſcheinung unſerer auße— —S 39 außeren Sinne uberhaupt, ſo wie der Raum die weſentliche Form derſelben. v Hier gerath nun der Philoſoph in ein Gedrange zwi— ſchen den Hornern eines gefährlichen Dilemms. Den erſte— ren Satz: daß der Raum ins Unendliche theilbar ſey, ab— zuleugnen, iſt ein leeres Unterfangen, denn Mathematik laßt ſich nichts wegvernunfteln; Materie aber als Diung an ſich ſelbſt, mithin den Raum als Eigenſchaft der Dinge an ſich ſelbſt anſehen, und dennoch jenen Satz ableugnen, iſt einerley. Er ſieht ſich alſo nothgedrungen, von der letzte— ren Behauptung, ſo gemein und dem gemeinen Verſtande gemaß ſie auch ſey, abzugehen, aber naturlicher Wetſa nur unter dem Beding, daß man ihn auf den Fall, daß er Ma— terie und Raum nur zur Erſcheinung (mithin letzteren nur zur Form unſerer außeren ſinnlichen Anſchauung, alſo beyde nicht zu Sachen an ſich, ſondern nur zu ſubjectiven Vor— ſtellungsarten uns an ſich unbekannter Gegenſtande) mach— te, alsdenn auch aus jener Schwierigkeit, wegen unendli— cher Theilbarkeit der Materie, wobey ſie doch nicht aus unendlich viel Theilen beſtehe, heraushelfe. Dieſes letztere laßt ſich nun ganz wohl durch die Vernunft denken, obgleich unmoglich anſchaulich machen und conſtruiren. Denn, was nur dadurch wirklich iſt, daß es in der Vorſtellung gegeben iſt, davon iſt auch nicht mehr gegeben, als ſo viel in der Vorſtellung angetroffen wird, d. ĩ. ſo weit der Progreſſus der Vorſtellungen reicht. Alſo von Erſcheinungen, deren Theilung ins Unendliche geht, kann man nur ſagen, daß der Theile der Erſcheinung ſo viel ſind, als wir deren nur geben, d. i. ſo weit wir nur immer theilen mogen. Denn die Theile, als zur Exiſtenz einer Erſcheinung gehorig, exi ſtiren nur in Gedanken, namlich in der Theilung ſelbſt. Run geht zwar die Theilung ins Unendliche, aber ſie iſt doch niemals als unendlich gegeben: alſo folgt daraus nicht, daß das Theilbare eine unendliche Menge Theile an ſich ſelbſt und außer unſerer Vorſtellung in ſich enthalte, darum, weil feine Theilung ins Unendliche geht. Denn es iſt nicht das C 4 Ding, 4s Ding, ſondern nur dieſe Vorſtellung deſſelben, deren Thei— lung, ob ſie zwar ins Unendliche fortgeſetzt werden kann, und im Ohjzecte (das an ſich unbekonnt iſt) da,u auch ein Grund iſt, dennoch niemals vollendet, foiglich ganz gege— ben werden kann, und atis arch keine wirtliche unendliche Menge im Objſecte (als die ein ausdrucklicher Wideripruch ſeyn wurde) bewetſet. Ein großer Maun, der, vielleicht mehr als ſonſt jemand, das Anſehen der Mathemattk in Deutſchland u erhalten beytragt, hat mehrmalen die me— turhyſiſchen Anm mungen, Lehrſatze der Geometrie von der unendlichen Theirbarkeit des Raums umzuſtoßen, durch die gegrundete Erinnerung abgewieſent daß der Raum nur zu der Erſcheinung außerer Dinge gehore; allein er iſt nicht verſtanden worden. Mau nahm dieſen Satz ſo, als ob er ſazgen wollte: der Raum erſcheine uns ſelbſt, ſonſt ſey er eine Sache oder Verhaliniß der Sachen an ſich. ſelbſt, der Mathemattker betrachtete ihn aber nur, wie er erſcheint; anſtatt daß ſie darunter hatten verſtehen ſollen, der Raum ſey gar keine Eigenſchaft, die irgend einem Din— ge außer unſeren Sinnen an ſich ankangt, ſondern nur die ſubjective Form unſerer Sinnlichkeit, unter welcher uns Gegenſtande außerer Sinne, die wir, wie ſie an ſich beſchaffen ſind, nicht kennen, erſcheinen, welche Erſchei— nung wir denn Materie nennen. Bey jener Misdeutuug: dachte man ſich den Raum itmmer noch als eine den Din— gen auch außer unſerer Vorſtellungskraft anhangende Be— ſchaffenheit, die ſich aber der Mathematiker nur nach ge— meinen Begriffen, d. i. verworren denkt, (denn ſo erklart man gemeinhin Erſcheinung) und ſchrieb alſo den mathe— matiſchen Lehrſatz von der unendlichen Thetlbarkeit der Ma— terie, einen Satz, der die hochſte Deutlichkeit in dem Be griffe des Raums vorausſetzt, einer verworrenen Vorſtel— lung vom Raume, die der Geometer zum Grunde legte, zu, wobey es denn dem Metaphyſiker unbenommen blieb, den Raum aus Puncten und die Materie aus einfachen Theilen zuſammenzuſetzen und ſo (ſeiner Meynung nach) Deut ar Deutlichkeit in dieſen Begriff zu bringen. Der Grund dieſer Verirrung liegt in einer ubelverſtandenen Monado— logie, die gar nicht zur Ertlarung der Naturerſcheinun— gen gehort, fondern ein von Leibnitzen ausgefuührter, an ſich richtiger platoniſcher Begriff von der Welt iſt, ſo fern ſie gar nicht als Gegenſtand der Sinne, ſondern als Ding an ſich ſelbſt betrachtet, blos ein Gegenſtand des Verſtan— des iſt, der aber doch den Erfcheinungen der Sinne zum Grunde liegt. Nun muß freylich das Zufammengeſetzte der Dinge an ſich ſelbſt aus dem Einfachen beſtehen; denu die Theile muſſen hier vor aller Zuſammenſetzung ge— geben ſeyn. Aber das Zuſammongeſetzte in der Er— ſcheinung beſteht nicht aus dem Einfachen, weil in der Erſcheiuung, die niemals andere als zuſummengeſetzt (aus— gedehnt) gegeben werden kann, die Theile nur durch Thei— lüng und alſo nicht vor dem Zuſammengeſetzten, ſondern nur in demſelben gegeben werden konnen. Daher war Leib— nitzens Meynung, ſo viel ich einſehe, nicht, den Nat.m durch die Ordnung einfacher Weſen neben einander zu er— klaren, ſondern ihm vielmehr dieſe als correſpondirend, aber zu einer blos intelligibeln (fur uns unbekannten) Welt ge— horig zur Seite zu ſetzenn, und nichts. anders. zu behaupten, als was anderwarts gezeigt worden, namlich, daß der Raum ſammt der Materie, davon er die Form iſt, nicht die Welt von Dingen an ſich ſelbſt, ſondern nur die Erſchei— nung derſelben enthalte, und felbſt nur die Form unſerer aufjern ſinnlichen Anfſchauung ſey. Lehrfatz 5. Die Moglichkeit der Materie erfodert eine. Anztehungskraft als die zweyte weſentliche, Grundkraft derſeiben. C5 Be— 42 —S Beweis. Die Undurchdringlichkeit, als die Grundeigen— ſchaft der Matecie, wodurch ſie ſich als etwas Rea— les im Raume unſeren äußeren Sinnen zuerſt offen— baret, iſt nichts, als das Ausdehnungsvermogen der Materie (Lehrſatzz. Nun kann eine eweſentliche bewe gende Kruaft, dadurch die Theile der Materie einan— der fliehen, erſtlich nicht durch ſich ſelbſt eingeſchrankt werven, weil die Materie dadurch vielmehr beſtrebt iſt, den Raum, den ſie erfullt, continuirlich zu erwei— tern; zweytens auch nicht durch den Raum allein auf eine gewiſſe Grenze der Ausdehnung geſetzt werden; denn dieſer kann zwar den Grund davon enthal— ten, daß bey Erweiterung des Volumens einer ſich aus— deoynenden Materie die ausdehnende Kraft im umgekehr— ten Verhaltniſſe ſchwacher werde, aber, weil von einer jeden bewegenden Kraft ins Unendliche kleinere Grade moglich ſind, niemals den Grund enthalten, daß ſie irgendwo aufhore. Alſo wurde die Materie durch ihre repulſive Kraft (welche den Grund der Undurchdring— lichkeit enthalt) allein, und, wenn ihr nicht eine andere bewegende Kraft entgegenwir?te, innerhalb keinen Grenzen der Ausdehnung gehalten ſeyn, d. i. ſich. ins Unendliche zerſtreuen, und in keinem anzugebenden Raume wurde eine anzugebende Quantitat Materie anzutreffen ſeyn. Folglich wurden bey blos repelliren— den Kraften der Materie alle Raume leer, mithin eigentlich gar keine Materie da ſeyn. Es erfodert alſo alle Materie zu ihrer Exiſtenz Krafte, die der ausdeh— nenden entgegengeſetzt ſind, d. i. zuſammendruckende Krafte. Dieſe konnen aber urſprunglich nicht wie— derum 43 derum in der Entgegenſtrebung einer anderen Materie geſucht werden; denn dieſe bedarf, damit ſie Materie ſey, ſelbſt einer zuſammendruckenden Kraft. Alſo muß irgendwo eine urſprungliche Kraft der Materie, welche in entgegengeſetzter Direction der repulſwen, mithin zur Annaherung wirkt, d. i. eine Anziehungs— kraft angenommen werden. Da nun dieſe Anziehungs— kraft zur Moglichkeit einer Materie, als Naterie, uberhaupr gehort, folglich vor allen Unterſchieden der— ſelben vorhergeht, ſo darſ ſie nicht blos einer beſonde— Dren Gattung derſelben, ſondern muß jeder Materie uberhaupt und zwar urſprunglich beygelegt werden. Alſo kommt aller Materie eine urſprungliche Anziehung, als zu ihrem Weſen gehorige Grundkraft, zu. Anmerkung. Bey dieſem Uebergange von einer Eigenſchaft der Max terie zu einer andern ſpecifiſch davon unterſchiedenen, die zum Begriffe der Materie eben ſowohl gehort, obgleich in demſelben nicht enthalten iſt, muß das Verhalten unſe— res Verſtandes in nahere Erwagung gezogen werden. Wenn Anziehungskraft ſelbſt zur Moglichkeit der Materie ur— ſprunglich erfodert wird, warum bedienen wir uns ihrer nicht eben ſowohl, als der Undurchdringlichkeit, zum erſten Keunnzeichen tiner Materie? warum wird die letztere unmit— telbar mit dem Begriffe einer Materie gegeben, die erſtere aber nicht in dem Begriffe gedacht, ſondern nur durch Schluſſe ihm beygefugt? Daß unſere Sinne uns dieſe Auziehung nicht ſo unmittelbar wahrnehmen lafſen, als die Zuruckſtoßung und das Widerſtreben der Undurchdringlich- keit, kann die Schwierigkeit noch nicht hinlanglich beant— worten. Denn, wenn wir auch ein ſolches Vermogen J hatten, ſo iſt doch leicht einzuſehen, daß unſer Verſtand fich 44 ſich nichts deſtoweniger die Erfullung des Raumes wahlen wurde, um dadurch die Subſtanz im Raunte, d. i. die Materie zu bezeichnen, wie denn eben in dieſer Erfullung, oder, wie man ſte ſonſt nennt, der Soliditat das Cha— raeteriſteſche der Materte, als eines vom Raume unterſchie— denen Dinges, geſetzt wird. Anziehung, weun wir ſie anch noch ſo gut empſanden, wurde uns doch niemals eine Materie von beſtimn.ten Volumen und Geſtalt offenba— ren, ſondern nichts als die Beſtrebung unſeres Organs, ſich einem Puncte außer uns (dem Mittelpunct des anzie— henden Koörpers) zu nahern. Denn die Anziehungskraft aller Thetle der Erde kann auf uns nichts mehr, auch nichts anderes wirken, als wenn ſie ganzlich in dem Mit— telpuncte derſelben vereinigt ware, und dieſer allern auf unſern Sinn einfloſſe, eben ſo die Anziehung eines Berges, oder jeden Steins c. Nun bekommen wir dadurch keinen beſtimmten Begriff von irgend einem Objecte im Raume, da weder Geſtalt, noch Große, ja nicht einmal der Ort, wo er ſich befande, in unſere Sinne fallen kann (die bloße Direction der Anziehung wurde wahrgenommen werden kon— nen, wie bey der Schwere: der anziehende Punct wurde unbekannt ſeyn, und ich ſehe nicht einmal wohl ein, wie er ſelbſt durch Schluſſe, ohne Wahrnehmung der Materie, ſo fern ſie den Raum erfullt, ſollte ausgemittelt werden). Alſo iſt klar: daß die erſte Anwendung unſerer Begriffe von Grroßen auf Materie, durch die es uns zuerſt moglich wird, unſere außeren Wahrnehmungen in dem Erfahrungs— begriffe einer Matetie als Gegenſtandes uberhaupt zu ver— wandeln, nur auf ihrer Eigenſchaft, dadurch ſie einen Rarm erfullt, gegründet ſey, welche, vermittelſt des Sin— nes des Gefuhls, uns die Große und Geſtalt eines ause gedehnten, mithin von einem beſtimmten Gegenſtande im Ranme einen Begriff verſchafft, der allem ubrigen, was man von dieſem Dinge Jagen kann, zum Grunde gelegt wird. Eben diefes iſt ohüe Zweifel die Urſache, weswegen man bey den klärſten anderweitigen Beweiſen, daß An zie— —S 45 ziehung eben ſo wohl zu den Grundkraften der Materie ge— horen muſſe, als Zurückſtoßung, ſich gietchwohl gegen die erſtere ſo ſehr ſtraubt, und gar keine bewegende Kraſt, als nur durch Stoß und Druck (beydes vermuttelſt der Un— durchdringlichteit) einräumen will. Denn, weodurch der Raum irfullet iſt, das iſt die Subſtanz, ſagt man, und das hat auch ſeine gute Richtigteit. Da aber dieſe Sub— ſtanz ihr Daſeyn uns nicht anders, ats durch den Ginn, wodurch wir ihre Undurchdringlichkeit wahrnehmen, nani— lich das Gefühl, offenbart, mithin nur in Beziehung auf Beruhrung, deren Anfang (in der Annaherung einer Ma— terte zur andern) der Stoß, die Fortdauer aber ein Druck heißt: ſo ſcheint es, als ob alle unmittelbare Wirkung einer Materie auf die andere niemals, was anders, als Druck, oder Stoß ſeyn konne, zwey Einfluſſe, die wir allein un— mittelbar empfinden konnen, dagegen Anziehung, die uns an ſich entweder gar keine Empfindung, oder doch keinen beſtimmten Gegenſtand derfelben geben kann, uns als Grundkraft ſo ſchwer in den Kopf will. Lehrſatz 6. Durch bloße Anziehungskraft, ohne Zu— ruckſtoßung, iſt keine Materie moglich. Beweis. Anziehungskraft iſt die bewegende Kraft der Ma— terie, wodurch ſie eine andere treibt, ſich ihr zu na— hern, folglich, wenn ſie zwiſchen allen Theilen der Materie angetroffen wird, iſt die Materie vermittelſt ihrer beſtrebt, die Entfernung ihrer Theile von einan— der, mithin auch den Raum, den ſie zuſammen ein— nehmen, zu verringern. Nun kann nichts die Wir— kung einer bewegenden Kraft hindern, als eine andere ihr entgegengeſetzte bewegende Kraft; dieſe aber, wel— che che der Attraction entgegengeſetzt iſt, iſt die repulſive Kraft. Alſo wurden, ohne repulſwe Krafte durch bloße Annaherung, alle Theile der Materie ſich ohne Hinderniß einander nahern, und den Raum, den dieſe einnimmt, verringern. Da nun in dem angenomme— nen Falle keine Entfernung der Theile iſt, in welcher eine großere Annaherung durch Anziehung vermit— telſt einer zuruckſtoßenden Kraft unmoglich gemacht wurde, ſo wurden ſie ſich ſo lange zu einander bewe— gen, bis gar keine Entfernung zwiſchen ihnen ange— troffen wurde, d. i. ſie wurden in einen mathemati— ſchen Punct zuſammenfließen, und der Raum wurde leer, mithin ohne alle Materie ſeyn. Dennoch iſt Materie durch bloße Anziehungskrafte ohne zuruckſto— ßende unmoglich. Zuſatz. Diejenige Eigenſchaſt, auf welcher als Bedin— gung ſelbſt die innere Moglichkeit eines Dinges beruht, iſt ein weſentliches Stuck derſelben. Alſo ge— ghort die Zuruckſtoßungskraft zum Weſen der Mate— rie eben ſo wohl, wie die Anziehungskraft, und kei— ne kann von der anderen im Begriff der Materie ge— trennt werden. Anmerkung. Weil uberall nur zwey bewegende Krafte im Raum gedacht werden konnen, die Zuruckſtoßung und Anziehung, ſo war es, um beyder Vereinigung im Begriffe einer Materie uberhaupt a priori zu beweiſen, vorher nothig, daß jede fur ſich allein erwogen wurde, um zu ſehen, was ſie, allein genommen, zur Darſtellung einer Materie leiſten kounte. Es zeigt ſich nun, daß, ſowohl wenn man keine von 47 von beyden zum Grunde legt, als auch wenn man blos eine von ihnen aunimmt, der Raum allemal leer bleibe und keine Materie in demſelben angetroffen werde. Erklarung 6. Beruhruug im phyſiſchen Verſtande iſt die unmittelbare Wirkung und Gegenwirkung der Undurchdringlichkeit. Die Wirkung erner Materie auf die andere außer der Beruhrung iſt die Wirkung in die Ferne (actio in chſlans). Dieſe Wirkung in die Ferne, die auch ohne Wer— mittelung zwiſchen inne liegender Materie mog— lich iſt, heißt die unmitteibare Wirkung in die Ferne, oder auch die Wirkung der Materie auf einander durch den leeren Raum. Anmerkung. Die Beruhrung in mathematiſcher Bedeutung iſt die gemeinſchaftliche Grenze zweyer Raume, die alſo weder innerhalb dem einen, noch dem anderen Raume iſt. Da— her konnen gerade. Linten einander nicht berühren, ſondern, wenn ſie einen Punct gemein haben, ſo gehort er ſowohl in— nerhalb die eine, als die andere dieſer Linien, wenn ſie fortgezogen werden, d. i. ſie ſchneiden ſich. Aber Cirkel und gerade Linie, Cirkel und Cirkel, beruhren ſich in einem Puncte, Flachen in einer Linie und Korper in Flachen. Die mathematiſche Beruhrung wird bey der phyſiſchen zum Grunde gelegt, abar ſie macht ſie allein noch nicht aus, zu ihr muß, damit die letztere daraus entſpringe, noch ein dy— namiſches Verhaltniß und zwar nicht der Anziehungskrafte, ſondern der zuruckſtoßenden, d. i. der Undurchdringlichkeit hinzugedacht werden. Phyſiſche Beruhrung iſt Wechſel— wirkung der repulſiven Krafte in der gemeinjchaftlichen Grenze zweyer Materien. Lehr Lehrſatz 7. Die aller Materie weſentliche Anzie— hung iſt eine unmittelbare Wirkung derſelben auf andere durch den leeren Raum. Beweis. Die urſprungliche Anziehungskraft enthalt ſelbſt den Grund der Moglichkeit der Materie, als desjeni— gen Dinges, was einen Raum in beſtimmtem Grade erfullt, mithin ſelbſt ſogar von der Moglichkeit einer phyſiſchen Beruhrung derſelben. Sie inuß alſo vor dieſer vorhergehen, und ihre Wirkung muß folglich von der Bedingung der Beruhrung unabhängig ſeyn. Nun iſt die Wirkung einer bewegenden Kraft, die von aller Beruhrung unabhängig iſt, auch von der Erful— lung des Raums zwiſchen dem bewegenden und dem be— wegten unabhangig, d. i. ſie muß auch, ohne daß der Raum zwiſchen beyden erfullt iſt, Statt ſinden, mit— hin als Wirkung durch den leeren Raum. Allſo iſt die urſprungliche und aller Materie weſentliche Anziehung eine unmittelbare Wirkung derſelben auf andere durch den leeren Raum. Anmerkung 1. i machen ſollte, iſt eine ganz unmogliche Foderung; denn ſit heißen eben darum Grundkrafte, weil ſie von ketner anderu abgeleitet, d. i. gar nicht begriffen werden konnen. Es iſt aber die urſprungliche Anziehungskraft nicht im mindeſten unbegreiflicher, als die urſprungliche Zuruckſtoßung. Gie bietet ſich nur nicht ſo unmittelbar den Sinnen dar, als die Undurchdringlichkeit, uns Begriffe von beſtimmten Ob— jecten J SJ 45 jeeten im Ruume zu liefern. Weil ſie alſo nicht gefuhlt, ſondern nur geſchloſſen werden will, ſo hat ſie ſo fern den Anſchein einer abgeleiteten Kraft, gleich als ob ſie nur ein verſtecktes Spiel der bewegenden Krafte durch Zuruckſtoßung ware. Raher erwogen ſehen wir: daß ſie gar nicht weiter irgend wovon abgeleitet werden konne, am wenigſten von der bewegenden Kraft der Materien durch ihre Undurchdring lichkeit, da ihre Wirkung gerade das Widerſpiel der letzte ren iſt. Der gemeinſte/ Einwurf wider die unmittelbare Wirkung in die Ferue iſt: daß eine Materie doch nicht da, wo ſie nicht iſt, unmittelbar wirken konne. Wun die Erde den Mond unmittelbar treibt, ſich ihr zu nahern, ſo wirkt die Erde auf ein Ding, das viele tauſend Meilen von ihr entfernt iſt, und dennoch unmittelbar; der Raum zwi ſchen ihr und dem Monde mag auch als vollig leer angeſehen werden. Denn obgleich zwiſchen beyden Korpern Ma terie lage, ſo thut dieſe doch nichts zu jener Anziehung. Gie wirkt alſo an einem Orte, wo ſie nicht iſt, unmittels bar: etwas, was dem Anſcheine nach widerſprechend iſt. Allein es iſt ſo wenig widerſprechend, daß man vielmeht. ſagen kann, ein jedes Ding im Raume wirkt auf ein andes res nur an einem Orte, wo das Wirkende nicht iſt. Denn ſollte es an demſelben Orte, wo es ſelbſt iſt, wirken, ſo wurde das Ding, worauf es wirkt, gar nicht außer ihtn, ſeyn; denn dieſes Außerhalb bedeutet die Gegenwart in einem Orte, darin das andere nichts iſt. Wenn Erde und Mond einander auch beruhrten, ſo ware doch der Punet der Beruhrung ein Ort, in dem weder die Erde noch der Mond iſt; denn beyde ſind um die Summe ihrer Halbuleſs ſer von einander entfernt. Auch wurde im Punete der Beruhrung ſo gar kein Theil, weder der Erde noch des Mondes, anzutreffen ſeyn, denn dieſer Punct liegt in der Grenze beyder erfulleten Raume, die keinen Theil wedert von dem einen noch dem anderen ausmacht. Dasß alſo Materien in einander in der Entfernung nicht unmittelbar wirken konnen, wurde ſo viel ſagen, als, ſie konnen in D elnan einander nicht unmittelbar wirken, ohne Vermittelung der Krafte der Undurchdringlichkeit. Nun wurde dieſes eben ſo viel ſeyn, als ob ich ſagte: die repulſiven Krafte ſind die etnzigen, damit Materien wirkſam ſeyn konnen, oder ſie ſind wenigſtens die nothwendigen Bedingungen, unter denen allein Materien auf einander wirken konnen, welches entweder die Anztehungskraft fur ganz unmoglich oder doch unmer von der Wirkung der repulſiven Krafte abhangig er— klaren wurde; beydes ſind aber Behauptungen ohne allen Grund. Die Verwechſelung der mathematiſchen Beruh— rung, der Raume und der phyſiſchen durch zurucktreibende Krafte macht hier den Grund des Miesverſtandes aus. Sich unmittelbar außer der Beruhrung anziehen, heißt jich einauder nach einem beſtandigen Geſetze nahern, ohne daß eine Kraft der Zuruckſtoßung dazu die Bedingung ent— halte, welches doch eben ſo gut ſich muß denken laſſen, als einander unmittelbar zuruckſteßen, d. i. ſich einander nach einem beſtandigen Geſetze fliehen, ohne daß die Auztehungs— kraft daran irgend einigen Anthetl habe. Denn beyde be— wegende Krafte ſind von ganz verſchiedener Art, und es iſt nicht der mindeſte Grund dazu, eine von der anderen ab— hangig zu machen, und ihr ohne Vermittelung der andern die Moglichkeit abzuſtreiten. Anmerkung 2. Aus der Anziehung in der Beruhrung kann ganz keine Bewegung entſpringen; deun die Beruhrung iſt Wechſel— wirkung der Undurchdringlichkeit, welche alſo alle Bewe— gung abhält. Alſo muß doch irgend zine unmittelbare An— ziehung außer der Beruhrung und mithin in der Entfernung angetroffen werden; denn ſonſt konnten ſelbſt die druckenden und ſtoßenden Kraſte, welche die Beſtrebung zur Annahe rung hervorbringen ſollen, da ſie in entgegengeſetzter Rich— tung mit der repulſiven Kraft der Materie wirken, keine, wenigſtens nicht in der Natur der Materie urſprunglich lie- gende, Urſache haben. Man kann diejenige Anziehund, die —S 51 die vhne Vermittelung der repulſiven Krafte geſchieht, die wahre Anziehung, diejenige, welche blos auf jene Art vor ſich geht, die ſcheinbare nennen; denn eigentlich ubt der Korper, dem ein anderer ſich blos darum zu nahern beſtrebt iſt, weil dieſer anderweitig durch Stoß zu ihm getrieben worden, gar keine Anziehungskraft auf dieſen aus. Aber ſelbſt dieſe ſcheinbaren Anziehungen muſſen doch zuletzt eine wahre zum Grunde haben, weil Materie, deren Druck oder Stoß ſtatt Anziehung dienen ſoll, ohne anziehende Krafte nicht einmal Materie ſeyn wurde Cehrſatz 5.) und folglich die Erklarungsart aller Phanomene der Aunaherung durch blos ſcheinbare Anziehung ſich im Cirkel herumdreht. Man halt gemeiniglich dafur, Newton habe zu ſeinem Gyſtem gar nicht noöthig gefunden, eine unmittelbare Attraction der NMaterien anzunehmen, ſondern, mit der ſtrengſten Enthalt— ſamkeit der reinen Mathematik, hierin den Phyſitern volle Freyheit gelaſſen, die Moglichkeit derſelben zu erklaren, wie ſie es gut finden mochten, ohne ſeine Satze mit ihrem Hy potheſenſpiel zu bemengen. Allein wie konnte er den Gatz grunden, daß die allgemeine Anziehung der Korper, die ſie in gleichen Entfernungen um ſich ausuben, der Quantitat ihrer Materie proportionirt ſey, wenn er nicht annahm, daß alle Materie, mithin blos als Materie und durch ihre we ſentliche Eigenſchaft, dieſe Bewegungskrafu ausube? Denn obgleich freylich zwiſchen zweyen Korpern, ſie mogen der Materie nach gleichartig ſeyn, oder nicht, wenn der eine den anderen zieht, die wechſelſeitige Annaherung (nach dem Geſetze der Gleichheit der Wechſelwirkung) immer in um— gekehrtem Verhaltniß der Quantitat der Materie geſchehen muß, ſo macht dieſes Geſetz doch nur ein Princip der Me— chaunik, aber nichi der Dynamik, d. i. es iſt ein Geſetz der Bewegungen, die aus anziehenden Kraften folgen, nicht der Proportion der Anziehungskrafte ſelbſt, und gilt vor allen bewegenden Kräften uberhaupt. Wenn daher ein Magnet einmal durch einen anderen gleichen Magnet, ein andermal durch eben denſelben, der aber in einer zweymal D 2 ſchwe 52 ſchwereren holzernen Buchſe eingeſchloſſen ware, gezogen wird, ſo wird dieſer im letzteren Falle dem erſteren mehr relative Bewegung ertheilen, als im erſteren, obgleich das Holz, welches die Quantitat der Materie des letzteren ver— mehrt, zur Anziehungstraäft deſſelben gar nichts— hinzuthut und keine magnetiſche Anziehung der Buchſe beweiſet. New ton ſagt: (Cor. 2. Prop. G. Lib. III. Princip. Phil. N.) „Wenn der Aether, oder irgend ein auderer Korper ohne „Schwere ware, ſo wurde, da jeuer von jeder anderen „Materie doch in nichts, als der Form, unterſchieden iſt, „er nach und nach durch allmalige Veranderung dieſer Form in eine Materie von der Art, wie die, ſo auf Erden die meiſte Schwere haben, rerwandelt werden koönnen, und „dieſe letztere alſo umgekehrt durch allmalige Veranderung „ihrer Form alle ihre Schwere verlieren konnen, welches „der Erfahrung zuwider iſt re.“ Er ſchleß alſo ſelbſt nicht den Aether (wieviel weniger andere Materien) vom Geſetzt der Anziehung aus. Was konnte ihm denn nun noch fur eine Materie ubrig bleiben, um durch deren Stoß die Ane naherung der Korper zu einander als bloße ſcheinbare Anzter hung anzuſehen? Alſo kann man dieſen großen Stifter der Arttrackionstheorie nicht als ſeinen Vorganger anfuhren, wenn man ſich die Freyheit nimmt, der wahren Anziehung, die dieſer behauptete, eine ſcheinbare zu unterſchieben, und die Nothwendigkeit des Autriebs durch den Stoß anzu— nehmen, um das Phanomen der Annaherung zu erklaren. Er abſtrahirte mit Recht von allen Hypotheſen, die Frage wegen der Urſache der allgemeinen Attraction der Materie zu beantworten; denn dieſe Frage iſt phyſiſch oder meta— phyſiſch, nicht aber mathematiſch, und ob er gleich in der Vorerinnerung zur zweyten Ausgabe ſeiuer Optik ſagt: ne quis gravitatem inter ęſentiales corporum proprietates me habere exiſtimet, quaeſtionem unam de ejus eauſa inveſtiganda ſubjeei, ſo merkt man wohl, daß der An— ſtoß, den ſeine Zeitgenoſſen, und vielleicht er ſelbſt, am Begriffe einer urſprunglichen Anziehung nahmen, ihn mit ſich 53 ſich ſelblſt unetnig machte: deun er konnte ſchlechterdings nicht ſagen, daß ſich die Anziehungskrafte zweyer Planeten, z. B. des Juptters und Saturns, die ſie in gleichen Ent— fernungen ihrer Trabanten (deren Maſſe man nicht kennt) beweiſen, wie die Quantitat der Materie jener Weltkorper verhalten, wenn er nicht annahm, daß ſie blos als Materie, mithin nach einer allgemeinen Eigenſchaft derſelben, andere Materie anzogen. Erklarung 7. Eine bewegende Kraft, dadurch Materien nur in der gemeinſchaftlichen Flache der Beruh— rung unmittelbar auf einander wirken konnen, nenne ich eine Flachenkraft; diejenige aber, wodurch eine Materie auf die Theile der an— „dern auch uber die Flache der Beruhrung hinaus unmittelbar wirken kann, eine durchdringen— de Kraft. Zu ſatz. Die Zuruckſtößungskraft, vermittelſt deren die Maierie einen Raum erfullt, iſt eine bloße Flachen— kraft. Denn die einander beruhrenden Theile begron— zen einer den Wirkungsraum der anderen, und die re— pulſive Kraft kann keiuen entferntern Theil bewegen, ohne vermittelſt der dazwiſchen liegenden, und eine quer durch dieſe gehende unmittelbare Wirkung einer Materie. auf eine andere durch Ausdehnungskrafte iſt unmoglich. Dagegen einer Anziehungskraft, vermit— telſt deren eine Materie einen Raum einnimmt, ohne ihn zu erfullen, dadurch ſie alſo auf andere entfernte wirkt durch den leeren Raum, deren Wirkung ſetzt keine Materie, die dazwiſchen liegt, Grenzen. So D 3 muß 54 ter muß nun die urſprungliche Anziehung, welche die Ma— terie ſelbſt moglich macht, gedacht werden, und alſo iſt ſie eine durchdringende Kraft, und dadurch allein jederzeit der Quantitat der Materie propoktionirt. Lehrſatz 8. Die urſprungliche Anziehungskraft, worauf ſelbſt die Moglichkeit der Materie, als einer ſol— chen beruht, erſtreckt ſich im Weltraume von jedem Theile derſelben auf jeden andern unmit— telbar ins Unendliche. Beweis. Weil die urſprungliche Anziehungskraft zum We— ſen der Materie gehort, ſo kommt ſie auch jedem Theil derſelben zu, namlich unmittelbar auch in die Ferne zu wirken. Setzet nun: es ſey eine Entfernung, uber welche heraus ſie ſich nicht erſtreckte, ſo wurde dieſe Begrenzung der Sphare ihrer Wirkſamkeit entweder auf der innerhalb dieſer Sphare liegenden Malerie, oder blos auf der Große des Raumes, auf welchen fie dieſen Einfluß verbreitet, beruhen. Das Erſteze findet nicht ſtatt; denn dieſe Anziehung iſt eine durch— dringende Kraft, und wirkt unmittelbar in der Ent— fernung, unerachtet aller dazwiſchen liegenden Mate— rien, durch jeden Raum, als einen leeren Raum. Das Zwehyte findet gleichfalls nicht ſtatt. Denn, weil eine jede Anziehung eine bewegende Kraft iſt, die einen Grad hat, unter dem ins Unendliche noch immer klei— nere gedacht werden konnen: ſo wurde in der große— ren Entfernung zwar ein Grund liegen, den Grad der Attrac 55 Attraction, nach dem Maaße der Ausbreitung der Kraft, in umgekehrtem Verhaltniſſe zu vermindern, niemals aber ſie vollig aufzuhebe n. Da nun alſo nichts iſt, was die Sphäre der Wirkſamkeit der ur— ſprunglichen Anziehung jedes Theils der Materie ir— gendwo begrenzte, ſo erſtreckt ſie ſich uber alle anzuge— bende Grenzen auf jede andere Materie, mithin im Weltraume ins Unendliche. Zuſatz 1. Aus dieſer urſprunglichen Anziehungskraft, als einer durchdringenden, von aller Materie, mithin in Proportion der Quantitat derſelben, ausgeubten, und auf alle Materie, in alle mogliche Weiten, ihre Wir— kung erſtreckenden Kraft, mußte nun; in Verbindung mit der ihr entgegenwirkenden, namlich zurucktreiben- den Kraft, die Einſchrankung der letzteren, mithin die Moglichkeit eines in einem beſtimmten Grade ere fulleten Raumes, abgeleitet werden konnen, und ſo wurde der dynamiſche Begriff der Materie, als des Beweglichen, das ſeinen Raum (in beſtimmtem Grade) erfullt, conſtruirt werden Aber hiezu bedarf may eines Geſetzes des Verhaltniſſes, ſowohl der urſprung- lichen Anziehung, als Zuruckſtoßung, in verſchiedenen Entfernungen der Materie und ihrer Theile von einander, welches, da es nun lediglich auf dem Un— terſchiede der Richtung dieſer beyden Krafte (da ein Punect getrieben wird, ſich entweder andern zu nahern, oder ſich von ihnen zu entfernen) und auf der Große des Raumes beruht, in den ſich jede dieſer Kraäfte in verſchiedenen Weiten verbreitet, eine reine mathema— D 4 tiſche 2 56 —S tiſche Aufgabe iſt, die nicht mehr fur die Metaphyſik gehort, ſelbſt nicht was die Verantwortung betrifft, wenn es etwa nicht gelingen ſollte, den Begriff der Ma— terie auf dieſe Art zu conſtruiren. Denn ſie verant— wortet blos die Richtigkeit der unſerer Vernunfterkennt— niß vergonneten Elemente der Conſtruction, die Unzu langlichkeit und die Schranken unſerer Wernunft in ber Ausfuhrung verantwortet ſie nicht. Zuſatz 2. Da alle gegebene Materie mit einem beſtimm- ten Grade der. repulſiven Kraft ihren Raum erfullen muß, um ein beſtimmtes materielles Ding auszuma— chen, ſo kann nur eine urſprungliche Anziehung im Confliet mit ber urſprunglichen Zuruckſtoßung einen beſtimmten: Grad:. der Erfullung des Raums, mithin Materie moglich machen; es mag nun ſeyn, daß der erſtere von der eigenen Anziehung der Theile der zuſam. mengedruckten. Materie unter einander, oder von der Vereinigung berſelben mit der Anziehung aller Welt. materie herruhre. J Die urſptüngliche Aujiehung iſt der Quantitae der Materie proportional und erſtreckt ſich ins Unendli. che. Alſo kann die dem Maaße nach beſtimmte Erful— lung eines Raumes durch Materie am Ende nur von der ins Unendliche ſich erſtreckenden Anzlehung derſel-. ben bewirkt, und jeder Materie nach dem Maaße ihrer Zuruckſtoßungskraft ertheilt werden. Die Wirkung von der allgemeinen Anziehung, die alle Materie auf alle und in allen Entfernungen unmittelbar ausubt, heißt die Gravitation; die Be— ſtra ——J— 57 ſtrebung in der Richtung der großeren Gravitation ſich zu bewegen „iſt die Schwere. Die Wirkung von der durchgangigen repulſiven Kraft der Theile jeder ge— gebenen Materie heißt dieſer ihre urſprungliche Ela ſticitat. Dieſe alſo und die Schwere machen die ein— zigen a priori einzuſehenden allgemeinen Charactere der Materie, jene innerlich, dieſe im außeren Ver— haltniſſe, aus; denn zuf den Grunden beyder beruht die Moglichkeit der Materie ſelbſt: Zuſammenhang, wenn er als die wechſelſeitige Anziehung der Materie, die lediglich auf die Bedingung der Beruhrung einge— ſchrankt iſt, erklart wird, gehort nicht zur Moglich— keit der Materie uberhaupt, und kann daher a priori als damit verbunden nicht erkannt werden. Dieſe Eigenſchafe wurde alſo nicht metaphyſiſch, ſondern phyſiſch ſeyn, und daher. nicht zu unſern gegenwartigen Betrachtungen gehoren. Anmerkung 12. Eine kleine Borerinnerung zum Behufe des Verſuchs einer folchen vielleicht moglichen Conſtruction kann ich doch nicht unterlaſſen beyzufugen. Von einer jeden Kraft, die in verſchiedene Welten unmittelbar. wirkt, und in Anſehung des Grades, wonut ſie auf.einen jeden in gewiſſer Weite gegebenen Punet bewe— gende Kraft ausubet, nur durch die Eiroße des Raunteb, in welchem ſie ſich ausbreiten muß, um auf jenen Punct gzu wirken, eingeſchrankt. wird, kann man fagen: daßt ſte in allen Raumen, in die ſie ſich verbreitet, fo klein odtr groß ſie auch ſeyn mogen, immer ein gleiches Quantum aus— macht, daß aber der Grad ihrer Wirkung auf jenen Punet in dieſem Raume jederzeit im umgekehrten Rerhältniß des Raumes ſtehe, in welchen fie fich hat verbreiten muſſen, um D5 auf 58 —S auf ihn wirken zu konnen. So breitet ſie z. B. von einem leuchtenden Puunct das Licht allerwarts in Kugelflachen aus, die mit den Quadraten der Entfernung immer wachſen, und das Quantum der Erleuchtung iſt in allen dieſen ins Unend liche großeren Kugelflachen im Ganzen immer daſſelbe, wor— aus aber folgt: daß ein in dieſer Kugelflache angenommener gleicher Theil dem Grade nach deſto weniger erleuchtet ſeyn muſſe, als jene Flache der Verbreitung eben deſſelben Licht— quantum großer iſt, und ſo bey allen anderen Kraften und Geſetzen, nach welchen ſie ſich entweder in Flachen, oder auch korperlichen Raum verbreiten muſſen, um ihrer Natur nach auf entfernte Gegenſtande zu wirken. Es iſt beſſer, die Verbreitung einer bewegenden Kraft aus einem Punct in alle Weiten ſo vorzuſtellen, als auf die gewohnliche Art, wie es unter andern in der Optik geſchieht, durch von einem Mittelpunct auseinanderlaufende Cirkelſtrahlen. Denn da auf ſolche Art gezogene Linien niemals den Raum, durch den ſie gehen, 'und alſo auch nicht die Flache, auf die ſie treffen, fullen konnen, ſo viel deren auch gezogen oder an— gelegt werden, welches die unvermeidliche Folge ihrer Di— vergenz iſt, ſo geben ſie nur zu beſchwerlichen Folgerungen, dieſe aber zu Hypotheſen Anlaß, die gar wohl vermieden werden konnten, wenn man blos die Große der ganzen Ku— gelflache in Betrachtung zoge, die von Derſelben Quantitat Licht gleichformig erleuchtet werden ſoll, und den Grad der Erleuchtung derſelben in jeder Stelle, wie naturlich, in umgekehrtem Verhaltniſſe ihrer Große zum Ganzen nimmt, und ſo bey aller anderer Verbreitung einer Kraft durch Rau me von verſchtedenet Große. 2) Wenn die Kraft eine unmittelbare Anziehung in der Ferne iſt, ſo muß um deſto mehr die Richtungslinie der Anziehung nicht, als ob ſie von dem ziehenden Puncte wie Strahlen ausliefln, ſondern ſo wie ſie von allen Puncten der umgebenden Kugelflache (deren Halbmeſſer jene gegebene Weite iſt) zum ziehenden Punct zuſammenlaufen, vorgeſtellt wer J 59 werden. Denn ſelbſt die Richtungslinie der Bewegung zum Puncte hin, der die Urſache und Ziel derſelben iſt, giebt ſchon den terminus a quo an, von wo die Linten anfangen müſſen, namlich von allen Puncten der Oberflache, von dem ſie zum ziehenden Mittelpunete und nicht umgekehrt thre Richtung haben: denn jene Große der Flache beſtimmt. allein die Menge der Linien, der Mittelpufttt laßt ſie uunbeſtimmt 3) Wenn x) Es iſt unmoglich, nach Linien, die ſich ſtrahlenweiſe aus einem Puncte ausbreiten, Flachen in gegebenen Entfer— „nungen als mit der. Wirkung derſelben, ſie ſey Erleuchs tung oder Anziehung, ganz erfullt vorzuſtellen. GSo wurde bey ſolchen auslaufenden Lichtſtrahlen die geringere Erleuchtung einer entfernten Flache blos darauf beru— hen, daß zwiſchen den erleuchteten Stellen unerleuchtete, und dieſe deſto großer, je weiter die Flache entfernt, uhrig bleiben. Eulers Hypotheſe vermeidet dieſe n— ſchicklichkeit, hat aber freylich deſto mehr Schmieiigleit, die geradlinigte Bewegung des Lichts begreiflich zu ma— chen. Dieſe Schwierigkeit aber ruhrt von einer gar wohl verineidlichen mathematiſchen Vorſtellung der Lichtmate— .Nrie, als einer Anhaufung von Kugelchen her, die freylich, nach ihrer verſchiedentlich ſchiefen Lage gegen die Rich— tung des Stoßes, Seitenbewegung des Lichts geben wur— de, da an deſſen Statt nichts hindert, dieſe Matertie als „ein urſprunglich Fluſſiges, und zwar durch und durch, ohne in feſte Korperchen zertheilt zu ſeyn, zu denken. Will der Mathematiker die Abnahme des Lichts bey zu— nehmender Entfernung anſchaulich machen, ſo bedurnt er ſich auslaufender Cirkelſtrahien, um auf der Kugeiflache ihrer Verbreitung die Große des Raumes, darin die— ſelbe Quantitat des Lichts zwiſchen dieſen Cirkelſtrahlen glerchformig verbreitet werden ſoll, mithin die Verringe— rung des Grades der Erleuchtung datzuſtellen; er will aber nicht, 60 3) Wenn die Kraft eine unmittelbare Zuruckſteltung iſt, dadurch ein Punct (in der blos mathematiſchen Dar— ſtellung) einen Raum dynamiſch erſullt, und es iſt die Frage, nach welchem Geſetze der unendlich kleinen Entfer— nungen (die hier den Beruhrungen gleich gelten) eine ur— ſprungliche repulſive Kraft (deren Einſchrankung folglich le— diglich auf dem Raum beruht, in dem ſie verbreitet wor— den) in verſchtedenen Entfernungen wirke: ſo kann man moch weniger dieſe Kraft durch divergirende Zuruckſtoßungs— ſtrahlen aus dem angenommeuen repellirenden Puncte vor— ſtellig machen, obgleich die Richtung der Bewegung ihn zum terminus a quo hat, weil der Raum, in welchem die Kraft verbreitet werden muß, um in der Entfernung zu wirken, ein korperlicher Raum iſt, der als erfullt gedacht werden ſoll (wovon die Art, wie namlich ein Punet durch bewegende Kraft dieſes, d. i. dynamiſch, einen Ralim kor— perlich erfullen konne, freylich keiner weiteren mathemati— ſchen Darſtellung fahig iſt) und divergirende Strahlen aus vinem Puncte die repellirende Kraft eines korperlichen erful leten nicht, daß man dieſe Strahlen als die einzig erleuchtenden anſehen ſolle, gleich als ob immer lichtleere Platze, die bey großerer Weite großer wurden, zwiſchen ihnen anzut treffen waren. Will man jebe ſolcher Flachen als durch aus erleuchtet ſich vorſtellen, ſo muß dieſelbe Quantitat der Erleuchtung, die die kleinere bedeckt, auf der großeren als gleichformig gedacht werden, und muſſen. alſe, um die geradlinigte Richtung anzuzeigen, von der Flache und allen ihren Puncten zu dem leuchtenden gerade Linien ge— zogen werden. Die Wirkung und ihre Große muß vor— her gedacht ſeyn und darauf die Urſache vorzeichnet wer den. Eben dieſes gilt von den Anziehungsſtrahlen, wenn man ſie ſo nennen will, ja von ullen Richtungen der Krafte, die von einem Puncte ans, einen Raum, und ware er auch ein korperlicher, erfullen ſollen. J leten Raumes unmoglich vorſtellig machen konnen: ſondern man wurde die Zuruckſtoßung, bey verſchiedenen unendlich kleinen Entfernungen dieſer einander treibenden Puncte, ſchlechterdings blos in umgekehrtem Verhaltniſſe der korper— lichen Raume, die jeder dieſer Puncte dynamiſch erfullt, mithin des Cubus der Entfernungen derſelben von einander. ſchatzen, ohne ſie conſtruiren zu konnen. 4) Alſo wurde die urſprungliche Anziehung der Ma— terie in umgekehrtem Verhaltniß der Quadrate der Entifer-n nung in alle Weiten, die urſprungliche Zuruckſtoßung in um— gekehrtem Verhaltniß der Wurfel der unendlich kleinen Ent— fernungen wirken, und durch eine ſolche Wirkung und Ge— genwirkung beyder Grundkrafte wurde Materie von einem beſtimmten Grade der Erfullung ihres Raumes moglich ſeyn; weil, da die Zurluickſtoßung bey Annaherung der Thei le in großerem Maaße wachſt, als die Anztehung die Gren— ze der Annaherung, uber die durch gegebene Anziehung kei— ne großere moglich iſt, mithin auch jener Grad der Zuſani— mendruckung beſtimmt iſt, der das Maaß der intenſiven Erfullung des Raumes ausmacht. Anmerkung 2. Ich ſehe wohl die Schwierigkeit dieſer Erklarungsart, der Moglichkeit einer Materie uberhaupt, die darin beſteht, daß, wenn ein Punet durch repulſive Kraft unmittelbar kei— nen anderen treiben kann, ohne, zugleich; den ganzen korper— lichen Raum bis zu der gegebenen Entfernung durch ſeine Kraft zu erfullen, dieſer alsdenn, wie zu folgen ſcheint, mehrere treibende Puncte enthalten mußte, welches der Vorausſetzung widerſpricht, und oben (Lehrſatz 4.), unter dem Namen einer Sphare der Zuruckſtoßung des Einfachen im Raume, widerlegt worden. Es iſt aber ein Unter— ſchied zwiſchen dem Begriffe eines wirklichen Raumes, der gegeben werden kann, und der bloßen Jdee von einem Rau— me, J 62 S J me, der lediglich zur Beſtimmung des Verhaltniſſes gege— bener Raume gedacht wird, in der That aber kein Raum iſt, zu machen. Jn dem angeführten Falle einer veimeyn— ten phyſiſchen Monadologte ſollten es wirkliche Raume ſeyn, welche von einem Puncte dynamiſch, namlich durch Zuruck— ſtozung, erfullt waren; deun ſie exiſtirten, als Puncte, vor aller daraus moglichen Erzeuguag der Materie, und beſtimmten durch die ihnen eigene Sphare ihrer Wirkſamkeit den Theil des zu erfullenden Raumes, der ihnen angehoren konite. Daher kann in gedachter Hypotheſe die Materie auch nicht als ins Unendliche theilbar und als Quantum conttnuum angeſehen werden; denn die Theile, die unmittelbat einander zurückſtogßen, haben doch eine beſtimmte Entfernung von einander (die Summe der Halbmeſſer der Sphare ihrer Zu ruckſtoßung); dagegen, wenn wir, wie es wirllich geſchieht, die Materte als ſtetige Große denken, ganz und gar keine Entfernung der einander unmittelbar zurückſtoßenden Theile Scatt findet, folglich auch keine großer oder kletner werdende Sphare ihrer unmittelbaren Wirkſamkett. Nun konnen ſich aber Praterten ausdehnen, oder zuſammengedrückt werden (wie die Lute), und da ſtellt man ſich eine Eutfernung ihrer nachſten Thetle vor, dee da wachſen und abnehmen konnen. Werl gber die uachſten Theile einer ſterigen Materie einan— der beruühren, ſie mag nun weiter ausgedehnt oder zuſam— mengedrückt ſeyn, ſo deutt man ſich jene Entfernungen von einander als unendlich-klein, und dieſen unendlich kleinen Raum als im großeren oder kleineren Grade von ihrer Zu— ruckſteßungskraft erfuüllt vor. Der unendlich kleine Zwi— ſchenraum iſt aber von der Beruhruirg gar nicht unterſchie— den, alſo nur die Jdee vom Raunie, die dazu dient, um die Erweiterung einer Materie, als ſtetiger Große, an— ſchaulich zu muchen, ob ſie zwar wirklich, ſo, gar nicht begriffen werden kann. Wenn es alſo heißt: die zuruckſto— ßenden Krafte der einander unmittelbar treibenden Theile der Materte ſtehen in umgekehrtem Verhaltniſſe der Wurfel ihrer Entfernungen, ſo bedrutet das nur: ſie ſtehen in umge —S 63 umgekehrtem Verhaltniſſe der korperlichen Raume, die man ſich zwiſchen Theilen denkt, die einander dennoch unmittel— bar beruhren, und deren Entfernung eben darum unendlich klein genannt werden muß, damit ſie von aller wirklichen Entfernung unterſchieden werde. Man muß alſo aus den Schwierigketten der Conſtruction eines Begriffs, oder viel— mehr aus der Misdeutung derſelben, keinen Einwurf wider den Begriff ſelber machen; denn ſonſt wurde er die mathe— matiſche Darſtellung der Proportion, mit welcher die An— ziehung in verſchiedenen Entfernungen geſchieht, eben ſo wohl, als diejenigen, wodurch ein jeder Punct in einem fich ausdehnenden oder zuſammengedruckten Ganzen von Materte den andern unmittelbar zuruckſtoßt, treffen. Das allgemeine Geſetz der Dynamik wurde in beyden Fallen die— ſes ſeyn: die Wirlung der hewegenden Kraft, die von einem Puncte auf jeden auderen außer ihm ausgeubt wird, verhalt ſich umgekehrt wie der Raum, in welchem daſſeibe Quantum der bewegenden Kraft ſich hat ausbreiten muſſen, um auf dieſen Puuct unmittelbar in der beſtimmten Entſer— nung zu wirken. Aus dem Gefetze der urſprunglich einander zuruckſto— ßenden Theile der Materie in umgekehrtem cubiſchen Ver— haltniſſe ihrer unendlich kleinen Entfernungen mußte aiſo nothwendig ein ganz anderes Geſetz der Ausdehnung und Zu— ſammendrüuckung derſelben, als das mariottiſche der Luft, folgen; denn dieſes beweiſet ftiehende Kräfte ihrer nächſten Theile, die in umgekehrtem Verhaltniſſe ihrer Entfernungen ſtehen, wie Newton darthut (Prinec. Ph. N. Lib. II. Propoſ. 23. Schol.). Allein man kann die Ausſpannungs-— ktaft der letzteren auch nicht als die Wirkung urſprünglich zuruckſtoßender Krafte anſehen, ſondern ſie beruht auf der ZWwarme, die nicht blos als eine in ſie eingedrungene Ma— terie, ſondern allem Anſehen nach durch ihre Erſchutterun— gen die eigentlichen Lufttheile (denen inan uberdem wirkliche Entfernungen von einauder zugeſtehen kann) nothigt, einan— der 64 —S der zu fliehen. Daß aber dieſe Bebungen der einander nach— ſten Theile eine Fliebkraft, die in umgekehrtem Verhaltniſſe ihrer Entfernungen ſteht, ertheilen muſſe, laßt ſich nach den Geſcetzen der Mittheilung der Bewegung durch Schwin— gung elaſteſcher Materien wohl begteiflich machen. Noch erkläre ich, daß ich nicht wolle, daß gegenwartige Expoſilivn des Geſetzes einer urſprützglichen Zuruckſtoßung als zur Abſicht imeiner metaphyſiſchen Behandlung der Ma— terie unothwendig gehorig angeſehen, noch die letztere (wel— cher es genug iſt, die Erfullung des Raums als dynamiſche Eigenſchaft derſeibentdargeſtellt zu haben) mit den Streitig— keiten und Zweifeln, welche die erſte treffen koöunten, bee mengt werde. Allgemeiner Zuſatz zur Dynamik. Wenn wir nach allen Verhandlungen derſelben zuruckſehen, ſo werden wir bemerten: daß darin zuerſt das Reelle im Raume, (ſonſt genannt das Solide) in der Erfullung deſſelben durch Zuruckſtoßungskraft, zweytens das, was in Anſehung des erſteren, als des eigentlichen Objects unſerer außeren Wahrnehmung, negativ iſt, namlich die Anziehungskraft, durch welche, ſo viel an ihr iſt, aller Raum wurde durch— drungen, mithin das Solide gänzlich aufgehoben wer— den, drittens die Einſchrankung der erſteren Kraft durch die zweyte und die daher ruhrende Beſtim- mung des Grades einer Erfullung des Raumes in Betrachtung gezegen, mithin die Qualitat der Mate— rie unter den Titeln der Realitat, Negation und ki— mitation, ſo viel es einer metaphyſiſchen Dynamik zukommt, vollſtandig abgehandelt worden. Alige— 5 Aligeme'ine Anmerkung iur Dynamit. g9 Vas allgemeine Princip der Dynamik der materiellen Na tur iſt: daß alles Reale der Gegenſtande außerer Sinne, die das, was nicht blos Beſtinmmung des Raums (Ort, Ausdehnung und Figur) iſt, als bewegende Kraft angeſehen werden muſſe; wodurch alſo das ſogenannte Solide, oder die abſolute Undurchdringlichkeit, als ein leerer Begriff, aus der Naturwiſſenſchaft verwieſen und an threr Statt zuruck— treibende Kraft geſetzt, dagegen aber die wahre und unmit—e telbare Anziehung gegen alle Veruunfteleyen etuer ſich ſelbſt misverſtehenden Metaphyſik vertheidigt, und, als Grund-— kraft, ſelbſt zur Moglichkeit des Begriffs von Materie fur nothwendig erklart wird. Hieraus entſpringt nun die Fol— ge: daß der Raum, wenn man es nothig finden ſolite, auch ohne leere Zwiſchenraume innerhalb der Materie auszu— ſtreuen, allenfalls durchgangig und gleichwohl in verſchiede— nem Grade erfullt angenommen werden konne. Denn es kann nach dem urſprunglich verſchiedenen Grade der repulſi— ven Krafte, auf denen die erſte Eigenſchaft der Materie, namlich die, einen Raum zu erfullen, beruht, ihr Verhalt— niß zur urſprunglichen Anziehung (es ſey einer jeden Ma— terie fur ſich ſelbſt, oder zur vereinigten Anziehung aller Materie des Univerſuums) unendlich verſchieden gedacht wer den; weil die Anziehung auf der Menge der Materie in einem gegebenen Raume beruht, da hingegen die expanſive Kraft derſelben auf dem Grade ihn zu erfullen, der ſpecifiſch ſehr unterſchieden ſeyn kaun; (wie etwa dieſelbe Quontitat Luft in demſelben Volumen nach ihrer großeren oder minde— ren Erwagung mehr oder weniger Elaſticitat beweiſet) wo von der allgemeine Grund dieſer iſt: daß durch wahre An— liehung alle Theile der Materie unmittelbar auf alle Thei le der andern, durch expanſive Kraft aber uur die in der E Verüh bs —S Beruhrungsflache wirken, woben es einerley iſt; ob hin ter dieſer vtel oder wentg von dieſer Materie angetroffen werde. Hieraus allein entſpringt nun ſchon ein großer Vor— theil fur die Naturwiſſenſchaſt, weil ihr dadurch die Laſt abgenommen wird, aus dem Volien und Leeren eine Welt blos nach der Phantaſie zu zimmekn, vielmehr alle Raume voll und doch in verſchiedenem Maaße erfullt gedacht wer— den konnen, wodurch der Fere Raum wenigſiens ſeine Nothwendigkeit verliert und auf den Werth einer Hypo— theſe zuruckgeſetzt wird, da er ſonſt, unter dem Vorwande einer zu Erklarung der verſchiedentlichen Grade der Erfullung des Raums nothwendigen Bedingung, ſich des Titels eines Grundſatzes anmaaßen konnte. Bey allem dieſem iſt der Vortheil einer hier metho— diſch-gebrauchten Metaphyſik, in Abſtellung gleichfalls metaphyſiſcher, aber nicht auf die Probe der Critik gebrach- ter Principien, augenſcheinlich nur negativ. Jndirect wird gleichwohl dadurch dem Naturforſcher ſein Feld erwei— tert; weil die Bedingungen, durch die er es vorher ſelbſt einſchrankte, und wodurch alle urſpruugliche Bewegungs krafte wegphiloſophirt wurden, jetzt ihre Gultigkeit verlie— ren. Man hute ſich aber uber das, was den allgemeinen Begriff einer Materie uberhaupt moglich macht, hinauszu gehen, und die beſondere oder ſo gar ſpeeifiſche Beſtimmung und Verſchiedenheit derſelben a priori erklaren zu wollen. Der Begriff der Materie wird auf. lauter bewegende Krafte zuruckgefuhrt, welches man auch nicht anders erwarten konnte, weil im Raume keine Thatigkeit, keine Veranderung, als blos Bewegung gedacht werden kann. Allein wer will die Moglichkeit der Grundkrafte einſehen? ſie konnen nur angenommen werden, wenn ſie zu einem Begriff, von dem es erweislich iſt, daß er ein Grundbegriff ſey, der von kei nem anderen weiter abgeleitet werden kann (wie der der Er— fullung des Raums), unvermeidlich gehoren, und dieſes ſind Zuruckſtoßungs und ihnen entgegenwirkende Augzie- hungs 67 hungskrafte uberhaupt. Von dieſer ihrer Verknupfung und Folgen können wir allenfalls noch wohl a priori urtheilen, welche Verhaltniſſe derſelben unter einander man ſich, ohne ſich ſelbſt zu widerſprechen, denken konne, aber ſich darum doch nicht anmaaßen, eine derſelben als wirklich anzuneh— men, weil zur Befugniß, eine Hypotheſe zu errichten, unnach— laßlich gefodert wird: daß die Moglichkeit deſſen, was man annimmt, vollig gewiß ſey, bey Grundkraften aber die Moglichkeit derſelben niemals eingeſehen werden kann. Und hierin hat die mathematiſch-mechaniſche Erklärungsart uber die metaphyſiſch-dynamiſche einen Vortheil, der ihr nicht abgewonnen werden kanu, namlich aus einem durch— gehends gleichartigen Stoffe, durch die mannigfaltige Ge— ſtalt der Theile, vermittelſt eingeſtreuter leerer Zwiſchen raume, eine große ſpecifiſche Mannigfaltigkeit der Materien, ſo wohl ihrer Dichtigkeit als Wirkungsart nach, (wenn freinde Krafte hinzukommen) zu Stande zu bringen. Denn die Moglichkeit der Geſtalten ſowohl als der leeren Zwi— ſchenraume laßt ſich mit mathematiſcher Evidenz darthun: dagegen, weun der Stoff ſelbſt in Grundkrafte verwandelt wird (deren Geſetze a priori zu beſtimmen, noch weniger aber eine Mannigfaltigkeit derſelben, welche zu Ertlarung der ſpecifiſchen Verſchiedenheit der Materie zureichte, zuver— laſſig anzugeben, wir nicht im Stande ſind), uns alle Mit— kel abgehen, dieſen Begriff der Materie zu conſtruiren, und, was wir allgemein dachten, in der Anſchauung als moglich darzuſtellen. Aber jenen Vortheil bußet dagegen eine blos mathematiſche Phyſik auf der anderen Geite dop pelt ein, indem ſie erſtlich einen leeren Begriff (der ab— ſoluten Undurchdringlichkeit) zum Grunde legen, zwey— tens alle der Materie eigene Krafte aiufgeben muß, und uberdem noch mit ihren urſprunglichen Confi— gurationen des Grundſtoffs und Einſtreuung der leeren Raume, nachdem es das Bedurfniß zu erklaren erfodert, der Einbildungskraft im Felde der Philoſophie mehr Frey— heit, ja gar rechtmaßigen Auſpruch verſtatten muß, als E 2 ſich 68 ſich wohl mit der Behutſamkeit der letzteren zuſammen— reimen laßt. Statt einer hinreichenden Erklärung der Moglichkeit der Materie und ihrer ſpecifiſchen Verſchiedenheit aus jenen Grundkraften, die ich nicht zu leiſten verniag, will ich die Momente, worauf ihre ſpecifiſche Verſchiedenheit ſich insge— ſammt a priori bringen (obgleich nicht eben ſo ihrer Mog— lichkeit nach begreifen) laſſen muß, wie ich hoffe, vollftan— dig darſtellen. Die zwiſchen die Definitionen geſchobenen An merkungen werden die Anwendung derſelben erlautern. 1. Ein Korper, in phyſiſcher Bedeutung, iſt eine Naterie zwiſchen beſtimmten Grenzen (die alſo eine Figur hat). Der Raum zwiſchen dieſen Grenzen, ſei— ner Große nach betrachtet, iſt dr Raumesinhalt (volumen). Der Grad der Erfullung eines Raumes von beſtimmtem Jnhalt heißt Dichtigkeit. (onſt wird der Ausdruck dicht auch abſolut gebraucht fur das, was nicht hohl (blaſicht, lochericht) iſt. Jn dieſer Bedeutung giebt es eine abſolute Dichtigkeit in dem Syſtem der abſo— luten Undurchdringlichkeit, und zwar, wenn eine Waterie gar keine leere Zwifchenraume enthalt. Nach dieſein Be— griffe von Erfullung des Raumes ſtellt man Vergleichungen an, und nennt eine Materie dichter als die andere, die we niger Leeres in ſich enthalt, bis endlich die, in der kein Theil des Raumes leer iſt, vollkommen dicht heißt. Des letzteren Ausdrucks kann man ſich nur nach dem blos mathe— matiſchen Begriffe der Materie bedienen, allein im dyuami— ſchen Syſtem einer blos relativen Undurchdringlichkeit giebt es kein Maximum oder Minimum derDichtigkeit, und gleich— wohl kann jede noch ſo dunne Materie doch vollig dicht hei— ßen, wenn ſie ihren Raum ganz erfuilt, ohne leere Zwi— ſchenraume zu enthalten, mithin ein Continuum, nicht ein Interruptum iſt; allein ſie iſt doch in Bergleichung mit einer andern weniger dicht, in dynamiſcher Bedeutung, wenn ſie ihren Raum zwar ganz, aber nicht in gleichem Grade er— fullt- fullt. Allein auch in dem letzteren Syſtem iſt es unſchick— lich, ſich ein Berhältniß der Materien ihrer Dichtigkeit uach zu denken, wenn man ſie ſich nicht unter einander als ſpeci— fiſch gleicharrig vorſtellt, ſo daß eine aus der andern durch bloße Zuſammendruckung erzeugt werden kann. Da nun das letztere nicht eben nothwendig zur NRatuc aller Materie an ſich erforderlich zu ſeyn ſcheint, ſo kann zwiſchen ungleich— artigen Materien keine Vergleichung in Anſehung ihrer Dich— tigkeit fuglich Statt finden, z. B. zwiſchen Waſſer und Queckſilber, ob zwar es im Gebrauche iſt. 2. Anziehung, ſo fern ſie blos als in der Be— ruhrung wirkſam gedacht wird, beißt Zufammen— hang (Zwar thut man durch ſehr gute Verſuche dar, daß dieſelbe Kraft, die in der Beruhrung Zuſammenhang heißt, auch in ſehr kleiner Entfernung wirkfam befunden werde; allein die Anziehung heißt doch nur Zuſammenhang, ſo fern ich ſie blos in der Beruhrung denke, der gemeinen Erfah— rung gemaß, bey welcher ſie in kleinen Entfernungen kaum wahrgenommen wird. Zufammenhang wird gemeinhin fur eine ganz allgemeine Eigenſchaft der Materie angenommen, nicht, als ob man zu ihr ſchon durch den Begriff einer Ma— terie geleitet wurde, ſondern weil die Erfahrung ſie aller— warts darthut. Altein dieſe Allgemeinheit muß nicht col— lertivs verſtanden werden, als ob jede Materie durch dieſe Art der Anziehung auf jede andere im Weltraume zugleich wirkte, dergletchen die der Gravitatton iſt ſondern blos disjunctiv, namlich auf eine oder die andere, von welcher Art Materien ſie auch ſeyn mag, die mit ihr in Bee ruhrung komnit. Um deswillen, und da dieſe Anziehung, wie es verſchiedene Beweisgrunde darthun konnen, nicht durchdringend, ſondern nur Flachenkraft iſt, da ſte ſelbſt als ſolche nicht einmal allerwarts nach der Dichtigkeit ſich richtet, da zur volligen Starke des Zuſammenhanges ein vor— hergehender Zuſtand der Fluſſigkeit der Materien und der nachmaligen Erſtarrung derfelben erfoderlich iſt und die aller— E3 Hee 70 genauſte Beruhrung gebrochener feſter Materien in eben den— ſelben Flachen, mit denen ſie vorher ſo ſtark zuſemmenhin— gen, z. B. eines Spiegelglaſes, wo es einen Razß hat, den— Mnoch bey weitem den Grad der Anziehung nicht mehr verſtat tet, den es von ſeiner Erſtarrung nach dem Fluſſe her hatte, ſo halte ich dieſe Attraction in der Beruhrung für keine Grundkraft der Materie, ſondern eine nur abgeleitete; wo— von weiter unten ein Mehreres). Eine Materie, deren Theile, unerachtet ihres noch ſo ſtarken Zuſammen— hanges unter einander, dennoch von jeder noch ſo kleinen bewegenden Kraft an einander konnen verſcho ben werden, iſt fluſſig. Theile einer Materie wer— den aber an einander verſchoben, wenn fie, ohne das Quantum der Beruhrung zu vermindern, uur genothigt werden, dieſe unter einauder zu verwech— jeln. Theile, mithin auch Materien, werden ge trennt, wenn die Beruhrung nicht blos mit andern verwechſelt, ſondern aufgehoben, oder ihr Quantum vermindert wird. Ein feſter beſſer ein ſtarrer Korper (eorpus rigidum) iſt der, deſſen Theile nicht durch jede Kraft an einander verſchoben werden kon nen die folglich mit einem gewiſſen Grade von Kraft dem Verſchieben widerſtehen. Das Hinderniß des Werſchiebens der Materien an einander iſt die Rei— bung. Der Widerſtand gegen die Trennung ſich be— ruhrender Materien iſt der Zuſammenhang. Fluſſige Ma— terien erleiden alſo in ihrer Theilung keine Reibung, ſon— dern, wo dieſe angetroffen wird, werden die Materien als ſtarr in großerem oder minderem Grade, deren die letzte Klebrigkeit (viſcoſitas) heißt, wenigſtens ihren kleineren Theilen nach, angenommen. Der ſtarre Korper iſt ſprode, wenn ſeine Theile nicht konnen an einauder verſchoben werden, ohne zu reiſſen mithin wenn der Zuſammenhang derſelben nicht kann verändert, ohne zugleich aufgehoben zu werden. (Man ſetzt ſehr unrichtig den Unu terſchied der fluſſigen und feſten Materien in dem verſchiede: nen 71 nen Grade des Zuſammenhanges ihrer Theile. Denn, um eine Materie flüſſig zu nennen, kommt es nicht auf den Grad des Widerſtandes an, den ſie dem Zerreiſſen, fondern nur dem Verſchieben threr Theile an einander entgegenſetzt. Jener kann ſo groß ſeyn, als man will, ſo iſt dieſer doch jederzeit in einer fluſſtgen Materie Zo. Man betrachte einen Tropfen Waſſer. Wenn ein Theilchen innerhalb dem— ſelben durch eine uoch ſo große Attraction der Nebenthelle, die es beruhren, nach der einen Seite gezogen wird, ſo wird eben daſſelbe doch auch gerade eben ſo viel nach der eutge— gengeſetzten gezogen, und, da die Attractionen beyderſeitig ihre Wirkungen aufheben, iſt das Partikelchen eben ſo leicht beweglich, als ob es im leeren Raume ſich befande, nem lich die Kraft, die es bewegen ſoll, hat keinen Zuſammeuü— hang zu uberwinden, ſondern nur die ſogenannte Tragheit, die ſie bey aller Materie, wenn ſie gleich gar nicht womit zuſammenhinge, uberwinden mußte. Daher wird ein klei nes mikroſtopiſches Thierchen ſich ſo leicht darin bewegen, als ob gar kein Zuſammenhang zu trennen ware. Denn es hat wirklich keinen Zuſammenhang des Waſſers aufzuheben und die Beruhrung deſſelben unter ſich zu vermindern, ſon— dern nur zu verandern. Denket euch aber eben dieſes Thier— chen, als ob es ſich durch die außere Oberflache des Tropfens durcharbeiten wollte, ſo iſt erſtlich zu merken, daß die wechſelſeitige Anziehung der Theile dieſes Waſſerklump— chens es macht, daß ſie ſich ſo lange bewegen, bis ſie in die großte Beruhrung unter einander, mithin in die kleinſte Be— ruhrung mit dem leeren Raum gekommen ſind, d. i. eine Kugelgeſtalt gebildet haben. Wenn nun das genannte Jn— ſect ſich uber die Oberflache des Tropfens hinaus zu arbei— ten beſtrebt iſt, ſo muß es die Kugelgeſtalt verandern, folglich mehr Beruhrung des Waſſers mit dem leeren Raum, und alſo auch weniger Beruhrung der Theile deſſelben un— ter einander bewirken, d. i. ihren Zuſammenhang vermin— dern, und da widerſteht ihm das Waſſer allererſt durch ſei— nen Zuſammenhang, aber nicht innerhalb dem Tropfen, wo Ea die 72 die Beruhrung der Theile unter einander gar nicht verminbett, ſondern nur in die Beruhrung mit andern Theilen verandert wird, mithin dieſe nicht im mindeſten getrennt, ſondern nur verſchoben worden. Auch kann man auf das mikroſcopiſche Thierchen und zwar aus ahnlichen Grunden anwenden, was Newton vom Lichtſtrahl fagt, daß er nicht durch die dichte Materie, ſondern nur durch den leeren Raum zurückgeſchla gen werde. Es iſt alſo klar: daß die Vergroßerung des Zuſammenhanges der Theile einer Materie ihrer Fluſſigkeit nicht den mindeſten Abbruch thue. Waſſer hangt in ſeinen Theilen weit ſtarker zuſammen, als man gemeiniglich glaubt, wenn man ſich auf den Verſuch einer von der Oberflache des Waſſers losgeriſſenen metallenen Platte verlaßt, welcher nichts entſcheidet, weil hier das Waſſer nicht in der ganzen Flache der erſten Beruhrung, ſondern in einer viel kleineren reißt, zu welcher es namlich durch das Verſchieben ſeiner Theile endlich gelaugt iſt, wie etwa ein Stab von weichem Vachſe ſich durch ein angehangtes Gewichte erſtlich dünner ziehen laßt, und alsdenn in einer weit kleineren Flache rei— ßen muß, als man anfanglich annahm. Was aber in Au— ſehung unſers Begriffs der Flufſigkeit ganz entſcheidend iſt, iſt dieſes: daß fluſſige Materien auch.als ſolche erklart werden konnen, deren jeder Punct nach alleu Directio nen mit eben derſelben Kraſt ſich zu bewegen trachtet, mit welcher er nach irgend einer gedruckt wird; eine Eigenſchaft, auf der das erſte Geſetz der Hydrodynamik beruht, die aber einer Anhauſung von glatten und dabey feſten Korperchen, wie eine ganz leichte Aufloſung ihres Drucks nach Geſetzen der zuſammengeſetzten Bewegung zei gen kann, niemals beygelegt werden kann, und dadurch die Originalitat der Eigenſchaft der Fluſſigkeit beweiſet. Wur— de nun die fluſſige Materie das mindeſte Hinderniß des Ver ſchiebens, mithin auch nur die kleinſte Reibung erleiden, ſo wurde dieſe mit der Starke des Druckes, womit die Theile derſelben an einander gepreßt werdrn, wachſen!und endlich rin Druck Statt finden, bey welchem die Theile dieſer Ma— terie 73 terie ſich nicht an einander durch jede kleine Kraft verſchicben laſſen, z. B. in einer gebogenen Rohre von zwey Schenkeln, deren der eine ſo weit ſeyn mag, als man will, der andere ſo enge als man will, außer, daß er nur nicht ein Haarrohrchen iſt wurde, wenn man beyde Schenkel eintige hundert Fuß hoch denkt, die flſſige Materie in der engen eben ſo hoch ſtehen, als in der weiten, nach Geſetzen der Hydroſtatik. Weil aber der Druck auf den Boden der Rohren und alſo auch auf den Theil, der beyde in Gemeinſchaft ſtehende Rohren verbindet, in Proportion der Hohen ins linendliche immer großer gedacht werden kann, ſo mußte, wenn die mindeſte Reibung zwiſchen den Theilen des Fluſſegen Statt fande, eine Hohe der Rohren gefunden werden konnen, bey der eine kleine Quantitat Waſſer in die engere Rohre gegof— ſen, das in der weiteren nicht aus ſeiner Lage verrucken, mithin die Waſſerſeule in dieſer hoher zu ſtehen kommen wurde, als in jener, weil ſich die unteren Theile, bey ſo großem Drucke derſelben gegen einander, nicht mehr durch ſo kleine bewegende Kraft, als das zugeſetzte Gewicht Waf— ſer iſt, verſchieben ließen, welches der Erfahrung und ſelbſt dem Begriffe des Fluffigen zuwider iſt. Eben daſſelbe gilt, wenn man ftatt des Drucks durch die Schwere den Zufam— menhang der Theile ſetzt, er mag ſo groß ſeyn, wie er will. Die angefuhrte zweyte Definition der Flüfſigkeit, worauf das Grundgeſetz der Hydroſtatik beruht, namtich daß ſie die Eigenſchaft einer Materie ſey, da ein jeder Theil derſelben ſich nach allen Seiten mit eben derſelben Kraft zu bewegen veſtrebt iſt, womit er in einer gegebenen Direetion gedruckt wird, folgt aus der erſten Definition, wenn man damit den Grundſatz der allgemeinen Dynamik verbindet, daß alle Materie urſprunglich elaſtiſch ſey, da denn dieſe nach jeder Seite des Raums, darin ſte zuſammengedruckt iſt, mit der— ſelben Kraft ſich zu erweitern, d. i. (wenn die Theile einer Materie ſich an einander durch jede Kraft ohne Hinderniß verſchieben lafſen, wie es bey der fluſſigen ſo wirklich iſt,) ſich zu bewegen beſtrebt ſeyn muß, womit ſder Druck in einer E5 jeden 74 rt jeden Richtung, welche es auch ſey, geſchiehet. Alſo ſind es eigentlich nur die ſtarren Materien, (deren Moglichkeit noch außer dem Zuſammenhange der Theile eines anderen Erklarungsgrundes bedarf) denen man Reibung beylegen darf, und die Reibung ſetzt ſchon die Eigenſchaft der Rigi— ditat voraus. Warum aber gewiſſe Materien, ob ſie gleich vielleicht nicht großere, vielleicht wohl gar kleinere Kraft des Zuſammenhanges haben, als andere fluſſige, dennoch dem Verſchieben der Theile ſo machtig widerſtehen, und daher nicht anders, als durch Aufhebung des Zuſammenhanges Aller Theile in einer gegebenen Flache zugleich, ſich trenuen laſſen, welches denn den Schein eines vorzüglichen Zuſam— menhanges giebt; wie alſo ſtarre Körper moglich ſeyn, das iſt immer noch ein unaufgeloſetes Problem, ſo leicht als auch die gemeine Naturlehre damit fertig zu werden glaubt. z. Elaſticitat (Springkraft) iſt das Vermogen einer Materie, ihre durch eine andere bewegende Kraft veranderte Große oder Geſtalt, bey Nachlaſſung der ſelben wiederum anzunehmen. Sie iſt entweder expan ſive, oder attractive Elaſticitat; jene, um nach der Zu— ſammendruckung das vorige großere, dieſe, um nach der Ausdehnung das vorige kleinere Volumen anzunehmen. (Die attractive Elaſticitat iſt, wie es ſchon der Ausdruck zeigt, offenbar abgeleitet. Ein eiſerner Drath, durch angehangte Gewichte gedehnt, ſpringt, wenn man das Band abſchnei— det, in ſein Volumen zuruck. Vermoge derſelben Attraction, die die Urſache ſeines Zuſammenhanges iſt, oder bey fluſſi— gen Materien, wenn die Warme dem Queckſilber plotzlich entzogen wurde, wuede die Materie deſſelben eilen, um das vorige kleinere Volumen wieder anzunehmen. Die Elaſti— citat, die blos in Herſtellung der vorigen Figur beſteht, iſt jederzeit attractiv, wie an einer gebogenen Degenklinge, da die Theile auf der convexen Flache aus einander gezerret, ihre vorige Nahheit anzunehmen trachten, und ſo kann auch ein kleiner Tropfen Queckſilber elaſtiſch genannt werden. Aber die U 19 die expanſive Elaſticitat kann eine urſprungliche, ſie kann aber auch eine abgeleitete ſeyn. So hat die Luft eine abge— leitete Elaſticitat, vermittelſt der Naterte der Warme, welche mit ihr innigſt vereinigt tſt, und deren Elaſticttat vtel- leicht urſprunglich iſt. Dagegen muß der Greundſtoff des Fluſſigen, welches wir Luft nenuen, dennoch als Materie überhaupt ſchon an ſich Elaſticitat haben, welche urſprung— lich heißt. Von welcher Art eine wahrgenommene Elaſtici— tat ſey, iſt in vorkommenden Fallen nicht moglich mit Ge— wißheit zu entſcheiden). 4. Die Wirkung bewegter Korper auf einan der durch Mittheilung ihrer Bewegung heißt mech a niſch; die der Materien aber, ſo fern ſie auch in Ruhe durch eigene Krafte wechſelſeitig die Verbin— dung ihrer Theile verandern, heißt chemiſch. Die— ſer chemiſche Einfluß heißt Aufloſung, ſo fern er die Trennung der Theile einer Materie zur Wirkung hat (die mechaniſche Theilung, z. B. durch einen Keil, der zwi— ſchen die Theile einer Materie getrieben wird, iſt alſo, weil der Keil nicht durch eigene Kraft wirkt, von einer chemiſchen ganzlich unterſchieden): derjenige aber, der die Abſonde— rung zweyer durch einander aufgeloſeter Materien zur Wir- kung hat, iſt die Scheidung. Die Aufloſung ſpecifiſch verſchiedener Materien durch einander, darin kein Theil der einen angetroffen wird, der nicht mit einem Theile der andern von ihr ſpecifiſch unterſchiedenen in derſelben Proportion, wie die Ganzen, vereinigt ware, iſt die abſvlute Aufloſung, und kann auch die chemiſche Durchdringung genannt werden. (Ob die aufloſenden Krafte, die in der Natur wirklich anzutreffen ſind, eine vollſtandige Auftoſung zu be wirken vermogen, mag unausgemacht bleiben. LKier iſt nur die Frage davon, ob ſich eine ſolche nur denken laſſe. Nun iſt offenbar, daßi, ſo lange die Theile einer aufgeloſe— ten Materie noch Klumpchen (moleculae) ſind, nicht min— der eine Aufloſung derſelben moglich ſey, als die der gro— ßeren, 76 ßeren, ja daß dieſe wirklich ſo lange fortgehen muſſe, wenn die aufluſende Kraft bleibt, bis kein Theil mehr da iſt, der nicht aus dem Aufloſungsmittel und der aufzuloſenden Ma— terie, in der Proportion, datin beyde zu einonder im Gan— zen ſtehen, zuſammengeſetzt ware. Weil alſo in ſolchem Falle kein Theil von dem Volumen der Aufloſung ſeyn kann, der nicht einen Theil des aufloſenden Mittels enthielte, ſo muß dieſes, als ein Continunm, das Volumen ganz er— fullen. Eben ſo, weil kein Theil eben deſſelben Volumens der Solution ſeyn kann, der nicht einen proportionulichen Theil der aufgeloſeten Materie enthielte, ſo muß dieſe auch als ein Continuum den ganzen Raum, der das Volumen der Miſchung ausmacht, erfullen. Wenn aber zwey Ma— terien, und zwar jede derſelben ganz, einen und denſelbeü Raum erfüllen, ſo durchdringen ſie einander. Alſo wur— de eine vollkommene chemiſche Aufloſung eine Durchdringung der Materien ſeyn, welche dennoch von der mechaniſchen gänzlich unterſchieden ware, indem bey ber letzten gedacht wird, daß bey der großern Aunaherung bewegter Materien die repulſive Kraft der einen die der andern ganzlich uberwie— gen, und eine oder beyde ihre Ausdehnung auf nichts bringen konnen; da hingegen hier die Ausdehnung bleibt, nur daß die Materien nicht außer einander, ſondern in einan der, d. i. durch Jntusſuſception (wie man es zu nennen pflegt,) zuſammen einen der Summe ihrer Dichtigkeit ge— maßßen Raum einnehmen. Gegen die Moglichkeit dieſer vollkemmenen Auftoſung und alſo der chemiſchen Durchdrin— guug iſt ſchwerlich etwas einzuwenden, obgleich ſie eine voliendete Theilung ins Unendliche enthalt, die in dieſem Falle doch keinen Widerſpruch in ſich faßt, weil die Aufloö— ſung ecine Zeit hindurch continuirlich, mithin gleichfalls durch eine unendliche Reihe Augenblicke mit Acceleration ge ſchieht, uberdem durch die Theilung die Summe der Ober— flachen der noch zu theilenden Materien wachſen, und, da die aufloſende Kraft continuirlich wirkt, die ganzliche Auflo— fung in einer anzugebenden Zeit vollendet werden kann. Die 77 Die Unbegreiflichkeit einer ſolchen chemiſchen Durchdringung zweher Materien iſt auf Rechnung der Unbegreiflichkeit der Theilbarkeit eines jeden Continuum uberhaupt ins Unendli— che zu ſchreiben. Geht man von dieſer vollſtandigen Auflo— ſung ab, ſo muß man annehmen, ſie ginge nur bis zu gewiſ— ſen kleinen Klumpen der aufzuloſenden Materie, die in dem Aufloſungsmittel in geſetzten Weiten von einander ſchwim— men, ohne daß man den mindeſien Grund angeben kann, warum dieſe Klumpchen, da ſie doch immer thetiibare Ma— terien ſind, nicht gleichfalls aufgeloſet werden. Denn, daß das Aufloſungsmittel nicht weiter wirke, mag immer in der Natur, ſo weit Erfahrung reicht, ſeine gute Nichtigkeit haben; es iſt hier aber nur die Rede von der Moglichleit einer aufloſenden Kraft, die auch dieſes Klumpchen und ſo ferner jedes andere, was noch ubrig bleibt, aufloſe, bis die Solution vollendet iſt. Das Volumen, was die Auf— loſung einnimmt, kann der Summe der Raume, die die einander aufloſenden Materien vor der Miſchung einnahmen, gleich, oder kleiner, oder auch großer ſeyn, nachdem die anziehenden Krafte gegen die Zurückſtoßungen in Verhaltniß ſtehen. Sie machen in der Aufloſung jedes fur ſich und beyde vereinigt ein elaſtiſches Medium aus. Dieſes kann auch allein einen hinreichenden Grund angeben, warum die aufgeloſete Materie ſich durch ihre Schwere nicht wiederum vom aufloſenden Mittel ſcheide. Denn die Anziehung des letzteren, da ſie nach allen Seiten gleich ſtark geſchieht, hebt ihten Widerſtand ſelbſt auf, und eine gewiſſe Klebrigkeit im Fluſſigen anzunehmen, ſtimmt auch gar nicht mit der gro— ßen Kraft, die dergleichen aufgeloſete Materien, z. B. die Gauren mit Waſſer verdunnt, auf metalliſche Korper aus— uben, an die ſie ſich nicht blos anlegen, wie es geſchehen mußte, wenn ſie blos in ihrem Medium ſchwammen, ſon— dern die ſie mit großer Anziehungskraft von einander tren— nen, und im ganzen Raume des Vehikels verbreiten. Ge— ſetzt auch, daß die Kunſt keine chemiſche Aufloſungskrafte Jieſer Art, die eine vollſtandige Auftoſung bewirkten, in ihrer 78 ihrer Gewalt hatte, ſo konnte doch vielleicht die Natur ſie in ihren vegetabiliſchen und animaliſchen Operationen bewei ſen, und dadurch vielleicht Materien erzeugen, die, ob ſie zwar gemiſcht ſind, doch keine Kunſt wiederum ſcheiden kann. Dieſe chemiſche Durchdringung konnte auch ſelbſt da angetroffen werden, wo die eine beyder Materien durch die andere eben nicht zertrennt und im buchſtablichen Sinne aufgeloſet wird, ſo wie etwa der Warmeſtoff die Korper durchdringt, da, wenn er ſich nur in leere Zwiſchenraume derſelben vertheilete, die feſte Subſtanz ſelbſt kalt bletben wurde, weil dieſe nichts von ihr einnehmen konnte. Jm— gleichen konnte man ſich ſo gar einen ſcheinbarlich freyen Durchgang gewiſſer Materien durch andere auf ſolche Weiſe denken, z. B. der magnetiſchen Materie, ohne ihr dazu offene Gänge und leere Zwiſcheuraume in allen, ſelbſt den dichteſten Materien vorzubereiten. Doch es iſt hier nicht der Ort, Hypotheſen zu beſonderen Erſcheinungen, ſondern nur das Princip, wornach ſie alle zu beurtheilen ſind, aus— fiadig zu machen. Alles, was uns des Bedurfuiſſes uber— hebt, zu leeren Raumen unſere Zuflucht zu nehmen, iſt wirk— licher Gewinn fur die Naturwiſſenſchaft. Denn dieſe geben gar zu viel Freyheit der Einbildungskraft, den Mangel der inneren Naturkenntniß durch Erdichtung zu erſetzen. Das ab ſolut Leere und das abſolut Dichte ſind in der Naturlehre ohngefahr das, was der blinde Zufall und das blinde Schick ſal in der metaphyſiſchen Weltwiſſenſchaft ſind, namlich ein Schlagbaum fur die herrſchende Vernunft, damit entweder Erdichtung ihre Stelle einnehme, oder ſie auf dem Pplſter dunkler Qualitäten zur Ruhe gebracht werde. Was nun aber das Verfahren in der Naturwiſſen— ſchaft in Anſehung der vornehmſten aller ihrer Aufgaben, namlich der Erklarung einer ins Unendliche moglichen ſpeci fiſchen Verſchiedenhekt der Materien betrifft, fo kann man dabey nur zwey Wege einſchlagen: den mechaniſchen, durch die Verbindung des Abſolutvollen mit dem Abſolutlee— ren, —S 79 ten, oder einen ihm entgegengeſetzten dynamiſchen Wea, durch die bloße Verſchiedenheit in der Verbindung der ur— ſprunglichen Krafte der Zuruclſtohung und Auztehung alle Verſchiedenheiten der Materien zu erklaren. Der erſte hat zu Materialten ſeiner Ableitung die Atomen und das Leere. Ein Atom iſt ein lleiner Theil der Materie, der phyſtſch un— theilbar iſt. Phyſiſch untheilbar iſt eine Materie, deren Theile mit einer Kraft zuſammenhängen, die durch keine in der Natur befindliche bewegende Kraft uberwaltigt werden kann. Ein Atom, ſo fern er ſich durch ſeine Figur von andern ſpecifiſch unterſcheidet, heißt ein erſtes Korperchen. Ein Korper (oder Korperchen), deſſen bewegende Kraft von ſeiner Figur abhangt, heißt Maſchine. Die Erklarungsart der ſpecifiſchen Verſchiedenheit der Materien durch die Beſchaffenheit und Zuſammenſetzung ihrer kleinſten Theile, als Maſchinen, iſt die mechaniſche Naturphiloſophje: diejenige aber, welche aus Materien, nicht als Maſchinen, d. i. bloßen Werkzeugen außerer bewegender Krafte, ſondern ihnen urſprunglich eigenen bewegenden Kraften der Anziehung und Zuruckſtoßung die ſpecifiſche Verſchiedenheit der Materte aAbleitet, kann die dynamiſche Naturphiloſophie genannt werden. Mie mechaniſche Erklurungsart, da ſie der Ma— thematik am fugſamſten iſt, hat unter dem Namen der Ato— miſtik oder Corpuſcularphiloſophie mit weniger Abande— rung vom alten Democrit an bis auf Carteſen und ſelbſt bis zu unſeren Zeiten immer ihr Anſehen und Einfluß auf die Principien der Naturwiſſenſchaft erhalten. Das We— ſentliche derſelben beſteht in der Voraueſetzung der abſolu— ten Undurchdringlichkeit der primitiven Materie, in der abſoluten Gleichartigkeit dieſes Stoffs und dem allein ubriggelaſſenen Unterſchiede in der Geſtalt, und in der ab— ſoluten Unuberwindlichkeit des Zuſammenhauges der Ma— terie in dieſen Grundkorperchen ſelbſt. Dies waren die Materialien zu Erzeugung der ſpecifiſch verſchiedenen Ma— rerien, um nicht allein zu der Unveranderlichkeit der Gattun— gen und Arten einen unveranderlichen und gleichwphl ver— ſchie— 14 80 S ſchledentlich geſtalteten Grundſtoff bey der Hand zu haben, ſon dern auch aus der Geſtalt dieſer erſten Theile, als Maſchi— uen, (denen nichts weiter, als eme außerlich eingedruckte Kraft fehlte) die mancherley Naturwirkungen mechaniſch zu erklaren. Die erſte und vornehmſte Beglaubiguug dieſes Syſtems aber beruht auf der vorgeblich unvermeidlichen Nothwendigkeit, zum ſpecifiſchen Unterſchiede der Dichtigkeit der Materien leere Raume zu brauchen, die man innerhalb der Materien und zwiſchen jenen Partikeln vertheilt, in einer Proportion, wie man ſie nothig fand, zum Behuf einiger Erſcheinungen gar ſo groß, daß der er— fullete Theil des Volumens, auch der dichteſten Materie, gegen den leeren beynahe fur nichts zu halten iſt, an— nahm. Unm nun eine dynamiſche Erklarungsart ein— zufuhren, (die der Expperimentalphiloſophie weit angemeſſe— ner und beforderlicher iſt, indem ſie geradezu darauf lei— tet, die den Materien eigene bewegende Krufte und deren Geſctze auszufinden, die Freyheit dagegen einſchrankt, leere Zwiſchenraume und Grundkorperchen von beſtimmten GSe ſtalten anzunehmen, die ſich beyde durch kein Exrperiment beſtimmen und ausfindig machen laſſen,) iſt es gar nicht nothig neue Hypotheſen zu ſchmieden, ſondern allein das Poſtulat der blos mechaniſchen Erklarungsart: daß es unmoglich ſey, ſich einen ſpecifiſchen Unterſchied der Dichügkeit der Materien ohne Beymiſchung leerer Raume zu denken, durch die bloße Anfuhrung einer Art, wie er ſich ohne Widerſpruch denken laſſe, zu widerlegen. Denn wenn das gedachte Poſtulat, worauf die blos mecha— niſche Erklarungsart füßet, nur erſt als Grundſatz fur un— gultig erklaret worden, ſo verſteht es ſich von ſelbſt, daß man es als Hypotheſe in der Naturwiſſenſchaft nicht auf— nehmen muſſe, ſo lange noch eine Moglichkeit ubrig bleibt, den ſpecifiſchen Unterſchied der Dichtigkeiten ſich auch ohne alle leere Zwiſchenraume zu denken. Dieſe Nothwendigkeit aber beruht darauf, daß die Materie nicht (wie blos me chaniſche Raturforſcher anuehmen) durch abſolute Undurch— drung J LT dringlichkeit ihren Raum erfullt, ſondern durch repulſive Kraft, die ihren Grad hat, der in verſchiedenen Materten verſchieden ſeyn kann, und, da er fur ſich nichts mit der Anziehungskraft, welche der Quantitat der Materie gemaß iſt, gemein hat, ſie bey einerley Anziehungskraft in verſchiedenen Materien dem Grade nach als urſprunglich ver— ſchieden ſeyn konne, folglich auch der Grad der Ausdeh— nung dieſer Materien bey derſelben Quautttat der Materie und umgekehrt die Quantitat der Materte unter demſelben Volumen, d. i. die Dichtigkett derſelben urſprunglich gar große ſpecifiſche Verſchiedenheiten zulaſſe. Auf dieſe Art wurde man es nicht unmoglich finden, ſich eine Materie zu denken, (wie man ſich eiwa den Aether vorſtellt) die ihren Nauni ohue alles Leere gaug erfullete und doch mit, ohne Ver gleichung minderer Quautttat der Materie unter gleichem Volumen, uls alle Korper/ die wir' unſeren Verſuchen un— teiwerfen: konnen. Die repulſive Kraft inuß ain Aether, in Verhaltnißittuf die eigene Anziehungskraft deſſelben, ohne Vergleichung grofßer gedacht werden, als an ällen andern uns bekannteli Materien. Und däs iſt denn auch das Einzige, was wir blos darum aunehmen, weil es ſich denken laßt, nur zum Widerſpiel einer Hypotheſe (der leeren Raume), die ſich alleiülauf das Vorgeben ſtutzt, daß ſich dergleichen ohne leexs Ruume nicht denken läſſe. Denu außer dieſem darf weder irgend ein Geſetz der anzieheüden, usch zurück— ſtoßenden Kraft, auf Muthmaßungen a ptiori gewagt, ſon— dern alles,“ ſelbſt die allgemeine Attractivn, als Ueſache der' Schweren, muß ſammt ihrem Geſetze aus Datis der Etfaährung geſchloſſen werden. Noch weniger wird dergleichen beh den chemiſchen Verwandtſchaften anders, als durch den Weg des Epyperinients verfucht werden durfen. Denn es iſt uberhaupt uber dem Geſichtskreis unſerer Vernunft' gelegen, urſprungliche Krafte a prioriĩ ihrer Moglichkeit nach einzuſehen, vielmehr beſteht alle Naturphiloſophie jn der Zuruckfuhrung gegebener, dem Anſcheine nach virſchiedener, Krafte auf vine geringere g Zahl 82 Zahl Krafte und Vermogen, die zu Erklarung der Wir— kungen der erſten zulangen, welche Reduction aber nur bis zu Grundtkraften fortgeht, uber die unſere Vernunft nicht hinaus kann. Und ſo iſt Rachforſchung der Me— taphyſik, hinter dem, was dem empiriſchen Begriffe der Materie zum Grunde liegt, nur zu der Abſicht nutzlich, die Naturphiloſophie, ſo weit als es immer mogtich iſt, auf die Erforſchung der dynamiſchen Erklarungs— grunde zu leiten, weil dieſe allein beſtimmte Geſetze, folglich wahren Vernunftzuſammenhang der Erklarungen, hoffen laſſen. Dies iſt nun alles, was Metaphyſik zur Con— ſtruction des Begriffs der Materie, mithin zum Behuf der Anwendung der Mathematik auf Naturwiſſenſchaft, in Anſehung der Eigenſchaften, wodurch Materie. einen Raum in beſtimmtem Maaße erfullet, nur immer, leiſten kann, namlich dieſe Eigenſchaften als dynamiſch anzu— ſehen und nicht als unbedigte urſprungliche Poſitionen, wie ſie etwa eine blos mathematiſche Behandlung poſtu— liren wurde. Den Beſchluß kann die bekannte Frage, wegen. der Zulaſſigkeit leerer Raume in der Weit, machen. Die Woglichkeit derſelben laßt ſich nicht ſtreiten. Denun. zu allen Kraften der Materie wird Raum erfodert, uind, da dieſer. auch die Bedingungen. der Geſetze. der Verbrei tung jener eothalt, nothwendig vor aller Materie vor— ausgeſetzt. So wird der Materie Attractionskraft bey— gelegt, ſo fern ſie einen Raum um ſich durch Auzie— hung einnimmt, ohne ihn gleichwohl zu erfullen, der alſo ſelbſit da, wo Materie wirkſam iſt, als leer gedacht werden katn, weil ſie da nicht durch. Zurſickſtoßungs— krafte wirkſam iſt und ihn alſo nicht erfullt. Allein leere Raume als wirklich anzunehmen, dazu. kann uns keine Erfahrung, oder Schluß aus derſelben, oder noth— wuven 7 83 wendige Hypotheſis, ſie zu erklaren, berechtigen. Denn alle Erfahrung giebt uns nur comparativ-leere Raume zu erkennen, welche, nach allen beliebigen Graden aus der Eigenſthaft der Materie, ihren Raum mit großerer oder bis ins Unendliche immer kleinerer Ausſpannungs— kraft zu erfullen, vollkommen erklart werden konnen, ohne leere Raume zu bedurfen. Drittes Haupftſtuck. Meettaphyſiſche Anfangsgrunde der Meſchanif. 91 Erklarung 1. Materie iſt das Bewegliche, ſo fern es, als ein ſolches, bewegende Kraft hat. 8 Anmerkung.e Dieſes iſt nun die dritte Definition von einer Materie. Der blos dynamiſche Begriff konnte die Materie auch als in Ruhe betrachten; die bewegende Kraft, die da in Erwa— gung gezogen wurde, betiaf blos die Erfullung eines gewiſ— ſen Raumes, ohne daß die Materie, die ihn erfullete, ſelbſt als bewegt angeſehen werden durfte. Die Züruckſtotzung war daher eine urſprunglich- bewegende Kraft, um Bewe— gung zu ertheilen; dagegen wird in der Mechanik die Kraft einer in Bewegung geſetzten Materie betrachtet, um dieſe Beweguing einer anderen. mitzutheilen. Es iſt aber klar; daß das: Bewegliche durch jeine Bewegung keine bewe F a gende 84 —S gende Kraft haben wurde, wenn es nicht urſprunglich-be wegende Krafte beſaße, dadurch es vor aller eigenen Bewe— gung in jedem Orte, da es ſich befindet, wirkſam iſt, und daß keine Materte eine andere, die ihrer Bewegung in der geraden Linie vor ihr im Wege liegt, gleichmaßige Bewe— gung eindrücken wurde, wenn beyde nicht urſprungliche Ge— ſetze der Zuruckſtoßung beſaßen, noch daß ſie eine andere durch ihre Bewegung nothigen konne, in der geraden Linie ihr zu folgen, (ſie nachſchieppen konnte) wenn beyde nicht Anziehungokrafte beſaßen. Alſo ſetzen alle mechaniſche Ge ſetze die dynamiſchen voraus, und eine Materte, als bewegt, kaun keine bewegende Kraft haben, als nur vermittelſt ihrer Zuruckſtoßung oder Anziehung, auf welche und mit welchen ſie in ihrer Bewegung uünmittelbar: wirkt und daduith ihre eigene Bewegung einer anderen mittheilt. Man wird es miur nachſehen, daß ich der Mittheilung der Bepegung durch Anziehung (z. Bo wenn etwa eiit Comet von ſtarkerem An ziehungsvermogen als die Erde im Vorbeygehen vor der— ſelben ſie nach ſich fortſchleppte) hier nicht weiter Erwah— nung thun werde, ſonderü nür der: Vermittelung der repul ſiven Krafte, alfo durch Druck, (wie veruittelſt. geſpauiffer Federn,) oder durch Stoß, da ohnebem die Anwendüng -der Geſetze der einen auf die der aideren nur in Anſehüng der Richtungslinie verſchieden, ubrigens aber in beyden Fallen einerley iſt. Erklarung 2. Die Quantitat der Materie iſt die Meuü— ge des Beweglichen in einem beſtimmten Raum. Dieſelbe, ſo fern alle ihre Theile in ihrer Bewe— gung als zugleich wirkenv (bewegend) beträchtet werden, heißt die Maſſe, und man ſagt, eine Materie wirke in Maſſe, wenn alle ihre Theile in einerley Richtung bewegt:außer ſich zugleich ihre bewegende Kraft ausuben. Eine Maſſe von be 91 et re 85 beſtimmter Geſtalt, heißt ein Korper (in mecha— niſcher Bedeutung). Die Große der Bewe— gung (mechaniſch geſchatzt) iſt diejenige, die durch die Quantitat der bewegten Materie und ihre Geſchwindigkeit zugleich geſchatzt wird; Phoronomiſch beſteht ſie blos in dem Grade der Geſchwindigkeit. Lehrſatz 1. Die Quantitat der Materie kann in Ver— gleichung mit jeder anderen nur durch die Quan— titat der Bewegung bey gegebener Geſchwindjg. keit geſchatzt werden. Beweis. Die Materie iſt ins Unendliche theilbar, folglich kann keiner ihre Quantitat durch eine Menge ihrer Theile unmittelbar beſtimmt werden. Denn wenn dieſes auch in der Vergleichung der gegebenen Mate— rie mit einer gleichartigen geſchieht, in welchem Falle die Quantitat der Materie der Große des Volumens proportional iſt, ſo iſt dieſes doch der Foderüng des Lehrſatzes, daß ſie in Vergleichung mit jeder anderen (auch ſpecifiſch verſchiedenen) geſchatzt werden ſoll, zu— wider. Alſe kann die Materie, weder unmittelbar, noch mittelbar, in Vergleichung mit jeder andern gultig geſchatzt werden, ſo lange man von ihrer eigenen Bewegung abſtrahirt. Folglich iſt kein anderes allge— mein gultiges Maaß derſelben, als die Quantitat ihrer Beweaung, ubrig. Jn dieſer aber kann der Unter— ſchied der Bewegung, der auf der verſchiedenen Quan— F 3 titat 86 titat der Materien beruht, nur alsdenn gegeben wer— den, wenn die Geſchwindigkeit unter den verglichenen Materien als gleich angenommen wird, folglich u. ſ. v. Zuſatz. Die Quantitat der Bewegung der Korper iſt in zuſammengeſetztem Verhaltniß aus dem der Quantitat ihrer Materie und ihrer Geſchwindigkeit, d. i. es iſt einerley, ob ich die Quantitat der Materie eines Kor— pers doppelt ſo groß mache, und die Geſchwindigkeit behalte, oder ob ich die Geſchwindigkeit verdoppele und eben dieſe Maſſe behalte. Denn der beſtimmte Be— griff von einer Große iſt nur durch die Conſtruction des Quantums moglich. Dieſe iſt aber in Anſehung des Begriffs der Quantitat nichts als die Zuſammen ſetzung des Gleichgeltenden; folglich iſt die Conſtruction der Quantitat einer Bewegung die Zuſammenſetzung vieler einander gleichgeltender Bewegungen. Nun iſt es nach den phoronomiſchen Lehrſätzen einerley, ob ich einem Beweglichen einen gewiſſen, Grad Geſchwin digkeit oder vielen gleich beweglichen alle kleinere Gra— de der Geſchwindigkeit ertheile, die aus der durch die Menge des Beweglichen dividirten gegebenen Ge— ſchwindigkeit herauskommen. Hieraus entſpringt zu erſt ein, dem Anſcheine nach, phoronomiſcher Begriff von der Quantitat einer Bewegung, als zuſammen geſetzt aus viel Bewegungen außer einander, aber doch in einem Ganzen vereinigter, beweglicher Puncte. Werden nun dieſe Puncte als etwas gedacht, was durch ſeine Bewegung bewegende Kraft hat, ſo entſpringt daraus der mechaniſche Begriff von der Quan r ĩñ 87 AQuantitat der Bewegung. Jn der Phoronomie aber iſt es nicht thunlich, ſich eine Bewegung als aus vie— len außerhalb einander befindlichen zuſammengeſetzt vorzuſtellen, weil das Bewegliche, da es daſelbſt ohne alle bewegende Kraft vorgeſtellt wird, in aller Zuſam— menſetzung mit mehreren ſeiner Art keinen Unterſchied der Große der Bewegung giebt, als die mithin blos in der Geſchwindigkeit beſteht. Wie die Quantitat der Bewegung eines Korpers zu der eines anderen, ſo ver halt ſich auch die Große ihrer Wirkung, aber wohl zu verſtehen, der ganzen Wirkung. Diejenigen, welche blos die Große eines mit Widerſtande erfulleten Raums (z. B. die Hohe, zu welcher ein Korper mit einer ge— wiſſen Geſchwindigkeit wider die Schwere: ſteigen, oder die Tiefe, zu der derſelbe in weiche Materien dringen kann,) zum Maaße der ganzen Wirkung annahmen, brachten ein anderes Geſetz der bewegenden Krafte bey wirklichen Bewegungen heraus, namlich das des zu— ſammengeſetzten Verhaltniſſes aus dem der Quantitat der Materien und der Quadrate ihrer Geſchwindigkei— ten; allein ſie uberſahen die Große der Wirkung in der gegebenen Zeit, in welcher der Korper ſeinen Raum mit kleinerer Geſchwindigkeit zurucklegt, und dieſe kann doch allein das Maaß einer durch einen ge— gebenen gleichformigen Widerſtand erſchopften Bewe— gung ſeyn. Es kann alſo auch kein Unterſchied zwi— ſchen lebendigen und todten Kraften Statt finden, wenn die bewegenden Krafte mechaniſch, d. i. als diejenigen, die die Korper haben, ſo fern ſie ſelbſt bewegt ſind, be— trachtet werden, es mag nun die Geſchwindigkeit ihrer Bewegung endlich oder unendlich klein ſeyn (bloße Be— ſtrebung zur Bewegung); vielmehr wurde man weit F 4 ſſchick. 88 —S ſchicklicher diejenigen Kräfte, womit die Materie, wenn man auch von ihrer eigenen Bewegung, auch ſo gar von der Beſtrebung ſich zu bewegen ganzlich abſtrahirt, in andere wirkt, folglich die urſprunglich bewegenden Krafte der Dynamik todte Krafte, alle mechaniſche, i d. i. durch eigene Bewegung bewegende Krafte dage— gen, lebendige Kräfte nennen konnen, ohne auf den J Unterſchied der Geſchwindigkeit zu ſehen, deren Grad auch unendlich klein ſeyn darf, wenn ja noch dieſe Be— nennungen todter und lebendiger Kraſte beybehalten zu werden verdienten. Anmerkung. Vir wollen, um Weitlauftigkeit zu vernieiden, die Erlauterung der vorſtehenden drey Satze in einer Anmer— kung zuſammenfaſſen. Daß die Quantitat der Materie nur als die Menge des Beweglichen (außerhalb einander) konne gedacht wer— den, wie die Definition es ausſagt, iſt ein mertwurdiger und Fundamentalſatz der allgemeinen Mechanik. Denn da— durch wird angezeigt: daß Materie keine andere Große habe, J als die, welche in der Menge des Maunigfaltigen außer— halb einander beſteht, folglich auch keinen Grad der be— wegenden Kraft mit gegebener Geſchwindigkeit, der von die— ſer Menge unabhangig ware und blos als intenſive Große betrachtet werden konnte, welches allerdings Statt finden wurde, wenn die Materie aus Monaden beſtande, deren Realität in aller Beziehung einen Grad haben muß, wel— cher großer oder kleiner ſeyn kann, ohne von einer Menge der Theile außer einander abzuhangen. Was den Begriff der Maſſe in eben derſelben Erklarung betrifft, ſo kann man ihn nicht, wie gewohnlich, mit dem der Quantitat fur einer— ley halten. Fluſſige Materien konnen durch ihre eigene Be— wegung 89 wegung in Maſſe, ſie konnen aber auch im Fluſſe wirken. Jm ſogenannten Waſſerhammer wirkt das anſtoßende Waſ— ſer in Maſſe, d. i. mit allen ſeinen Theilen zugleich; eben das geſchieht auch im Waſſer, welches, in einem Gefaße eingeſchloffen, durch ſein Gewicht auf die Wagſchale, dar— auf es ſteht, druckt. Dagegen wirkt das Waſſer eines Muhlbachs auf die Schaufel des unterſchlagigen Waſſerra des nicht in Maſſe, d. i. nut allen ſeinen Theilen, die ge— gen dieſe anlaufen, zugleich, ſondern nur nach einander. Wenn alſo hier die Quantitat der Materie, die, mit einer gewiſfen Geſchwindigkeit bewegt, die bewegende Kraft, hat, beſtimmt werden ſoll, ſo muß man allererſt den Waſſer korper, d. i. diejenige Quantitat der Materie, die, wenn ſie in Maſſe mit einer gewiſſen Geſchwindigkeit wirkt (mit ihrer Schwere), dieſelbe Wirkung hervorbringen kann, ſu— chen. Daher verſteht man auch gewohnlich unter dem Worte Maſſe die Quantitat der Materie eines feſten Kor pers Gas Gefaß, darin ein Fluſſiges eingeſchloſſen iſt, vertritt auch die Stelle der Feſtigkeit deſſelben). Was end— lich den Lehrſatz mit dem augehangten Zuſatz zuſammen be— trifft, ſo liegt darin etwas Befreindliches: daßß, nach dem erſteren, die Quantitat der Materie durch die Quantitat der Bewegung mit gegebener Geſchwindigkeit, nach dem zweyten aber wiederum die Quantitat der Bewegung (eines Korpers; denn die eines Puncts beſteht blos aus dem Gra— de der Geſchwindigkeit) bey derſelben Geſchwindigkeit durch die Quantitat der bewegten Materie geſchatzt werden muiſſe, welches im Cirkel herum zu gehen und weder von einem noch Dem anderen einen beſtinmten Begriff zu verſprechen ſcheint. Allein dieſer vermeynte Cirkel wurde es wirklich ſeyn, wenn er eine wechſelſeitige Ableitung zweyer identiſchen Begriffe von einander ware. Nun aber euthalt er nur einerſeits die Erklarung eines Begriffs, andererſeits die der Anwendung deſſelben auf Erfahrung. Die Quantttat des Beweglichen im Raume iſt die Quantitat der Materie; aber diefe Quan— titat der Materie (die Menge des Beweglichen) beweiſet F5 ſich 90 ſich in der Erfahrung nur allein durch die Quantitat der Bewegung bey gleicher Geſchwindigkeit (z. B. durchs Gleichgewicht). Noch iſt zu merken, daß die Quantitat der Materie die Quautitat der Subſtanz im Beweglichen ſey, folglich nicht die Große einer gewiſſen Qualttat derſelben (der Zu— ruckſiobung, oder Anziehung, die in der Dynamik angefuhrt werden), und daß das Quantum der Subſtanz hier nichts anderes, als die bloße Menge des Beweguichen bedeute, welches die Materie ausmacht. Denn nur dieſe Menge des Bewegten kann bey derſelben Geſchwindigkeit einen Un— terſchied in der Quanuitat der Bewegung geben. Daß aber die bewegende Kraft, die eine Materie in ihrer eigenen Bewegung hat, allein die Quantitat der Subſtanz be— weiſe, beruht auf dem Begriffe der letzteren als dem letzten Subject (as weiter kein Pradicat von einem andern iſt) im Raume, welches eben darum keine andere Große haben kann, als die der Menge des Gleichartigen außerhalb ein— ander. Da nun die eigene Bewegung der Materie ein Pradicat iſt, welches ihr Subject (das Bewegliche) be— ſtimmt, und an einer Materie, als einer Menge des Be weglichen, die Vielheit der bewegten Subjecte (bey gleicher Geſchwindigkeit auf gleiche Art) angiebt, welches bey dyna miſchen Eigeuſchaften, deren Große auch die Große der Wirkung von einem einzigen Subjecte ſeyn kann (z. B. da ein Lufttheilchen mehr oder weniger Elaſticitat haben kaun), nicht der Fall iſt, ſo erhellet daraus, wie die Quantitat der Subſtanz an einer Materie nur mechaniſch, d. i. durch die Quantttat der eigenen Bewegung derſelben, und nicht dy— namiſch, durch die Große der urſprunglich bewegenden Krafte, geſchatzt werden muſſe. Gleichwohl kann die ur ſprungliche Anziehung, als die Urſache der allgemeinen Gravitation, doch ein Maaß der Quantitat der Materie und ihrer Subſtanz abgeben (wie das wirklich in der Ver— gleichung der Materien durch Abwiegen geſchieht), obgleich hier te 91 hier nicht eigene Bewegung der anziehenden Materie, ſont dern ein dynamiſches Maaß, namlich Anziehungskraft, zum Grunde gelegt zu ſeyn ſcheint. Aber, weil bey dieſer Kraft die Wirkung einer Materie mit allen ihren Theilen unmittel— bar, auf alle Theile einer andern, geſchieht, und alfo (bey gleichen Entfernungen) offenbar der Menge der Theile pro— portionirt iſt, dereziehende Korper ſich dadurch auch ſelbſt eine Geſchwindigkeit der eigenen Bewegung ertheilt (durch den Widerſtand des Gezogenen), welche, in gleichen auße— ren Umſtanden, gerade der Menge ſeiner Theile proporttonirt iſt, ſo geſchieht die Schatzung hier, ob zwar nur indirect, doch in der That mechaniſch. Lehrſatz 2. Erſtes Geſetz der Mechanik. Bey allen Veranderungen der korperlichen Natur bleibt die Quantitat der Materie im Ganzen dieſelbe, un— vermehrt und unvermindert. Beweis. (Aus der allgemeinen Metaphyſik wird der Satz zum Grunde gelegt, daß bey allen Veranderungen der Natur keine Subſtanz weder entſtehe noch vergehe, und hier wird nur dargethan, was in der Materie die Subſtanz ſey). Jn jeder Materie iſt das Bewegliche im Raume das letzte Subject aller der Materie inhari— renden Accidenzen, und die Menge dieſes Bewegli— chen außerhalb einander die Quantitat der Subſtanz. Alſo iſt die Große der Materie, der Subſtanz nach, nichts anders, als die Menge der Subſtanzen, daraus ſie beſteht. Es kann alſo die Quantitat der Materie nicht vermehrt oder vermindert werden, als dadurch, daß neue Subſtanz derſelben entſteht oder vergeht. Nun 92 Nun entſteht und vergeht bey allem Wechſel der Ma— terie die Suhſtanz niemals; alſo wird auch die Quan— titat der Materie dadurch weder vermehrt, noch ver— mindert, ſondern bleibt immer dieſelbe und zwar im Ganzen, d. i. ſo, daß ſie irgend in der Welt in der— ſelben Quantitat fortdauert, obgleich dieſe oder jene Materie durch Hinzukunft oder Abſonderung der Theile vermehrt oder vermindert werden kann. Anmerkung. Das Weſentliche, was in dieſem Beweiſe der Sub ſtanz, die nur im Raume und nach Bedingungen deſſelben, folglich als Gegenſtand außerer Sinne moglich iſt, cha— racteriſiret, iſt, daß ihre Große nicht vermehrt oder ver— mindert werden kann, ohne daß Subſtanz entſtehe, oder vergehe, darum, weil alle Große eines blos im Raum moglichen Objects aus Theilen außerhalb einander be— ſtehen muß, dieſe alſo, wenn ſie real (etwas Bewegliches) ſind, nothwendig Subſtanzen ſeyn muſſen. Dagegen kann das, was als Gegenſtand des inneren Sinnes betrachtet wird, als Subſtanz eine Große haben, die nicht aus Theilen außerhalb einander beſteht, deren Theile alſs auch nicht Subſtanzen ſind, deren Entſtehen oder Vergehen folglich auch nicht ein Entſtehen oder Vergehen einer Sub— ſtanz ſeyn darf, deren Vermehrung oder Verminderung da— her, dem Grundſatze von der Beharrlichkeit der Subſtanz unbeſchadet, moglich iſt. So hat namlich das Bewußt ſeyn, mithin die Klarheit der Vorſtellungen meiner Seele, und, derſelben zu Folge, auch das Vermogen des Bewußt ſeyns, die Apperception, mit dieſem aber ſelbſt die Sub— ſtanz der Seele einen Grad, der großer oder kleiner werden kann, ohne daß irgend eine Subſtanz zu dieſem Behuf ent— ſtehen oder vergehen durfte. Weil aber, bey allmaliger Verminderung dieſes Vermogens der Apperception, endlich ein —SJ 93 J ein ganzliches Verſchwinden derſelben erfolgen mußte, ſo wurde doch ſelbſt die Subſtanz der Seele einem allmaligen Vergehen unterworfen ſeyn, ob ſie ſchon einfacher Natur ware, weil dieſes Verſchwinden ihrer Grundktaft nicht durch Zertheiluung (Abſonderung der Subſtanz von einem Zuſam— mengeſetzten), ſondern gierchſam durch Erloſchen, und auch dieſes nicht in einem Augenblicte, ſondern duich allinalige Nachlaſſung des Grades derjelben, es ſey aus welcher Ur— ſuche es wolle, erfolgen konnte. Das Jch, das allgemei ne Correlat der Apperception und ſelbſt vlos ein Gedanke, bezeichnet, als ein bioßes Vorwort, ein Ding von unbe— ſtunmter Bedeutung, namlich das Subjzect aller Pradicate; ohne irgend eine Bedingung, die djeſe Voirſtellung des GSobjectsẽ von dem eines Etwas ujberhaupi unterſchiede, Alſo Subſtanz von der man, was ſie ſey, durch diefen Ausdruck keinen Begriff hat. Dagegen det Begriff einer Materie rals Süubſtanz der Begriff des Beweglichen im Raume iſt. Es iſt daher: kein Wunder, wenn von der letzteren die Beharrlichkeit der Subſtanz“bewieſen werden kann;. von der erſteren aber, nicht, weil. bey der Materie ſchon aus ihrem Begriffe, namlich daß ſie. das Bewegliche ſey das nur im Vaume moglich iſt, fließt, daß das, was in ihr Große. hat, eine Vielheit des Realen außer eitian der, 'üiuthin der Subſtanzen, enthalte, und folglich bie Quüanititat derſelben uur durch Zertheilung, welche kein Ver ſchiinden iſt, vermindert werden konne, und das letztere in ihr nach- dem Geſetze. der. Stetigkeit auch, unmoglich ſeyn wurde. Der Gedanke. Jch iſt dagegen gar kein Begriff, ſondern; nur innere Wahrnehmung, aus ihm kann alſo auch gar nichts, (außer der ganzliche Unterſchied eines Gegen— ſtandes des inneren Sinnes von dem, was blos als Ge— genſtand außerer Sinne gedacht wird) folglich auch nicht die Beharrlichteit der Seele, als Subftanz, gefolgert werden. e udo 2 Lehr— 94 Lehrſatz 3. Zweytes Geſetz der Mechanik. Alle Veranderung der Materie hat eine außere Ur— ſache. (Ein jeder Korper beharrt in ſeinem Zu ſtande der Ruhe oder Bewegung, in derſelben Richtung und mit derſelben Geſchwindigkeit, wenn er nicht durch eine außere Urſache genothigt wird, dieſen Zuſtand zu verlaſſen.) 1 Beweis. (Aus der allgemeinen Metaphyſik wird der Satz zum Grunde gelegt, daß alle Veranderung eine Ur— ſache habe; hier ſoll von der Materie nur bewieſen wer den, daß ihre Veranderung jederzeit eine außere Ur ſache haben muſſe). Die Materie, als bloßer Gegen ſtand außerer Sinne, hat keine andere Beſtimmungen, ais die der außeren Verhaltniſſe im Raume, und. er: leidet alſo auch keine Veranderungen, als durch Be— wegung. Jn Anſehung dieſer, als Wechſels einer Bewegung mit einer andern, oder derſelben mit der Ruhe, und umgekehrt, muß eine Urſache derſelben angetroffen werden (nach Princ. der. Metaph.). Diefe Urſache aber kann nicht, innerlich ſeyn, denn die Ma— terie hat keine ſchlechthin innere Beſtimmungen und Beſtimmungsgrunde. Allſo iſt alle Veranderung einer Materie auf außere Urſache gegrundet (d. i.? ein Kor- per beharret, u. ſ. w.) Anmettuns. Dieſes mechaniſche Geſetz muß allein das Geſetz der Tragheit (ex inertiae) genanut werden, das Geſetz der riner epν 95 einer jeden Wirkung entgegengeſetzten gleichen Gegenwirkung kann dieſen Namen nicht fuhren. Deann dieſes ſagt, was die Materie thut, jenes aber nur, was ſie nicht thut, wel— ches dem Ausdrucle der Tragheit beſſer angemeſſen iſt. Die Tragheit der Materie iſt und bedeutet nichts anders, als ihre Lebloſigkeit, als Materie an ſich ſeibſt. Leben heißt das Vermogen einer Subſtanz, ſich aus einem inneren Princip zum Handeln, einer endlichen Subſtauz ſich zur Veranoerung, und einer materiellen Subſtanz ſich zur Bewegung oder Ruhe, als Veranderung ihres Zuſtandes, zu beſtimmen. Nunm kennen wir kein anderes inneres Prin— cipreiner Subſtanz, ihren. Zuſtand zu verandern, als das Beẽgehren, und uberhaupt keine andere innere Thatigkeit, als Denken, mit dein,“ was davon abhangt, Gefüuhl der Luſt oder Unluſt und Begierde oder Willen. Dieſe Beſtinimungsgrunde aber und Handlungen gehoren gar nicht zu den Vorſtellungen außerer Sinne und alſo auch nicht zu den Beſtimmungen der Materie als Materie. Aliſo iſt alle Materie als ſolche leblos. Das ſagt der Gatz der Trag heit, uſid nichts meht. Wenn wir die Urſache irgend einer Veranderung der Mterie im Leben ſüchen, ſo wetden wir es auch .ſo ſort in einer onderen, von der Materie verſchie— denen, vb zwar init ihr verbundenen Subſtanz zu ſuchen Puben:. Denn in der Naturkenntniß iſt es nothig, zuvor bie Geſetze der Materie uls einer ſolchen zu kennen und ſie von vem Beytritte aller anderen wirkenden Urſachen zu lau— terhz ehe man ſie damit verknuüpft, um wohl zu uuterſchei— den, was; und wie jede: derſelben fur ſich allein wirke. Auf dem Geſetze der Tragheit (neben dem der Beharrlichkeit der Subſianz) beruht die Moglichkeit etner eigentlichen Na turwiſſenſchaft ganz und gar. Das Gegentheil des erſtern, und daher auch der Tod aller Naturphiloſophie, ware der Hilozoiſm. Aus eben demſelben Begriffe der Tragbheit, uls btoßer Lebloſigkeit, fließt von ſelbſt, daß ſie nicht ein poſitives Beſtreben feinen Zuſtand zu erhalten bedeute. Nut debende Weſen werden in dieſem letzteren Verſtande trag gee 96 genannt, weil ſie eine Vorſtellung von einem anderon Zu ſtande haben, den ſie verabſcheuen, und ihre Kraft dage— gen anſtrengen. Lehrſatz 4. Drittes mechaniſches Geſetz. Jn aller Mittheilung der Bewegung ſind Wirkung und Gegenwirkung einander jederzeit gleich. Beweis. Qlus der allgemeinei. Metaphyſik muß der Sutz entlehnt werden, daß alle außere Wirkung in der Welt Wechſelwirkuug ſey. Hier ſoll, um in den Schranken der Mechanik zu bleiben, nur gezeigt wer den, daß dieſe Wechſelwirkung (actio mutua) zugkeich Gegenwirkung (reactio) ſey ʒrallrin ich kann;hne der Vollſtandigkeit der Einſicht Abbruch zu thunz:jenb metaphyſiſche Geſetz? det Gemeinſchaft hier vvth nicht gänz weglaſſen). Alle thatige Verhaltniſſe ver Ma terien im Raume und alle Veranbtrungen dieſer Per hältniſſe, ſo. fern! ſie. Urſachen von gewiſſen Wirkjnr gen ſeyn konnen, muſſen jederzeit als wechſelſeitig ·vort geſtellt werden, d. i. weil alle Veranderung derfelbeu Bewegung iſt, ſo kann keine Bewegung eines Kor pers in Beziehung auf einen. abſolut ruhigen,/ ber dadurch auch in Bewegung geſetzt werden ſoll, gedacht werden, vielmehr inuß dieſer nür als relätih?rühig in Anſehung des Raums, quf ben nian ihn bejitht. zuſammt dieſem Raume aber in eutgegengeſetzier Rich tung als mit. eben derſelben, Quautitut. der Bewegung im abſoluten Raume bewmegt. moegeſtelit vrden;:. as ber Bewegte in eben demſelben gegen ihn hat. Denu die νν 97 die Veranderung des Verhaltniſſes (mithin die Bewe— gung) iſt zwiſchen beyden durchaus wechſelſeitig; ſo viel der eine Korper jedem Theile des anderen naher kommt, ſo viel nahert ſich der andere jedem Theil des erſteren, und, weil es hier nicht auf den empiriſchen Raum, der beyde Korper umgiebt, ſondern nur auf die Linie, die zwiſchen ihnen liegt, ankommt, (indem dieſe Kor— per lediglich in Relation auf einander, nach dem Ein-s fluſſe, den die Bewegung des einen auf die Verande— rung des Zuſtandes des anderen, mit Abſtraction von aller Relation zum empiriſchen Raume, haben kann, betrachtet werden,) ſo wird ihre Bewegung als blos inz abſoluten Raume beſtimmbar betrachtet, in welchem jeder der beyden Korper an der Bewegung, die dem einen im relativen Raume beygelegt wird, gleichen An— theil haben muß, indem kein Grund da iſt, einem von beyden mehr davon, als dem anderen, beyzulegen. Auf dieſem Fuß wird die Bewegung eines Korpers A. gegen einen anderen ruhigen B, in Anſehung deſſen er dadurch bewegend ſeyn kann, auf den abſoluten Raum reducirt, d. i, als Verhaltniß wirkender Urſachen bloß auf einander bezogen, ſo betrachtet, wie beyde an der Bewegung, welche in der Erſcheinung dem Korper A allein beygelegt wird, gleichen Antheil haben, welches nicht anders geſchehen kann, als ſo, daß die Geſchwin— digkeit, die im relativen Raume blos dem Korper A beygelegt wird, unter/A und h in umgekehrtem Ver— haltniß der Maſſen, dem A allein die ſeinige im abſo luten Raume, dem P dagegen zuſammt dem relati ven Raume, worin er ruht, in entgegengeſetzter Rich- tung ausgetheilt werde, wodurch dieſelbe Erfcheinung der Bewegung vollkonnmen beybehalten, die Wirkung G atpet 98 aber in der Gemeinſchaft beyder Korper auf folgende Art conſtruirt wird. v 53 O Es ſey ein Korper A mit einer Geſchwindigkeit AB in Anſehung des relativen Raumes gegen den Korper B, der in Auſehung eben deſſelben Raums ruhig iſt, im Anlaufe. Man theile die Geſchwindigkeit Abß in zwey Theile, Ac und ße, die ſich umgekehrt wie die Maſſen Z und A gegen einander verhalten, und ſtlle ſich Amit der Geſchwindigkeit Ac im abſoluten Rau— me, B aber mit der Geſchwindigkeit Be in entgegenge— ſettter Richtung zuſammt dem relativen Raume be— wegt vor: ſo ſind beyde Bewegungen einander entge— gengeſetzt und gleich, und, da ſie einander wechſelſeitig aufheben, ſo verſetzen ſich beyde Korper beziehungs— weiſe auf einander, d. i. im abſoluten Raume, in Ruhe. Nun war aber B mit der Geſchwindigkeit Bc in der Richtung BA, die der des Korpers A. namlich AB, gerade entgegengeſetzt iſt, zuſammt dem relati— ven Raume in Bewegung. Wenn alſo die Bewe— gung des Korpers B durch den Stoß aufgehoben wird, ſo wird darum doch die Bewegung des relativen Raums nicht aufgehoben. Alſo bewegt ſich nach dem Stoße der relative Raum in Anſehung beyder Kor— per A und B, (die nunmehr im abſoluten Raume ru— hen,) in der Richtung BA mit der Geſchwindigkeit Be, oder, welches einerley iſt, beyde Korper bewegen ſich nach dem Stoße mit gleicher Geſchwindigkeit bBd Bec in 99 in der Richtung des Stoßenden Ab. Nun iſt aber, nach dem vorigen, die Quantitat der Bewegung des Korpers B in der Richtung und mit der Geſchwindig— keit Bc, mithin auch die in der Richtung Bd mit der— ſelben Geſchwindigkeit, der Quantitat der Bewegung des Korpers A mit der Geſchwindigkeit und in der Richtung Ac gleich: folglich iſt die Wirkung, d. i. die Bewegung Ed, die der Korper B durch den Stoß im relativen Raume erhalt, und alſo auch die Handlung des Korpers Anmit der Geſchwindigkeit Ac der Gegen— wirkung Bo jederzeit gleich. Da eben daſſelbe Geſetz (wie die mathematiſche Mechanik lehrt) keine Abande— rung erleidet, wenn, anſtatt des Stoßes auf einen ruhigen, ein Stoß deſſelben Korpers auf einen gleich— falls bewegten Korper angenommen wird, imgleichen die Mittheilung der Bewegung durch den Stoß von der durch den Zug nur in der Richtung, nach welcher die Materien einander in ihren Bewegungen widerſte— hen, unterſchieden iſt: ſo folgt, daß in aller Mitthei— lung der Bewegung Wirkung und Gegenwirkung einander jederzeit gleich ſeyn (daß jeder Stoß nur ver— mittelſt eines gleichen Gegenſtoßes, jeder Druck ver— mittelſt eines gleichen Gegendrucks, imgleichen jeder Zug nur durch einen gleichen Gegenzug die Bewegung eines Korpers dem andern mittheilen konne.) G 2 Zu— 2) Jn der Phoronomie, da die Bewegung eines Korpers blos in Anſehung des Raums, als Veranderung der Relation in demſelben, betrachtet wurde, war es ganz gleichgultig, ob ich den Korper im Raume, oder, an ſtatt deſſen, dem relativen Raume eine gleiche, aber entt toe Zuſatz 1. Hieraus folgt das, fur die allgemeine Mechanik nicht unwichtige, Naturgeſetz: daß ein jeder Korper, wie groß auch ſeine Maſſe ſey, durch den Stoß eines ieden anderen, wie klein auch ſeine Maſſe oder Ge— ſchwindigkeit ſeyn mag, beweglich ſeyn muſſe. Denn der Bewegung von A in der Richtung Ab correſpon— dirt nothwendiger Weiſe eine entgegengeſetzte gleiche Bewegung von B in der Richtung BA. Bende Be— wegun entgegengeſetzte Bewegung zugeſtehen wollte: beydes gab vollig einerley Erſcheinung. Die Quantitat der Bewegung des Raums war blos die Geſchwindigkejt, und daher die des Korpers gleichfalls nichts, als ſeine Geſchwindigkeit (weswegen er als ein bloßer beweglicher Punct betrachtet werden konnte). Jn der Mechenik aber, da ein Korper in Bewegung gegen einen anderen betrachtet wird, gegen den er durch ſeine Bewegung ein Caußalverhaltniß hat, namlich das, ihn ſelbft zu bewegen, indem es entweder bey ſeiner Annaherung durch die Kraft der Undurchdringlichkeit, oder ſeiner Entfernung durch die Kraft der Anziehung, mit ihm in Gemeinſchaft kommt, da iſt es nicht mehr gleichgultig, ob ich einem dieſer Korper, ober dem Raume eine ent gegengeſetzte Bewegung zueignen will. Denn nunmehrv kommt ein anderer Begriff der Quantitat der Bewegung ins Spiel, namlich nicht derjenigen, die blos in An— ſehung des Raumes gedacht wird, und allein in der Get ſchwindigkeit beſteht, ſondern derjenigen, wobey zualeich die Quantitat der Subſtanz (als bewegende Urſache) in Anſchlag gebracht werden muß, und es iſt hier nicht mehr beliebig, ſondern nothwendig, jeden der bey— den Korper als bewegt anzunehmen, und zwar mit glei— cher ror wegungen heben durch den Stoß einander im abſolu— ten Raume auf. Dadurch aber erhalten berde Kor— per eine Geſchwindigkeit bd—Be in der Richtung des Stoßenden, folglich iſt der Korper ßZ fur jede noch fo kleine Kraft des Anſtoßes beweglich. Zuſatz 2. Dies iſt alſo das mechaniſche Geſetz der Gleich— heit der Wirkung und Gegenwirkung, welches darauf G 3 be— cher Quantitat der Bewegung in entgegengeſetzter Rich tung; wenn aber der eine relative in Anſehung des Rau mes in Ruhe iſt, ihm die erfoderliche Bewegung zu— ſammt dem Raume beyzulegen. Denn einer kann auf den anderen durch ſeine eigene Bewegung nicht wir— ken, als entweder bey der Annaherung vermittelſt der Zuruckſtoßungskraſt, oder bey der Entfſernung vermittelſt der Anziehung. Da beyde Krafte nun jederzeit beyder— ſeitig in entgegengeſetzten Richtungen und gleich wirken, ſo kann kein Korper vermittelſt ihrer durch ſeine Bewegung auf einen anderen wirken, ohne gerade ſo viel, als der an dere mit gleicher Quantitat der Bewegung enigegenwirkt. Alſo kann kein Korper einem ſchlechthinzruhigen durch ſeine Bewegung Bewegung ertheilen, ſondern dieſer muß gerade mit derſelben Quantitat der Bewegung (zuſammt dem Raume) in entgegengeſetzter Nichtung bewegt ſeyn, als diejenige iſt, die er durch die Bewe— gung des erſteren und in der Richtung deſſelben erhalten ſoll. Der Leſer wird leicht inne werden, daß, nuerachtet des etwas Ungewohnlichen, welches dieſe Vorſtellungsart der Mittheilung der Bewegung an ſich hat, ſie ſich dennoch in das helleſte Licht ſtellen laffe, wenn man die Weitlauftigkeit der Erlanterung nicht ſcheuet. 102 —SJ beruht: daß keine Mittheilung der Bewegung Statt finde, außer ſofera eine Gemeinſchaft dieſer Bewe— gungen vorausgeſetzt wird, daß aljo kein Korper einen anderen ſtoße, der in Auſehung ſeiner ruhig iſt, ſon— dern, iſt dieſer es in Auſehung des Raums, nur ſo ſern er zuſammt dieſem Rauine in gleichem Maaße, aber in entgegengeſetzter Richtung bewegt, mit der Be— wegung, die alsdenn dem erſteren zu ſeinem relativen Antheil fallt, zuſammen, allererſt die Quantitat der Bewegung gebe, die wir dem erſten im abſoluten Rau— me beylegen wurden. Denn keine Bewegung, die in Anſehung eines anderen Korpers bewegend ſeyn ſoll, kann abſolut ſeyn: iſt ſie aber relativ in Anſehung des letzteren, ſo giebts keine Relation im Raume, die nicht wechſelſeitig und gleich ſeny. Es giebt aber noch ein anderes, namlich ein dynamiſches Geſetz der Gleichheit der Wirkung und Gegenwirkung der Ma— terien, nicht ſo fern eine der anderen ihre Bewegung mittheilt, ſondern dieſer urſprunglich ertheilt und durch deren Widerſtreben zugleich in ſich hervorbringt. Dieſe laßt ſich auf ahnliche Art leicht darthun. Denn, wenn die Materie A die Materie B zieht, ſo nothigt ſie dieſe, ſich ihr zu nahern, oder, welches einerley iſt, jene widerſteht der Kraft, womit dieſe ſich zu ent— fernen trachten mochte. Weil es aber einerley iſt, ob B ſich von A, oder A von B entferne: ſo iſt dieſer Widerſtand zugleich ein Widerſtand, den der Korper B gegen A ausubt, ſo fern er ſich von ihm zu entfernen trachten mochte, mithin ſind Zug und Gegenzug einan— der gleich. Eben ſo, wenn A die Materie B zuruck— ſtoßt, ſo widerſteht A der Annaherung von BH. Da es aber einerley iſt, ob ſich h dem A, oder A dem B nahere, 103 nahere, ſo widerſteht B auch oben ſo viel der Annahe— rung von A; Druck und Gegendruck ſind alſo auch jederzeit einander gleich. Anmerkung 1. Dies iſt alſo die Conſtruction, der Mittheilung der Bewegung, welche zugleich das Geſetz der Gleichheit der Wirkung und Gegenwirtkung, als nothwendige Bedingung derſelben, bey ſich fuhret, welches Newton ſich gar nicht getrauete a priori zu beweiſen, ſondern ſich deshalb auf Erfahrung berief, welchem zu Gefallen andere eine be— ſondere Kraft der Materie, unter dem von Keplern zuerſt angefuhrten Namen der Tragheitskraft (ris inertiae), in der Naturwiſſenſchaft einfuhreten, und alſo im Grunde es auch von Erfahrung ableiteten, endlich noch andere in dem Begriffe etner bloßen Mittheilung der Bewegung ſetzten, weiche ſie, wie einen allmaligen Uebergang der Bewegung des einen Korpers in den andern anſahen, wobey der bewe— gende gerade ſo viel einbußen muſſe, als er dem bewegten ertheilt, bis er dem letzteren keine weiter eindruckt, (wenn er namlich mit dieſem ſchon bis zur Gleichheit der Geſchwin— digkeit in derſelben Richtung gekommen iſt,) H wodurch ſie G 4 im Die Gleichheit der Wirkung mit der in dieſem Falle falſchlich ſogenannten Gegenwirknng kommt eben ſo wohl heraus, wenn man bey der Hypotheſe der Transfu— ſion der Bewegungen aus einem Korper in den ande— ren, den bewegten Korper A dem ruhigen in einem Aut genblicke ſeine ganze Bewegung uberliefern laßt, ſo, daß er nach dem Stoße ſelber ruhe, weicher Fall unausbleib— lich war, ſo bald man beyde Korper als abſoluto-hart (welche Eigenſchaft von der Elaſticitat unterſchieden wer— den muß) dachte. Da dieſes Beweaungsgeſetz aber weder mit der Erfahrung, noch mit ſich ſelbſt in der An— went 104 im Srunde alle Gegenwirkung aufhoben, d. i. alle wirklich entgegenwirkende Kraft des geſtoßenen gegen den ſtoßenden (der etwa vermogend ware, eine Spritigfeder zu ſpannen), und außerdem, daß ſie das nicht beweiſen, was in dem ge— nannten Geſetze eigentlich gemeynt iſt, die Mittheilung der Bewegung ſelbſt, ihrer Moelichkeit nach, gar nicht erklat— ten. Denn der Name vom Uebergang der Bewegung von einem Korper auf den andern erklart nichts, und, wenn man ihn nicht etwa (dem Grundſatze accidentia non migrant e ſubſtantiis in ſubſtantias zuwide.) buchſtablich nehmen will, als wenn Bewegung von einem Korpet in einen an— deren, wie Waſſer aus einem Giaſe in das andere, gegoſſen wurde, ſo iſt es hier eben die Aufgabe, wie dieſe Moglich— Zeit begreiflich zu machen ſey, deren Erklarung nun gerade auf wendung zuſammenſtimmen wollte, ſo wußte man ſich nicht anders zu helfen, als dadurch, daß man die Exi— ſtenz abſolut, harter Korper leugnete, welches ſo viel Hieß, als die Zufalligkeit dieſes Geſetzes zugeſtehen, in dem es auf der beſondeten Qualitat der Materie beruhen ſollte, die einander bewegen. Jn unſeter Darſtellung dieſes Geſetzes iſt es dagegen ganz einerley, ob man die Korper, die einandet ſtoßen, abfolut-hart oder nicht denken will. Wie aber die Transfuſioniſten der Bewegung die. Bewegung elaſt iſcher Korper durch den Stoß nach ihrer Art erklaren wollen, iſt mir ganz unbegreiflich. Denn da iſt klar, daß der ruhende Kort per nicht als blos ruhend Bewegung bekomme, die der Stoßende einbußt, ſondern, daß er im Stoße wirk—e liche Kraft in entgegengeſetzter Richtung gegen den Sto— ßenden ausube, um gleichſam die Feder zwiſchen beyt den zuſammenzudrücken, welches von ſeiner Seite eben ſo wohl wirkliche Bewegung (aber in entgegengeſetzter Richtung) erfodert, als der bewegende Korper ſeinert ſeits dazu nothig hat: 105 auf demſelben Grunde beruht, woraus das Gecctz der— Gleichheit der Wirkung und Gegenwirkung abgeleitet wird. Man kann ſich gar nicht denken, wie die Bewequng etnes Korpers Anmit der Bewegung eines andern B nothwendig verbunden ſeyn muſſe, als ſo, daß man ſich Krafte an bey— den denkt, die ihuen (dynamiſch) vor aller Bewegung zukom— men, z. B. Zuruckſtoßung, und nun beweiſen kann, daf die Bewegung des Korpers A durch Annaherung gegen B, mit der Annaherung von B gegen A, und, wenn B als tuhig angeſchen wird, mit der Bewegung deſſelben, zuſammt ſeinem Raume gegen Annothwendig verbunden ſeh, ſo fern ein Korper mit ihren (urſprünglich) bewegenden Krafren blos relativ auf einander in Bewegung betrachtet werden. Dieſes letztere kann vollig a priori dadurch eingeſehen wer— den, daß, es mag nun der Korper Bin Anſehung des em— piriſch kennbaren Raumes ruhig, oder bewegt ſeyn, er doch in Anſehung des Korpers A nothwendig als bewegt, und zwar en entgegengeſetzter Richtung als bewegt, angeſehen werden muſſe; weil ſonſt kein Einfluß deſſelben auf die re— pulſive Kraft beyder Statt finden wurde, ohne welchen ganz und gar keine mechauiſche Wirkung der Matecrien auf einan— der, d. i. keine Mittheilung der Bewegung durch den Etoß, Anmerkung 2. Die Benennung der Tragheitskraft (vis inertiae) muß alſo, unerachtet des beruhmten Namens ihres Urhe— bers, aus der Naturwiſſenſchaft ganzlich weggeſchafft wer— den, nicht allein weil ſie einen Widerſpruch im Ausdruckte ſelbſt bey ſich fuhrt, oder auch derwegen, weil das Geſeü der Tragheit Eebloſigkeit) dadurch leicht mit dem Geſetze der Gegenwirkung in jeder mitgetheilten Berdegung verwrchſelt werden konnte, ſondern vornemlich, weil dadurch die irrige Vorſtellung derer, die der mechaniſchen Geſetze nicht recht kundig ſind, erhalten und beſtarkt wird, nach melcher die Gegenwirkung der Korper, von der unter dem Namen der 35 Trag 106 Tragheitskraft die Rede iſt, darin beſtehe, daß die Bewe— gung dadurch du der Welt aufgezehrt, vermindert oder ver— tuigt, nicht aber die blore Peittheilung derſelben dadurch be— wirkt werde, indem namlich der bewegende Korper einen Theil ſeiner Bewegung blos dazu aufwenden müßte, um die Tragheit des ruhenden zu uberwinden (welches denn reiner Verluſt ware), mit dem ubrigen Theile allein konne er den letzteren in Bewegung ſetzen; bliebe ihm aber nichts ubrig, ſo wurde er durch ſeinen Stoß den letzteren, ſeiner großen Maſſe wegen, gar nicht in Bewegung bringen. Einer Be— wegung kann nichts widerſtehen, als eutgegengeſetzte Bewe— gung eines anderen, keinesweges aber deſſen Ruhe. Hier iſt alſo nicht Tragheit der Materie, d. i. bloßes Unvermogen ſich von ſelbſt zu bewegen, die Urſache eines Widerſtandes. Eine beſondere ganz eigenthumliche Kraft, blos un zu wi— derſtehen, ohne einen Korper bewegen zu konnen, ware un— ter dem Namen einer Tragheitskraft ein Wort ohne alle Be— deutung. Man konnte alſo die drey Geſetze der allgemeinen Mechanik ſchicklicher ſo benennen: das Geſetz der Selbſt ſtandigkeit, der Tragheit und der Gegenwirkung der gnaterien dex Subſiſtentiae, Inertiae et Antagoniſmi) bey allen ihren Veranderungen derſelben. Daß dieſe, mithin die geſammten Lehrſatze gegenwartiger Wiſſenſchaft, den Categorten der Subſtanz, der Caußalitat und der Gemeinſchaft, ſo fern dieſe Begriffe auf, Materie ange— wandt werden, genau antworten, bedarf keiner weiteren Erorterung. Allgeme'ine Anmerkung iur Mechanif. Vie Mittheilung der Bewegung geſchieht nur vermittelſt 8 ſolcher bewegenden Krafte, die ciner Materie auch in Ruhe beywohnen (Undurchdringlichkeit und Anziehung). Die Wir 107 Wirkung einer bewegenden Kraft auf einen Korper in einem Augenblicke iſt die Solicltation deſſelben, die gewirkte Geſchwindigkeit des letzteren durch die Solicitation, ſo fern ſie in gleichem Verhaltniß mit der Zeit wachſjen kann, iſt das Moment der Acceleration. (Das Moment der Acce— leration muß alſo uur eine unendlich kleine Geſchwindigkeit enthalten, weil, ſonſt der Korper durch daſſelbe in einer ge— gebenen Zeit eine unendliche Geſchwindigkeit erlangen wurde, welche unmoglich iſt. Uebrigens beruht die Moglichkeit der Beſchleunigung uberhaupt, durch ein fortwahrendes No— ment derſelben, auf dem Geſetze der Tragheit). Die Soli— citation der Materie durch expanſive Kraft (z. B. einer zu— ſammengedrückten Luft, die ein Gewichte tragt) geſchieht jederzeit nnt einer endlichen Geſchwindigkeit, die Geſchwin— digkeit aber, die dadurch einem anderen Korper eingedruckt (oder entzogen) wird, kann nur unendlich klein ſeyn; denn jene iſt nur eine Flachenkraft, oder, welches einerley iſt, die Bewegung eines unendlich kleinen Quantums von Materie, die folglich mit endlicher Geſchwindigkeit geſchehen muß, um, der Bewegung eines Korpers von endlicher Maſſe mit unend lich kleiner Geſchwindigkeit (einem Gewichte) gleich zu ſeyn. Dagegen iſt die Anziehung eine durchdringende Kraft und als mit einer ſolchen ubt ein endliches Quantum der Materie auf ein gleichfalls endliches Quantum einer andern bewegen— de Kraft aus. Die Soltcitation der Anztehung muß alſo unendlich klein ſeyn, weil ſie dem Monteut der Acceleration (welches jederzeit unendlich klein ſeyn muß) gleich iſt, wel— ches bey der Zuruckſtoßung, da ein unendlich kleiner Theil der Materie einem endlichen ein Moment eindrucken ſoll, der Fall nicht iſt. Es laßt ſich keine Anziebung mit einer endli— chen Geſchwindigkeit denken, ohne daß die Materie durch ihre eigene Anziehungskraft ſich ſelbſt durchdringen mußte. Denn die Anziehung, welche eine endliche Quantitat Mate— rie auf eine endliche mit einer endlichen Geſchwindigkeit ausubt, muß eine jede endliche Geſchwindigkeit, womit die Mattrie durch ihre Undurchdringlichkeit, aber nur mit einem un— ros ν unendlich kleinen Theil der Quantitat ihrer Materie entgegen— wirkt, in allen Puncten der Zuſammenorückung uberlegen ſeyn. Wenn die Auziehung nur eine Flächenkraft iſt, wie man ſich den Zuſammenhang denkt, ſo wurde das Gegen— thell von dieſem erfolgen. Allein es iſt unmoglich, ihn ſo zu denken, wenu er wahre Anziehung (und nicht blos außere Zuſammendruckung) ſeyn ſoll. Ein abſolut-harter Korper wurde derjenige ſeyn, deſ— ſen Theile einander ſo ſtark zogen, daß ſie durch kein Ge— wicht getrennt, noch in ihrer Lage gegen einander verun— dert werden konnten. Weil nun die Theile der Materie eines ſolchen Korpers ſich mit einem Moment der Accelera— tion ziehen mußten, welches gegen das der Schwere unend— lich, der Maſſe aber, welche dadurch getrieben wird, end— lich ſeyn wurde, ſo mußte der Widerſtand durch Undurch— dringlichkeit, als erpanſive Kraft, da er jederzeit mit einer unendlich- kleinen Quantitat der Materie geſchteht, mit mehr als endlicher Geſchwindigkeit der Solicitation geſche— hen, d. i. die Materie wurde ſich mit unendlicher Geſchwin— digkeit auszudehnen trachten, welches unmoglich iſt. Alſo iſt ein abſolut-harter Korper, d. i. ein ſolcher, der einem mit endlicher Geſchwindigkeit bewegten Korper im Stoße einen Widerſtand, der der ganzen Kraft deſſelben gleich ware, in einem Augenblick entgegenſetzte, unmoglich. Folglich leiſtet eine Materie durch ihte Undurchdiinglichkeit oder Zuſammenhang, gegen die Kraft eines Korpers in end licher Bewegung, in einem Augeublicke nur unendlich klei— Inen Widerſtand. Hieraus folgt nun das mechaniſche Ge— ſetz der Stetigkeit ex continui mechanica), namlich: an keinem Korper wird der Zuſtand der Ruhe, oder der Be— wegung, und an dieſer, der Geſchwindigkeit oder der Rich— rung, durch den Stoß in einem Augenblicke verandert, ſon— dern nur in einer gewiſſen Zeit, durch eine unendliche Reihe von Zwiſchenzuſtanden, deren Unterſchied von einander klei— ner iſt, als der des erſten und letzten. Ein bewegter Kor— per, 109 per, der auf eine Materie ſtoßt, wird alſo durch deren Widerſtand nicht auf einmal, ſondern nur durch continuir— liche Retardation zur Ruhe, oder der, ſo in Ruhe war, nur durch continuirrliche Acceleration in Beweguna, eder aus einem Grade Geſchwindigkeit tir einen andern nur nach derſelben Regel verſetzt; ungleichen wird die Richtung ſet— ner Bewegung in etne ſolche, die mit jener einen Wincel macht, nicht anders als vermittelſt aller moglichen dazwi— ſchen liegenden Richtungen, d. i. vermirtelſt der Bewegung in einer krummen Linie, verandert (welches Geſetz aus einem ahnlichen Grunde auch auf die Veranderung des Zu— ſtundes eines Korpers durch Anziehung erweitert werden kann). Dieſe lex continni grundet ſich auf das Geſetz der Tragheit der Malerie, da hingegen das metaphyſijche Geſetz der Stetigleit. auf alle Veranderung (innere ſo wohl als außere) uberhaupt ausgedehnt ſeyn mußte, und alſo auf den bloßen Begriff einer Veranderung uberhaupt, als Große, und der Erzeugung derſelben, (die nothwendig in einer gewiſſen Zeit continuirlich, ſo wie die Zeit ſelbſt, vor ginge,) gegrundet ſeyn wurde, hier alſo keinen Platz findet. 110 Viertes Hauptſtuck. Metaphyſiſche Anfangsgrunde der Phanomenologie. Erklarung. cον Wi aterie iſt das Bewegliche, ſo fern es, als ein ſolches, ein Gegenſtand der Erfahrung ſeyn kann. Anmerkung. Bewegung iſt, ſo wie alles, was durch Sinne vot— geſtellt wird, nur als Erſcheinung gegeben. Damit ihre Vorſtellung Erfahrung werde, dazu wird noch erfodert, daß etwas durch den Verſtand gedacht werde, nämlich zu der Art, wie die Borſtellung dem Subjecte inharirt, noch die Beſtinmung eines Objects durch dieſelbe. Alſo wird das Bewegliche, als ein ſolches, ein Gegenſtand der Er— fahrung, wenn ein gewiſſes Object (hier alſo ein materiel— les Ding) in Anſehung des Pradicats der Bewegung als beſtimmt gedacht wird. Nun iſt aber Bewegung Veran— derung der Relation im Raume. Es ſind alſo hier immer zweh Correlata, deren einem in der Erſcheinung erſtlich eben ſo gut wie dem anderen die Veranderung beygelegt, und daſſelbe entweder, oder das andere bewegt genannt wer— den kann, weil beydes gleichgultig iſt, oder zweytens, deren eines in der Erfahrung mit Ausſchließung des ande— ren als bewegt gedacht werden muß, oder drittens, deren bende nothwendig durch Vernunft als zugleich bewegt vor— geſtellt werden muſſen. Jn der Erſcheinung, die nichts als die Relation in der Bewegung (ihrer Veranderung nach) enthalt, iſt nichts von dieſen Beſtimmungen enthalten; wenn aber li 111 aber das Bewegliche, als ein ſolches, namlich ſeiner Be— wegung nach, beſtimmt gedacht werden ſoll, d. i. zum Be— huf einer moglichen Erfahrung, iſt es nothig, die Bedin— gungen anzuzeigen, unter welchen der Gegenſtand (die Ma— terie) auf eine oder andere Art durch das Pradicat der Be— wegung beſtimmt werden muſſe. Hier iſt nicht die Rede von Verwandlung des Scheins in Wahrheit, ſondern der Erſcheinung in Erfahrung; denn beym Scheine iſt der Ver— ſtand mit ſeinen einen Gegenſtand beſtimmenden Urtheilen jederzeit im Spiele, ob zwar er in Gefohr iſt, das Sub— jective fur objectiv zu nehmen; in der Erſcheinung aber iſt gar kein Urtheil des Verſtandes anzutrefſen; welches nicht blos hier, ſondern in der ganzen Philoſophie anzumerken nothig iſt, weil man ſonſt, wenn von Erſcheinungen die Rede iſt, und man nimmt dieſen Ausdruck fur einerley der Bedeutung nach mit dem des Scheins, jederzeit uhel ver— ſtanden wird. Lehrſatz 1. Die geradlinigte Bewegung einer Materie in Anſehung eines empiriſchen Raumes iſt, zum Unterſchiede von der entgegengeſetzten Bewegung des Raums, ein blos mogliches Praodicat. Eben daſſelbe in gar keiner Relation auf eine Materie außer. ihr, d. i. als abſolute Bewe— gung gedacht, iſt unmoglich. Beweis. t Ob ein Korper im relativen Raume bewegt, die— ſer aber ruhig genannt werde, oder, umgekehrt, die— ſer in entgegengeſetzter Richtung gleich geſchwinde be— wegt, dagegen jener ruhig genannt werden ſolle, iſt kein Streit uber das, was dem Gegenſtande, ſondern nur 112 nur ſeinem Verhaltniſſe zum Subject, mithin der Er— ſcheinung und nicht der Erfahrung, zukommt. Denn, ſtellt ſich der Zuſchauer in demſelben Raume als ruhig, ſo heißt ihm der Korper bewegt; ſtellt er ſich (wenig— ſtens in Gedanken) in einem andern und jenen umfaſ— ſenden Raum, in Anſehung deſſen der Korper gleich falls ruhig iſt, ſo heißt jener relative Raum bewegt, Alſo iſt in der Erfahrung (einer Erkenntniß, die das Object fur alle Erſcheinungen gultig beſtimmt,) gar kein Unterſchied zwiſchen der Bewegung des Korpers im relativen Raume, oder der Ruhe des Korpers im abſoluten und der entgegengeſetzten gleichen Bewegung des relativen Raums. Nun iſt die Vorſtellung eines Gegenſtandes durch eines von zweyen Pradicaten, die in Anſehung des Objects gleichgeltend ſind und ſich nur in Anſehung des Subjects und ſeiner Vorſtellungsart von einander unterſcheiden, nicht die Beſtimmung rach einem disjunctiven, ſondern blos die Wahl nach einem alternativen Urtheile, (deren das erſtere von zweyen objectiv entgegengeſetzten Pradicaten eines mit Ausſchließung des Gegentheils, das andere aber von objektiv zwar gleichgeltenden, ſubjectiv aber einander entgegengeſetzten Urtheilen, ohne Ausſchließung des Gegentheils vom Object, alſo durch bloße Wahl eines zur Beſtimmung deſſelben annimmt) das Hheißt: durch den Begriff der Bewegung, als Gegen— ſtandes der Erfahrung, iſt es an ſich unbeſtimmt, mit— hin Von dieſem Unterſchiede der disjunetiven and altornati ven Entgegenſetzung ein Mehreres in dar allgemeinen Anmerkung zu dieſem Hauptſtücke. 113 hin gleichgeltend, ob ein Korper im relativen Raume, oder dieſer in Anſehung jenes als bewegt vorgeſtellt werde. Nun iſt dasjenige, was in Anſehung zweyer einander entgegengeſetzter Pradicate an ſich unbeſtimmt iſt, ſo fern blos moglich. Alſo iſt die geradlinigta Bewegung einer Materie im empiriſchen Raume, zum Unterſchiede von der entgegengeſetzten gleichen Bewe— gung des Raumes, in der Erfahrung ein blos mogli— ches Pradicat; welches das erſte war. Da ferner eine Relation, mithin auch eine Ver anderung derſelben, d. i. Bewegung, nur ſo fern ein Gegenſtand der Erfahrung ſeyn kann, als beyde Cor— relate Gegenſtande der Erfahrung ſind; der reine Raum aber, den man auch, im Gegenſatze gegen den relativen (empiriſchen), den abſoluten Raum nennt, kein Gegenſtand der Erfahrung und uberall nichts iſt: ſo iſt die geradlinigte Bewegung ohne Beziehung auf irgend etwas Empiriſches, d. i. die abſolute Bewegung, ſchlechterdings unmoglich, welches das zweyte war. Anmerkung. Dieſer Lehrſatz beſtimmt die Modalitat der Bewegung in Anſehung der Phoronomie. Lehrſätz 2. Die Kreisbewegung einer Materie iſt, zum Unterſchiede von der entgegengeſetzten Bewegung des Raums, ein wirkliches Pradicat derſelben; dagegen iſt die entgegengeſetzte Bewegung eines relativen Raums, ſtatt der Bewegung des Kor 9H pers 114 ESre pers genommen, keine wirkliche Bewegung des letzteren, ſondern, wenn ſie dafur gehalten wird, ein bloßer Schein. Beweis. Die Kreisbewegung iſt (ſo wie jede krummlinigte) eine continuirliche Veranderung der geradlinigten, und, da dieſe ſelbſt eine continuirliche Veranderung der Rie— lation in Anſehung des außeren Raumes iſt, ſo iſt die Kreisbewegung eine Veranderung, der Veranderung dieſer außeren Verhaltniſſe im Raume, folglich ein continuirliches Entſtehen neuer Bewegungen. Weil nun nach dem Geſetze der Tragheit eine Bewegung, ſo fern ſie entſteht, eine außere Urſache haben muß, J gleichwohl aber der Korper in jedem Puncte dieſes Kreiſes (nach eben demſelben Geſetze) fur ſich in der den Kreis beruhrenden geraden Linie fortzugehen be— ſtrebt iſt, welche Bewegung jener außeren Urſache ent— gegenwirkt, ſo beweiſet jeder Korper in der Kreisbe— wegung durch ſeine Bewegung eine bewegende Kraft. Nun iſt die Beweguna des Raumes, zum Unterſchie— de der Bewegung des Korpers vios phoronomiſch, und hat keine bewegende Kraft. Folgüch iſt das Ur. theil, daß hier entweder der Rorper, oder der Raum, in entgegengeſetzter Richtung bewegt ſey, ein disjuncti— ves Urtheil, durch welches, wenn das eine Glied, namlich die Bewegung des Korpers, geſetzt iſt, das andere, namlich die des Raumes, ausgeſchloſſen wird; alſo iſt die Kreisbewegung eines Korpers, zum Unter— ſchiede von der Bewegung des Raums, wirrkliche Bewegung, folglich die letztere, wenn ſie gleich der Erſchei S 115 Erſcheinung nach mit der erſteren ubereinkommt, den noch im Zuſammenhange aller Erſcheinungen, d. i. der moglichen Erfahrung, dieſer widerſtreitend, alſo nichts als bloßer Schein. Anmerkung. Dieſer Lehrfatz beſtimmt die Modalitat der Bewegung in Anſehung der Dynamik; denn eine Bewegung, die nicht ohne den Einfluß einer continuirlich wirkenden außern bewegenden Kraft Statt fiden kann, beweiſet mittelbar oder unmittelbar, urſprungliche Bewegkraſte der Materie, es ſey der Anziehung oder Zuruckſtohung. Uebrigens kann Newtons Scholium zu den Definttionen, die er ſei— nen Prine. Phil. Nat. Math. vorausgeſetzt hat, gegen das Ende, hieruber nachgeſehen werden, aus welchem erhellet, daß die Kreisbewegung zweher Korper um einen gemein— ſchaftlichen Mittelpunect (mithin auch die Achſendrehung der Erde) ſelbſt im leeren Raume, alſo ohne alle durch Erfah— rung mogliche Vergleichung mit dem außeren Raume, dennoch vermittelſt der Erfahrung konne erkannt werden, daß alſo eine Bewegung, die eine Veranderung der außeren Ver— haltniſſe im Raume iſt, emptiriſch gegeben werden konne, obgleich dieſer Raum ſelbſt nicht empiriſch gegeben uns kein Gegenſtand der Erfahrung iſt, weiches Paradoxron aufge— loſet zu werden verdient. Lehrſatz 3. Jn jeder Bewegung eines Korpers, wo—— durch er in Anſehung eines anderen bewegend iſt, iſt eine entgegengeſetzte gleiche Bewegung des letzteren nothwendig. Ha Be— 116 S3 Beweis. Nach dem dritten Geſetze der Mechanik (Lehrſ. 4.) iſt die Mittheilung der Bewegung der Korper nur durch die Gemeinſchaft ihrer urſprunglich bewegenden Krafte, und dieſe nur durch beyderſeitige entgegenge— ſetzte und gleiche Bewegung moglich. Die Bewegung beyder iſt alſo wirklich. Da aber die Wirklichkeit die— ſer Bewegung nicht (wie im zweyten Lehrſatze) auf dem Einfluſſe außerer Krafte beruht, ſondern aus dem Begriffe der Relation des Bewegten im Raume zu jedem anderen dadurch Beweglichen unmittelbar und unvpermeidlich folgt, ſo iſt die Bewegung des letz teren nothwendig. Anmerkung. Dieſer Lehrſatz beſtimmt die Modalltat der Bewegung in Anſehung der Mechanik. Daß ubrigens dieſe drey Lehrſatze die Bewegung der Materie in Auſehung ihrer Moglichkeit, Wirklichkeit und Nothwendigkeit, mit— xin in Anſehung aller dreyen Categorien der Modalitat beſtimmen, fallt von ſelbſt in die Augen. Allgemeine Anmerkung zjur Phanomenologie. Es zeigen ſich alſo hier drey Begriffe, deren Gebrauch in det allgemeinen Naturwiſſenſchaft unvermeidlich, deren Hgenaue Beſtimmung um deswillen nothwendig, obgleich eben nicht ſo leicht und faßlich iſt, namlich der Begriff der Bewegung im relativen (beweglichen) Raume,— zwey 117 zweytens der Begriff der Bewegung im abſoluten (un— beweglichen) Raume, drittens der Veariff der relati— ven Bewegung uberhaupt, zum Unterſchiede von der ab— ſoluten. Allen wird der Begriff des abſoluten Raunies zum Grunde gelegt. Wie kommen wir aber zu dieſem ſon— derbaren Begriffe, und worauf beruht die Rothwendigkeit feines Gebrauchs? Er kann kein Gegenſtand der Erfahrung ſeyn; denn der Raum ohne Materie iſt kein Objeet der Wahrneh— mung, und dennoch iſt er ein nothwendiger Vernunftbegriff, mithin nichts weiter, als eine bboße Jdee. Denn, damit Bewegung auch nur als Erſcheinung gegeben werden konne, dazu wird eine empiriſche Vorſtellung des Raums, in An— ſelbung deſſen das Bewegliche ſein Berhaltniß verandern ſoll, erfodert; der Raum aber, der wahrgenommen werden ſoll, muß matertal, mithin, dem Begriffe einer Materie uber haupt zu Folge, ſelbſt beweglich ſeyn. Um ihn nun be— wegt zu denken, darf man ihn nur als in einem Raume von großerem Umfange enthalten denken und dieſen als ruhig annehmen. Mit dieſem aber laßt ſich eben daſſelbe in An— ſehung eines noch mehr erweiterten Raumes veranſtalten und ſo ins Unendliche, ohne jemals zu einem unbeweglichen cunmateriellen) Raume durch Erfahrung zu gelangen, in Anſehung deſſen irgend einer Materte ſchlechthin Bewe— gung oder Ruhe beygelegt werden konne, ſondern der Be— griff dieſer Berhaltnißbeſtimmungen wird beſtandig abgean Dert werden muſſen, nachdem man das Bewegliche mit einem oder dem anderen dieſer Raume in Verhaltniß be— rtrachten wird. Da nun die Bedingung, etwas als ruhig vder bewegt anzuſehen, im relativen Raume ins Unendliche immer wiederum bedingt iſt, ſo erhellet daraus erſtlich: daß alle Bewegung oder Ruhe blos relativ und keine abſolut ſeyn konne, d. i. daß Materie blos in Verhaltniß auf Ma— terie, niemals aber in Bnſehung des bloßen Raumes ohne Materie, als bewegt oder ruhig gedacht werden konne, mit— H 3 bin 118 hin abſolute Bewequng, d. i. eine ſolche, die ohne alle Be— ziehung einer Materie auf eine andere gedacht wird, ſchlechthin unmoglich ſey; ziveyteus, daß auch eben darum kein fur alle Erſcheinung guitiger Begriff von Be wegung oder Ruhe im relativen Ramme moglich ſey, ſon dern man ſich einen Raum, in welchem dieſer ſelbſt als be wegt gedacht werden., konne, der aber feiner Beſtimmung nach weiter von keinem anderen empiriſchen Raume abhangt und daher nicht wiederum bedingt iſt, d. i. einen abſoluten Raum, auf den alle relatire Bewegungen bezogen werden konnen, denken muſſe, in welchem alles Empiriſche be— weglich iſt, eben darum, demit in demſelben alle Bewe— gung des Materiellen, als blos relativ gegen einander, als alternativ- wechſelſeitig“), keine aber als abſolute Bewe gung oder Ruhe (da, indem das eine bewegt heißt, das andere, worauf in Zeziehung jenes bewegt iſt, gleichwohl als ſchlechthin ruhig vorgeſtellt wird) gelten moge. Der abſolute Raum iſt alſo nicht als ein Begriff von einem wirklichen Object, ſondern als eine Jdee, welche zur Re— gel Jn der Logik bezeichnet das Entweder-Oder jeder— zeit ein disjunetives Urtheit; da denn, wenn das eine wahr iſt, das andere falſch ſeyn muß. Z. B. ein Korper iſt entweder bewegt, oder nicht bewegt, d. i. in Ruhe. Denn man redet da lediglich von dem Verhaltt niß des Erkenntniſſes zum Objecte. Jn der Erſcheinungtt lehre, wo es auf das Verhaltniß zum Dubject ankommt. um darnach das Verhaltniß der Objrcte zu beſtimmen, iſt es anders. Denn da iſt der Satz: der Lorper iſt ente weder bewegt und der Raum ruhig, oder umgekehrt, nicht ern disjunctiver Satz in objectiver, ſondern nur in ſubzectiver Beziehung, und beyde darin enthaltene Urtheile gelten alternativ. Jn eben derſelben Phat nomenologie, wo die Bewegung nicht blos phoronomiſch, ſondern vielmehr dynamiſch belrachtet wird, iſt dagegen der n 119 gel dienen ſoll, alle Bewegung in ihm blos als relativ zu betrachten, nothwendig, und alle Bewegung und Ruhe muß auf den abſoluten Raum reducirt werden, wenn die Erſcheinung derſelben in einen beſtimmten Erfahrungs— begriff (der alle Erſcheinungen vereinigt) verwandelt wer— den ſoll. So wird die geradlinigte Bewegung eines Korpers im relativen Raume auf den abſoluten Raum reducirt, wenn ich den Korper als an ſich ruhig, jenen Raum aber im abſoluten (der' nicht in die Sinne fallt) in ent— gegengeſetzter Richtung bewegt, und dieſe Vorſtellung als diejenige denke, welche gerade dieſelbe Erſcheinung giebt, wodurch denn alle mogliche Erſcheinungen geradltnigter Bewegungen, die ein Korper allenfalls zugleich haben mag, auf den Erfahrungsbegriff, der ſie insgeſammt vereinigt, namlich den der blos relativen Bewegung und Ruhe, zuruckgefuhrt werden. Ha4 Die der disjunctive Satz in objectiver Bedeutung zu nehmen; d. i. an die Stelle der Umdrehung eines Korpers kann ich nicht die Ruhe deſſelben und dagegen die entgegenge— ſetzte Bewegung des Raums annehmen. Wo aber die Bewegung ſogar mechaniſch betrachtet wird, (wie weenn ein Korper gegen einen dem Scheine nach ruhigen aulauft) iſt ſogar das der Form nach disjunctwe Urtheil in Anſehung des Objects diſtributiv zu gebrauchen, ſo daß die Bewegung nicht eütweder dem einen oder dem andern, ſondern einem jeden ein gleicher Antheil daran beygelegt werden muß. Dieſe Unterſcheidung der alternativen, disjunctiven und diſtributi— ven Beſtimmung eines Begriffs, in Anſehung entge— gengeſetzter Pradicate, hat ihre Wichtigkeit, kann aber hier nicht weiter erortert werden. 126 S J Die Kreisbewegung, weil ſie, nach dem zweyten kehrſatze, auch ohne Bezichung auf den außeren empitiſch— gegebenen Raum als wirkliche Bewegung in der Erfahrung gegeben werden kann, ſcheint doch in der That abſolute Bewegung zu ſeyn. Denn die relative in Anſehung des außeren Raums (z. B. die Achſendrehung der Erde relativ auf die Sterne des Himmels), iſt eine Erſcheinung, an deren Stelle die entgegengeſetzte Bewegung dieſes Raums (des Himmels) in derſeiben Zeit, als jener vollig gleichgeltend geſetzt werden kann, die aber nach dieſem Lehrſatze in der Erfahrung durchaus nichi an deren Stel— le geſetzt werden darf, mithin auch jene Kreisdrehung uicht als außerlich relativ vorgeſtellt werden ſoll, wel— ches ſo lautet, als ob dieſe Art der Bewegung fur ab— folut anzunehmen ſey. Allein es iſt wohl zu merken: daß hier von der wah ren Cwirklichen) Bewegung, die doch nicht als ſolche er ſcheint, die alſo, wenn man ſie blos nach empiriſchen Verhaltniſſen zum Raume beurtheilen wollte, fur Ruhe könnte gehalten werden, d. i. von der wahren Bewe gung, zum Unterſchiede vom Schein, nicht aber von ihr als abſoluten Bewegung im Gegenſatze der relativen die Rede ſey, mithin die Kreisbewegung, ob ſie zwar in der Erſcheinung keine Stellen-Veranderung, d. i. keine phoro nomiſche, des Verhaltniſſes des Bewegten zum (empiri— ſchen) Raume, zeigt, dennoch eine durch Erfahrung er— weisliche continuirliche dynamiſch. Veranderung des Ver— haltniſſes der Materie in ihrem Raume, z. B. eine be ftandige Verminderung der Anziehung durch eine Beſtrebung zu entfliehen, als Wirkung der Kreisbewegung, zeige und dadurch den Unterſchied derſelben vom Sehein ſicher be— zeichne. Man kann ſich z. B. die Erde im unendlichen lee— ren Raum, als um die Achſe gedreht, vorſtellen, und dieſe Bewegung auch durch Erfahrung darthun, obgleich weder das Verhaltnifj der Theile der Erde unter einander, noch zum 121 zum Raume außer ihr, phoronomiſch, d. i. in der Erſchei nung verandert wird. Denn in Anſchung des erſteren als empiriſchen Raumes verandert nichts auf und in der Erde ſeine Stelle, und in Beztehung des zweyten, der ganz leer iſt, kann uberall kein außeres verandertes Verhaltniß, mit— hin auch keine Erſcheinung einer Bewegung Statt finden. Allein, wenn ich mir eine zum Mittelpunct der Erde hinge— hende tiefe Hole vorhzelle, und laſſe einen Stein darin fallen, finde aber, daß, ob zwar in jeder Weite vom Mittelpuncte die Schwere immer nach dieſem hingerichtet iſt, der fallende Stein dennoch von ſeiner ſenkrechten Richtung im Fallen tcontinuirlich und zwar von Weſt nach Oſt abweiche, ſo ſchließe ich, die Erde ſey von Abend gegen Morgen um die Achſe gedreht. Oder wenn ich auch außerhalb den Stein von der Oberflache der Erde weiter entferne, und er bleibt nicht uber demfelben Puncte der Oberflache, ſondern entfernt ſich von demſelben von Oſten nach Weſten, ſo werde ich auf eben dieſelbe vorhergenannte Achſendrehunz der Erde ſchlie— Fen und beyderley Wahrnehmungen werdben zum Beweiſe der Wirklichkeit dieſer Bewegung hinreichend ſeyn, wozu die Veranderung des Verhaltniſſes zum außeren Raume (dem beſtirnten Himmeh nicht hinreicht, weil fie bloße Er— ſcheinung iſt, die von zwey in der That entgegengeſetzten Grunden herruhren kann und nicht ein aus deit Erklarungs— grunde aller Erſcheinungen dieſer Veranderung abgelcite— tes Erkenntniß, d. i. Erfahrung, iſt. Daß aber dieſe Be— wegung, ob ſie gleich keine Veranderung des Verhaltniſſes zum empiriſchen Raume iſt, dennoch keine abſolute Bewe— gung, ſondern continuirliche Veranderung der Relationen der Materien zu einander, ob zwar im abſoliten Naume vorgeſtellt, mithin wirklich nur relative und ſogar darum allein wahre Bewegung ſey, das beruht auf der Vorſtel— Jung der wechſelfettigen continuirlichen Entfernung eines jeden Theils der Erde C(außerhalb der Achſe) von jedem andern ihm in gleicher Entfernung vom Mittelpuncte im Diameter gegenuber liegenden. Denn dieſe Bewegung iſt H5 im 122 im abſoluten Raume wirklich, indem dadurch der Abgang der gedachten Entfernung, den die Schwere kur ſich allein dem Korper zuztehen wurde, und zwar ohne alle dynamiſche zurucktreibende Urſache (wie man aus dem von Newton Prine. Ph. N. pag. 10. Edit. 1714. gewahlten Bey— ſpiele erſehen kann), mithin durch wirkliche, aber auf den innerhalb der bewegten Materie (namlich des Centrum derſelben) beſchloſſenen, nicht aber auf den außeren Raum bezogene Bewegung, continuirlich erſetzt wird. Was den Fall des dritten Lehrſatzes anlangt, ſo bedarf es, um die Wabrheit der wechſelſeitig- entgegenge— ſetzten und gleichen Bewegungs beyder Korper auch ohne Ruckſicht auf den empiriſchen Raum zu zeigen, nichi ein mal des im zweyten Fall nothigen, durch Erfahrung gege— benen thatigen dynamiſchen Einfluſſes (der Schwere, oder eines geſpannten Fadens), ſondern die bloße dynamiſche Moglichkeit eines ſolchen Einfluſſes, als Eigenſchaft der Materie, (die Zuruckſtohung oder Anziehung) fuhrt, bey der Bewegung der einen, die gleiche und entgegengeſetzte Bewegung der andern zugleich mit ſich, und zwar aus blo— ßen Begriffen einer relativen Bewegung, wenn ſie im abſo luten Ee ſagt daſelbſt: Motus quidem veras corporum ſingulorum cognoſcere et ah apparentibus aetu diieriminare diffieillimum eſt: propterea, quod partes ſpatii illius immobilis, in quo eorpora vere meventur, non incurrunt in ſenſus. Cauſſa tamen non eſt prorſus deſperata. Hierauf laßt er zwey durch einen Faden verknupfte Kugeln ſich um ihren gemeinſchaftlichen Schwerpunct im leeren Raume drehen, und zeigt, wie die Wirklichkeit ihrer Bewegung ſammt der Richtung derſelben dennoch durch Erfahrung koönne gefunden wer— den. Jch habe dieſes auch an der um ihre Achſe bet wegten Erde unter etwas veranderten Umſtanden zu zei gen geſucht. —S 123 luten Raume, d.i. nach der Wahrheit betrachtet wird, und iſt daher, wie alles, was aus bloßen Begriffen hinreichend erweislich iſt, ein Geſetz einer ſchlechterdings nothwendigen Gegenbewegung. Es iſt alſo auch keine abſolute Bewegung, wenn gleich ein Korper im leeren Raume in Anſehung eines anderen als bewegt gedacht wird; die Bewegung beyder wird hier nicht relativ auf den ſie umgebenden Raum, ſondern nur auf den zwiſchen ihnen, welcher ihr autzeres Verhaltniß unter einan— der allein beſtimmt, als den abſoluten Raum betrachtet, und iſt alſo wiederum nur relativ. Abſolute Bewegung wurde alſo nur diejenige ſeyr, die einem Korper ohne ein Verhaltniß auf irgend eine andere Materie zukame. Eint ſolche ware allein die geradlinigte Bewegung des Weltgan zen, d. i. des Gyſtems aller Veaterie. Denn, wenn außer einer Materie noch irgend eine andere, ſelbſt dureh den lee— ren Raum getrennte Materie ware, ſo wurde die Bewegung ſchon relativ ſeyn. Um deswillen iſt ein jeber Beweis eines Bewegungsgeſttzes, der darauf hinauslauft, daß das Ge— gentheil deſſelben eine geradlinigte Bewegang des ganzen Weltgebaudes zur Folge haben mußte, ein apodictiſcher Beweis der Wagrheit deſſelben; blos weil daraus abſolute Bewegung folgen wurde, die ſchlechterdings unmoglich iſt. Von der Art iſt das Geſetz des Antagoniſi.tz in aller Ge— meinſchaft der Materie durch Bewegung. Den eine jede Abweichung von demſelben wurde den Zereinſehaftlichen Mittelpunct der Ecywere aler Materie, mith.a das ganze Weltgebaude aus der Stelle rurLen, welches dagegen, wenn man dieſes ſich als um ſeine Tahſe ocdreht vor“cen wollte, nicht geſchthen arde, welche Zewegun; alſo immer noch zu denken rn glieh, ob zwar anzunehmen, ſo viel man ab— ſehen kann, ganz ehne begre. Lichen Nutzen ſeyn wurde. Auf die verſ.hiedenen Begriffe der Bewegang und be— wegenden Krafte haben auch die verſchiedenen Begriffe vom leeren Raume ihre Beziehung. Der leere Raum in pho ronomiſecher Ruckſicht, der auch der abſolute Raum heißt, ſollte 124 S ſollte billig nicht ein leerer Raum genannt werden; denn er iſt nur die Jdee von einem Raume, an welchem ich von aller beſonderen Materie, die ihn zum Gegenſtande der Erfahrung macht, abſtrahire, um in ihm den materiellen, oder jeden empiriſchen Raum, noch als beweglich und dadurch die Bewegung nicht. blos einſeitig, als abfolutes, ſondern jeder— zeit wechſelſeitig, als blos relatives Pradicat zu denken. Er iſt alſo gar nichts, was zur Exiſtenz der Dinge, ſon— dern blos zur Beſtimmung der Begriffe gehort, und ſo fern exiſtirt kein lerrer Raum. Der leere Raum in dynami ſcher Ruckſicht iſt der, der nicht erfullet iſt, d. i. worin dem Eindringen des Beweglichen nichts anderes Bewegli— ches widerſteht, folglich keine repulſive Kraft wirkt, und er kann entweder der leere Raum in der Welt (vacuum mun— danurn), oder, wenn dieſe als begrenzt vorgeſtellt wird, der leere Raum außer der Welt (vacuum extramundamum) ſeyn; der erſtere auch entweder als zerſtreuter (vacuum diſſeminatum, der nur einen Theil des Volumens der Materie ausmacht), oder als gehaufter leerer Raum cvacuum coaceruatum, der die Korper, z. B. Weltkor per, von einander abſondert) vorgeſtellt werden, welche Unterſcheidung, da ſie nur auf den Unterſchied der Platze, die man dem leeren Raum in der Welt anweiſet, beruht, eben nicht weſentlich iſt, aber doch in verſchiedener Abſicht gebraucht wird, der erſte, um den ſpecifiſchen Unterſchied der Dichtigkeit, der zweyte, um die Moglichkeit einer von allem außeren Widerſtande freyen Bewegung im Weltraume daven abzuleiten. Daß den leeren Raum in der erſteren Abſichr anzunehmen nicht nothig ſey, iſt ſchon in der all— gemeinen Anmerkung zur Dynamik gezeigt worden; daß er aber uiimoglich ſey, kann aus ſeinem Begriffe allein, nach dem Satzre des Widerſpruchs, keinesweges bewieſen werden. Gleichwohl, wenn hier auch kein blos logiſcher Grund der Verwerfung deſſelben anzutreffen ware, konnte doch ein all— gemeiner phyſiſcher Grund, ihn aus der Naturlehre zu ver— weiſen, namlich der von der Moglichkeit der Zuſammen— ſetzung ν 145 ſetzung einer Materie uberhaupt, da ſeyn, wenn man die letztere nur beſſer einſahe. Denn, wenn die Anziehung, die man zur Erklarung des Zuſammenhanges der Materte annimmt, nur ſcheinbare, nicht wabre Anziehung, viel— mehr etwa blos die Wirkung einer Zuſammendrückung durch außere im Weltraume allenthalben verbreitete Matette (den Aether), welche ſelbſt nur durch eine allgemeine und urſpruugliche Anziehung, namlich die Gravitatton, zu die— ſem Drucke gebracht wird, ſeyn ſollte, welche Meynung manche Grunde fur ſich hat; ſo wurde der leere Raum in— nerhalb den Materien, wenn gleich nicht legiſch, doch dy— namiſch und alſo phyſiſch uninoglich ſeyn, weil jede Materie ſich in die leeren Raume, die man innerhalb derſelben an— nahme, (da ihrer expanſiven Kraft hier nichts widerſteht) von ſelbſt ausbreiten und ſie jederzeit erfüllet erhalten wurde. Ein leerer Raum außer der Welt wurde, wenn man un— ter dieſer den Jnbegriff aller vorzuglich attractiven Materien (der großen Weltkorper) verſteht, aus eben denſelben Grun— de unmoglich ſeyn, weil nach dem Maaße, als die Entfer— nung von dieſen zuninimt, auch die Anziehungskraſt auf den Aether, Oder jene Korper alle einſchließt und, von jener ge— trieben, ſie in ihrer Dichtigkeit durch Zuſammeundruckung erhalt) in umgekehrtem Verhaltniſſe abnimit, dieſer alſo ſelbſt nur ins Unendliche an Dichtigkeit abnehmen, nirgend Haber den Raum ganz leer laſſen wurde. Daß es indeſſen mit dieſer Wegſchaffung des leeren Raums ganz hypothetifch zugeht, darf niemand befremden; geht es doch mit der Be— hauptung deſſelben nicht beſſer zu. Diejenigen, welche dieſe Streitfrage dogmatiſch zu entſcheiden wagen, ſie mögen es bejahend oder verneinend thun, ſtutzen ſich zuletzt auf lauter metaphyſiſche Vorausſetzungen, wie aus der Dynamik zu erſehen iſt, und es war wenigſtens nothig, hier zu zeigen, daß dieſe uber gedachte Aufgabe gar nicht entſcheiden konne. Was drittens den leeren Raum in mechaniſcher Abſicht betrifft, ſo iſt dieſer das gehaufte Leere innerhalb dem Welt— oanzen, um den Weltkorpern frehe Bewegung zu verſchaffen. Man 126 Man ſiehet leicht, daß die Moglichkeit oder Unmoglichkeit deſſelben nicht auf metaphyſiſchen Grunden, ſondern dem ſchwer aufzuſchließenden Naturgeheimniſſe, auf welche Art die Materie ihrer eigenen ausdehnenden Kraft Schranken ſetze, beruhe. Gleichwohl, wenn das, was in der allge— meinen Anmukung zur Dynamik von der ins Unendliche moglichen großeren Ausdehnung ſpecifiſch verſchiedener Stoffe, bey derſelben Quantitat der Materie (ihrem Ge— wichte nach) geſagt worden, eingeraumt wird; ſo mochte wohl, um der freyen und daurenden Bewegung der Welt— korper willen, einen leeren Raum anzunehmen, unnothig ſeyn, weil der Widerſtand, ſelbſt bey ganzlich erfulleten Raumen, alsdenn doch ſo klein, als man will, ge— dacht werden kann. Uno ſo endigt ſich die metaphyſiſche Korperlehre mit dem Leeren und eben darum Unbegreiflichen, worin ſie einerley Schickſal nut allen übrigen Verſuchen der Vernunft hat, wenn ſie im Zuruckgehen zu Principien den erſten Grun den der Dinge nachſtrebt, da, weil es ihre Natur ſo mit ſich bringt, niemals etwas anders, als ſo fern es unter gegebenen Bedingungen beſtimmt iſt, zu begreifen, folglich ſie weder beym Bedingten ſtehen bleiben, zoch ſich das Unbedingte faßlich machen kann, ihr, wenn Wißbegierde ſie auffodert, das abſolute Ganze aller Bedingungen zu faſ— ſen, nichts ubrig bleibt, als von den Gegenſtanden auf ſich ſelbſt zuruckzukehren, um, anſtatt der letzten Grenze der Dinge, die letzte Grenze ihres eigenen ſich ſelbſt uberlaſſe uen Vermogens zu erforſchen und zu beſtinmmen.
https://openalex.org/W2269572354	https://pubblicazioni.unicam.it/bitstream/11581/441612/1/FRONT%20CELL%20INFECT%20MICROBIOL.pdf	English	null	Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota	Frontiers in cellular and infection microbiology	2,016	cc-by	10,427	Salmonella enterica Serovar Typhimurium Exploits Inﬂammation to Modify Swine Intestinal Microbiota Rosanna Drumo 1, 2 †, Michele Pesciaroli 1, 3 †, Jessica Ruggeri 4, Michela Tarantino 1, 1 Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy, 2 Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy, 3 VISAVET Health Surveillance Centre, Universidad Complutense Madrid, Madrid, Spain, 4 Department of Veterinary Diagnostic, Istituto Zooproﬁlattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy, 5 Research and Development Area, Istituto Zooproﬁlattico Sperimentale dell’Umbria e della Marche, Perugia, Italy, 6 Department of Experimental Medicine, University of Perugia, Perugia, Italy, 7 Department of Biology, University of Roma Tor Vergata, Rome, Italy Citation: Drumo R, Pesciaroli M, Ruggeri J, Tarantino M, Chirullo B, Pistoia C, Petrucci P, Martinelli N, Moscati L, Manuali E, Pavone S, Picciolini M, Ammendola S, Gabai G, Battistoni A, Pezzotti G, Alborali GL, Napolioni V, Pasquali P and Magistrali CF (2016) Salmonella enterica Serovar Typhimurium Exploits Inﬂammation to Modify Swine Intestinal Microbiota. Front. Cell. Infect. Microbiol. 5:106. doi: 10.3389/fcimb.2015.00106 Drumo R, Pesciaroli M, Ruggeri J, Tarantino M, Chirullo B, Pistoia C, Petrucci P, Martinelli N, Moscati L, Manuali E, Pavone S, Picciolini M, Ammendola S, Gabai G, Battistoni A, Pezzotti G, Alborali GL, Napolioni V, Pasquali P and Magistrali CF (2016) Salmonella enterica Serovar Typhimurium Exploits Inﬂammation to Modify Swine Intestinal Microbiota. Front. Cell. Infect. Microbiol. 5:106. doi: 10.3389/fcimb.2015.00106 Keywords: Salmonella Typhimurium, microbiota, inﬂammation, immune response, pig, salmonellosis ORIGINAL RESEARCH published: 22 January 2016 doi: 10.3389/fcimb.2015.00106 Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inﬂamed intestinal environment exploiting inﬂammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modiﬁcations are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inﬂammation and a reduction of speciﬁc protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inﬂammatory response, was unable to successfully sustain competition with the microbiota. Edited by: D. Scott Merrell, Uniformed Services University, USA Uniformed Services University, USA Reviewed by: Richard E. Isaacson, University of Minnesota, USA Johanna R. Elfenbein, North Carolina State University, USA Correspondence: Paolo Pasquali paolo.pasquali@iss.it; Chiara F. Magistrali c.magistrali@izsum.it †These authors have contributed equally to this work. Reviewed by: Richard E. Isaacson, University of Minnesota, USA Johanna R. Elfenbein, North Carolina State University, USA Correspondence: Paolo Pasquali paolo.pasquali@iss.it; Chiara F. Magistrali c.magistrali@izsum.it †These authors have contributed equally to this work. Received: 12 September 2015 Accepted: 28 December 2015 Published: 22 January 2016 Received: 12 September 2015 Accepted: 28 December 2015 Published: 22 January 2016 Accepted: 28 December 2015 P bli h d J Keywords: Salmonella Typhimurium, microbiota, inﬂammation, immune response, pig, salmonellosis INTRODUCTION During the past few years, there has been an expanding interest concerning the role played by intestinal microbiota in the susceptibility to enteric pathogens. Microbiota contributes to the digestion of dietary substances and to the synthesis of essential food supplements such as vitamins, and to the development or maintenance of the mucosal immune system (Littman and Pamer, 2011). Moreover, it acts as a barrier against invading bacteria both physically, blocking pathogen access to the epithelial layer, and also by outcompeting for nutrients reducing the survival and invasiveness of enteric pathogens (Hallstrom and McCormick, 2011; Sassone-Corsi and Raﬀatellu, 2015). However, it has been known that S. Typhimurium requires intestinal inﬂammation to circumvent “colonization resistance” provided by the intestinal microbiota (Santos et al., 2009). It has been shown that Salmonella can alter the normal composition of the gut microbiota, and this inﬂuence is associated with Salmonella virulence factors that induce inﬂammatory mucosal host responses (Barman et al., 2008). Furthermore, animals with disrupted microbiota have an increased susceptibility to infection (Barman et al., 2008; Juricova et al., 2013). Most of the studies examining salmonellosis have been carried out in murine models that naturally do not develop gastroenteritis. To resemble the disease in humans, mice can be subjected to antibiotic treatment in order to eliminate microbiota and to develop colitis (Ahmer and Gunn, 2011). Therefore, due to the lack of an intact microbiota, murine models are not suitable for the comprehension of the mechanisms used by Salmonella to thrive in the gastrointestinal environment (Elfenbein et al., 2013). To circumvent this limitation, we chose the pig as a model for our study. The advantage of the pig lies in the great similarity between humans and pigs in the gastrointestinal tract and in the disease caused by Salmonella as well as being a natural host of Salmonella (Zhang et al., 2013). We hypothesized that the Salmonella virulence degree is a determining factor in inﬂuencing the capability of the pathogen to overcome protective microbiota. To explore this, we compared the eﬀects on the intestinal microbiota of S. Typhimurium wild type to that of an attenuated Salmonella strain lacking the ZnuABC transporter. Our ﬁndings through the reduction of microbiota members normally involved in the intestinal homeostasis and in the inhibition of pathogen growth. INTRODUCTION Nontyphoidal salmonellae (NTS) as Salmonella enterica serovar Typhimurium are a leading cause of acute food-borne zoonoses worldwide being responsible for hundreds of millions of cases of gastroenteritis and bacteremia annually (Hohmann, 2001). Pigs are important reservoir of infection for humans as they are asymptomatic carriers of broad host-range serovars of Salmonella (Funk and Gebreyes, 2004; Pires et al., 2011). The intestine is considered to be the biological niche of January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Salmonella Alters Gut Microbiota in Piglets Drumo et al. Salmonella with the intestinal mucosa having a central role in regulating the immune response to bacteria (Hallstrom and McCormick, 2011). However, Salmonella has developed strategies to overcome and cope with most of the immune defenses developed by the host (Behnsen et al., 2015). Examples of the strategies used by Salmonella to evade mucosal innate immunity include the ability to resist to the reactive oxygen species generated during inﬂammation (Bogomolnaya et al., 2013), in order to produce energy by an anaerobic respiration chain which uses an electron acceptor speciﬁcally generated in the gut under oxidative stress (Winter et al., 2010) and to resist to the sequestration of essential nutrients such as iron and zinc (Raﬀatellu et al., 2009; Liu et al., 2012). As a matter of fact, the ability to resist to the antimicrobial host responses characterizing gut inﬂammation promotes the growth of Salmonella in the intestinal lumen over the competing microbiota. During the past few years, there has been an expanding interest concerning the role played by intestinal microbiota in the susceptibility to enteric pathogens. Microbiota contributes to the digestion of dietary substances and to the synthesis of essential food supplements such as vitamins, and to the development or maintenance of the mucosal immune system (Littman and Pamer, 2011). Moreover, it acts as a barrier against invading bacteria both physically, blocking pathogen access to the epithelial layer, and also by outcompeting for nutrients reducing the survival and invasiveness of enteric pathogens (Hallstrom and McCormick, 2011; Sassone-Corsi and Raﬀatellu, 2015). However, it has been known that S. Typhimurium requires intestinal inﬂammation to circumvent “colonization resistance” provided by the intestinal microbiota (Santos et al., 2009). Animals and Samples Collection p Thirty-one post weaned piglets old 28 days, from Salmonella-free sows (routinely monitored with microbiological and serological tests), were used in the experiment. Group A (9 piglets) received sterile sodium bicarbonate buﬀer and it was used as naïve control group. Groups B and C (11 piglets each) were orally infected with 20 ml of sterile 10% sodium bicarbonate buﬀer containing 2 × 109 CFU of STM1znuABC (Group B) or 2 × 109 CFU of STMwt (Group C). At 0, 1, 2, 7, and 12 days post infection (dpi), rectal temperature was recorded and serum and fecal samples were collected to evaluate TNF-α, IL1-α, haptoglobin, and CRP production and to detect fecal excretion of Salmonella, respectively. Four piglets of group A and 5 for groups B and C were sacriﬁced at 1 dpi, while 5 piglets of group A and 6 for groups B and C at 12 dpi. Portions of spleen, ileum, cecum, colon, ileocecal lymph nodes, and tonsil of the soft palate were taken for microbiological analysis, histology, and for mRNA isolation. Feces and cecal and colonic contents were collected to analyze the microbiota composition. All the experiments were authorized by national authority and conducted according to European Directive (2010/63/EU; approval number 54/2012). Salmonella spp. Cultures The wild-type strain S. Typhimurium ATCC 14028 (hereafter STMwt) and its isogenic attenuated znuABC mutant (hereafter STM1znuABC; Ammendola et al., 2007), were used throughout the study. Strains were grown overnight at 37◦C in Brain Heart Infusion broth (Oxoid Ltd., Basingstoke, UK), harvested by centrifugation and washed twice in ice-cold phosphate buﬀer solution (PBS) (Sigma-Aldrich, Milan, Italy). INTRODUCTION It has been shown that Salmonella can alter the normal composition of the gut microbiota, and this inﬂuence is associated with Salmonella virulence factors that induce inﬂammatory mucosal host responses (Barman et al., 2008). Furthermore, animals with disrupted microbiota have an increased susceptibility to infection (Barman et al., 2008; Juricova et al., 2013). Most of the studies examining salmonellosis have been carried out in murine models that naturally do not develop gastroenteritis. To resemble the disease in humans, mice can be subjected to antibiotic treatment in order to eliminate microbiota and to develop colitis (Ahmer and Gunn, 2011). Therefore, due to the lack of an intact microbiota, murine models are not suitable for the comprehension of the mechanisms used by Salmonella to thrive in the gastrointestinal environment (Elfenbein et al., 2013). To circumvent this limitation, we chose the pig as a model for our study. The advantage of the pig lies in the great similarity between humans and pigs in the gastrointestinal tract and in the disease caused by Salmonella as well as being a natural host of Salmonella (Zhang et al., 2013). We hypothesized that the Salmonella virulence degree is a determining factor in inﬂuencing the capability of the pathogen to overcome protective microbiota. To explore this, we compared the eﬀects on the intestinal microbiota of S. Typhimurium wild type to that of an attenuated Salmonella strain lacking the ZnuABC transporter. Our ﬁndings provide evidence that the microbiota modiﬁcations induced Salmonella with the intestinal mucosa having a central role in regulating the immune response to bacteria (Hallstrom and McCormick, 2011). However, Salmonella has developed strategies to overcome and cope with most of the immune defenses developed by the host (Behnsen et al., 2015). Examples of the strategies used by Salmonella to evade mucosal innate immunity include the ability to resist to the reactive oxygen species generated during inﬂammation (Bogomolnaya et al., 2013), in order to produce energy by an anaerobic respiration chain which uses an electron acceptor speciﬁcally generated in the gut under oxidative stress (Winter et al., 2010) and to resist to the sequestration of essential nutrients such as iron and zinc (Raﬀatellu et al., 2009; Liu et al., 2012). As a matter of fact, the ability to resist to the antimicrobial host responses characterizing gut inﬂammation promotes the growth of Salmonella in the intestinal lumen over the competing microbiota. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbiology gy Fecal shedding and organs colonization of STMwt and STM1znuABC were determined according to the ISO 6579: 2002/Amendment 1:2007 protocol. Brieﬂy, samples were weighed and homogenized in nine parts of Buﬀered Peptone Water (BPW) (Oxoid Ltd., UK). This initial solution was then used to perform a decimal dilution series carried out by systematically transferring an aliquot of 0.5 ml of each successive dilution in 4.5 ml of BPW. All BPW-diluted samples were incubated at 37◦C for 18 ± 3 h. 0.1 ml of cultures were subsequently seeded on Modiﬁed Semisolid Rappaport- Vassiliadis (MSRV) agar plates (Oxoid Ltd., UK) and incubated at 41.5◦C for 24 h for the selective enrichment of Salmonella. A loopful of growth from each MSRV plate was streaked onto Xylose-Lysine-Desoxycholate Agar (Oxoid Ltd., UK) and Brilliant Green Agar (Oxoid Ltd., UK) plates and hence incubated at 37◦C overnight. Suspect Salmonella colonies were subjected to biochemical identiﬁcation by the BBL Enterotube II January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 2 Salmonella Alters Gut Microbiota in Piglets Drumo et al. (BD Franklin Lakes, USA) and serological identiﬁcation using Salmonella group-speciﬁc antisera (Remel, Lenexa, USA). This is a semi-quantitative approach that allows the quantiﬁcation of Salmonella in a sample within a tenfold band (detection limit 1 CFU/g feces). singletons and to normalize the data by sequencing depth. Alpha- and beta- diversity were determined by QIIME using Shannon’s and Fisher’s indices for alpha diversity, unweighted Unifrac and Bray-Curtis for beta diversity, respectively. Histology Tissue samples of cecum were ﬁxed in formalin, embedded in paraﬃn wax and stained with hematoxylin, and eosin according to standard procedures. q q-PCR was performed using bacterial groups-speciﬁc 16S rRNA primers (see Supplementary Table 2) to determine the amount of bacteria in the study groups. However, this method is an approximation of microbial abundance as a great amount of bacteria features many copies of the 16S gene. Therefore, both variation in the abundance of organisms and genomic copy number variation can inﬂuence the quantitative prediction of 16S gene abundances. Real time PCRs were carried out on SensiMix SYBR Low-ROX Kit (Bioline). The ampliﬁcation program started with an initial step at 95◦C for 10 min, followed by 40 cycles of 15 s each at 95◦C, 15 s at 55◦C-63◦C (depends on the Tm of primers), and 15s at 72◦C. The 16S gene copy numbers per µl of DNA, from each sample, were determined by using standard curves generated from fragments of 16S rRNA genes of reference bacteria speciﬁc for each group cloned into plasmid (Promega) as templates. The plasmid was puriﬁed by using the Wizard Plus SV Minipreps DNA puriﬁcation kit (Promega) and its concentration was quantiﬁed by using a NanoDrop R⃝ND- 1000 Spectrophotometer. With the molecular weight data of the plasmid and insert sequences, the copy number (g/molecule) was calculated according to the equation deﬁned by Whelan et al. (2003). For each microbial population, the corresponding plasmid was chosen to create a 10-fold standard curve ranged from 108 to 102 copies. Copy numbers of 16S rRNA genes per µl of sample (feces, caecal, and colonic contents) were transformed into logarithms to achieve normal distribution, and the mean ± standard deviation was calculated. To estimate the copy number of Enterobacteriaceae other than Salmonella, for each sample the Salmonella 16S gene copy number was subtracted from the total Enterobacteriaceae 16S gene copy number. Immune Mediators Production TNF-α, IL1-α, haptoglobin, and C-reactive Protein (CRP) production was measured in serum samples from animals bled at 1 and 12 dpi using a sandwich ELISA (Porcine Quantikine ELISA Kit, R&D System, Mn, USA), according to the producer’s instructions. TNF-α, IL1-α, haptoglobin, and C-reactive Protein (CRP) production was measured in serum samples from animals bled at 1 and 12 dpi using a sandwich ELISA (Porcine Quantikine ELISA Kit, R&D System, Mn, USA), according to the producer’s instructions. Next-Generation Sequencing q g Bacterial genomic DNA (gDNA) was extracted from feces, cecal, and colonic contents using QIAmp DNA Stool Mini Kit (Qiagen, Hilden, Germany). Fifty nanograms of gDNA were used to amplify by PCR the hypervariable V3-V4 regions of the 16S rDNA using bacteria/archeal primers 515F/806R with Illumina overhang adapters (Caporaso et al., 2012). One nanogram of PCR amplicon was used for each sample to prepare the sequencing library according to the Illumina Nextera XT DNA Sample Preparation Kit. During this procedure, using a limited cycle PCR, Illumina sequencing adapters, and dual-index barcodes were added to the amplicon. All the libraries were subsequently normalized and pooled by 24 prior to sequencing according to manufacturer’s instructions (Illumina Nextera XT DNA Library Preparation Guide). Each pool of 24 samples was sequenced on Illumina MiSeq using a 2 × 250 paired-end (PE) setting on a standard MiSeq ﬂow cell. Sequencing reads were trimmed and all the reads with a quality score below the Q20 parameters were discarded from the analysis. Then, all the PE reads were joined using the join_paired_ends scripts of QIIME utilities (Caporaso et al., 2010) to create longer fragments. The Lederhosen pipeline (based on UCLUST software and green genes v 13.5 16S database) was used to create the OTU table for each sample. The OTU tables were provided as input for the MatR package to remove Gene Expression Total RNA was extracted from sections of the cecum, colon, and ileocecal lymph nodes using the PureLink RNA Mini Kit (Ambion, Life Technologies). Reverse transcription of 1 µg of RNA was performed for each individual sample using Tetro cDNA Synthesis Kit (Bioline) and 5 µl of cDNA were used for real-time reaction using SensiMix II Probe Kit (Bioline). Primers for cytokines (RPL-32, IL-1α, IL-1β, TNF-α, and IFN-γ) were designed using PrimerQuest Design Tool (Integrated DNA Technologies, IDT; see Supplementary Table 1). Fold changes in gene expression were calculated using the 11Ct method in comparison to the results for the reference housekeeping gene RPL32. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Fecal 16S rDNA Metagenomics Next-Generation Sequencing RESULTS degree of colonization than piglets infected with STM1znuABC (group B) in the gut organs (p < 0.05) at 1 dpi (Figure 3) and in the colon (p < 0.05) at 12 dpi (Supplementary Figure 1). Organs samples taken from naïve animals (group A) were negative. Pathogenicity of Salmonella Typhimurium is Positively Correlated to Bacterial Virulence g These ﬁndings conﬁrm that STMwt and STM1znuABC have a diﬀerential colonization eﬃciency. Moreover, STM1znuABC did not show a signiﬁcant systemic inﬂammation. We could infer that these results are a direct consequence of the intrinsic incapability of STM1znuABC to induce an inﬂammatory response but, in alternative, they could be due to the lower colonization of STM1znuABC which is not suﬃcient to give rise to a systemic immune response. Piglets infected with STM1znuABC (group B) and STMwt (group C) had a transient increase in body temperature at 1 dpi compared with naïve controls (group A). At 2 dpi, only the group C (STMwt) continued showing a signiﬁcantly higher body temperature than group A (Figure 1A). Moreover, diﬀerences in the levels of Salmonella fecal shedding were observed among the study groups. Animals infected with STMwt and STM1znuABC started to shed bacteria the day after experimental infection and reached the peak of excretion at 2 dpi. However, unlike group C (STMwt) that continued shedding a similar amount of bacteria throughout the whole period of observation, group B (STM1znuABC) showed a sharp decline over time (Figure 1B). To further assess the inﬂammatory response induced by STMwt and STM1znuABC, piglets were bled at diﬀerent time points and haptoglobin, CRP, IL1-α, and TNF-α levels were measured in sera. Group C (STMwt) had an early immune response characterized by a signiﬁcant increase of haptoglobin and IL1-α at 2 dpi, and TNF-α at 2 and 7 dpi, followed by a late production of CRP which reached a signiﬁcant level at 12 dpi. Conversely, group B (STM1znuABC) did not show any diﬀerent production of haptoglobin, CRP, IL1-α, and TNF-α when compared with the naïve (group A; Figure 2). Piglets of diﬀerent groups were euthanized at 1 and 12 dpi to assess bacterial colonization of organs. As shown in Figure 3, colonization occurred as early as 1 dpi, either in gut or in systemic organs. However, piglets infected with STMwt (group C) showed a signiﬁcant higher Histology We compared the cecum histopathological ﬁndings from control, STM1znuABC and STMwt-infected piglets at 1 and 12 dpi. At 1 dpi, sections from control piglets did not show inﬂammatory inﬁltrate (Figure 4A); conversely, piglets infected with STM1znuABC and STMwt showed neutrophilic inﬁltrate in the lamina propria and submucosa (Figures 4B,C). The neutrophilic inﬁltrate appeared moderate and multifocal in the STM1znuABC (Figure 4B), with crypt abscess formation, whereas marked and diﬀused in the STMwt infected piglets (Figure 4C). On the other hand, the neutrophil inﬂammation was mild at 12 dpi and present in a multifocal pattern in piglets infected with STM1znuABC, while inﬂammation was mild and diﬀuse in piglets infected with STMwt (data not shown). Overall, a histological investigation indicated the presence of inﬂammatory inﬁltrate only in STMwt and STM1znuABC. A higher degree of inﬂammation was observed in piglets infected with STMwt. FIGURE 1 \| STM1znuABC (group B) shows a lower virulence in piglets compared to the STMwt (group C). (A) Mean values and standard deviation (SD) bars of body temperature of study groups in different time points. In the table on the bottom the levels of signiﬁcance were reported among groups at different time points. Different letters at each time point represent signiﬁcant different results (P ≤0.05, Dunn’s test). (B) Mean values and SD bars of CFU/g of STM1znuABC and STMwt shed in feces. Results of piglets infected with STM1znuABC were compared to results of STMwt and differences were statistically signiﬁcant when P ≤0.05, Mann–Whitney test. FIGURE 1 \| STM1znuABC (group B) shows a lower virulence in piglets compared to the S f b d t t f t d i diff t ti i t I th t bl th b tt th l l FIGURE 1 \| STM1znuABC (group B) shows a lower virulence in piglets compared to the STMwt (group C). (A) Mean values and standard deviation (SD) bars of body temperature of study groups in different time points. In the table on the bottom the levels of signiﬁcance were reported among groups at different time points. Different letters at each time point represent signiﬁcant different results (P ≤0.05, Dunn’s test). (B) Mean values and SD bars of CFU/g of STM1znuABC and STMwt shed in feces. Statistical Analysis Statistical Analysis Statistical analysis was performed using GraphPad 6.0 software for Windows (GraphPad Software Inc.; San Diego; CA). Microbiota analysis by q-PCR were estimated using one- way analysis of variance (One-way ANOVA). Fecal shedding, organs colonization, and cytokines expression were analyzed using non-parametric Mann–Whitney test. Diﬀerences in body temperature and diﬀerences between groups in the TNF- α, IL1-α, haptoglobin, and CRP production were estimated using non-parametric Dunn’s test. Moreover, non-parametric Kruskal–Wallis was used to test the presence of signiﬁcant diﬀerences among the sample groups analyzed for each diﬀerent taxonomical level considered (Phylum, Family, Genus) and Benjamini-Hochberg FDR was applied to correct multiple testing. A P ≤0.05 was considered statistically signiﬁcant. Non- parametric Dunn’s test was also used to estimate diﬀerences in alfa diversity. January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3 Salmonella Alters Gut Microbiota in Piglets Drumo et al. Histology Results of piglets infected with STM1znuABC were compared to results of STMwt and differences were statistically signiﬁcant when P ≤0.05, Mann–Whitney test FIGURE 1 \| STM1znuABC (group B) shows a lower virulence in piglets compared to the STMwt (group C). (A) Mean values and standard deviation (SD) bars of body temperature of study groups in different time points. In the table on the bottom the levels of signiﬁcance were reported among groups at different time points. Different letters at each time point represent signiﬁcant different results (P ≤0.05, Dunn’s test). (B) Mean values and SD bars of CFU/g of STM1znuABC and STMwt shed in feces. Results of piglets infected with STM1znuABC were compared to results of STMwt and differences were statistically signiﬁcant when P ≤0.05, Mann–Whitney test. January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 4 Drumo et al. Salmonella Alters Gut Microbiota in Piglets FIGURE 2 \| (A–D) S. Typhimurium induces an inﬂammatory response correlated to the virulence of the bacterial strain. Haptoglobin, TNF-α, IL1-α, and C-reactive protein levels in serum of animals were determined by ELISA. The asterisks indicate statistical signiﬁcance: P ≤0.05 and *P ≤0.01 (multiple comparisons-Dunn’s test). FIGURE 2 \| (A–D) S. Typhimurium induces an inﬂammatory response correlated to the virulence of the bacterial strain. Haptoglobin, TNF-α, IL1-α, and C-reactive protein levels in serum of animals were determined by ELISA. The asterisks indicate statistical signiﬁcance: P ≤0.05 and *P ≤0.01 (multiple comparisons-Dunn’s test). Inﬂuence of Salmonella Infection on the Expression of Pro-Inﬂammatory Cytokines Inﬂuence of Salmonella Infection on the Expression of Pro-Inﬂammatory Cytokines Pro-inﬂammatory (IL1-α, IL1-β, TNF-α) and regulatory (IFN- γ) cytokines were observed so as to evaluate the early immune response in the ileocecal lymph nodes, colon, and cecum at 1 and 12 dpi (Figures 5A–H; Supplementary Figures 2A–H, 3A,B). At 1 dpi, we observed a tendency of the pro-inﬂammatory cytokines to increase in all organs analyzed; however, only the increase of IL1-β (p < 0.05) in the cecum and in the colon, and IL1-α (p < 0.05) in the lymph nodes of group C (STMwt) were statistically signiﬁcant (Figures 5A–C; Supplementary Figures 2A–C, 3A–C). At 12 dpi, overall expression of IL1-α, IL1-β, and TNF-α returned to baseline levels (Figures 5E–G; Supplementary Figures 2E–G, 3E–G). Histology Moreover, TNF-α (p < 0.01), IL1-β (p < 0.01), and IL1-α (p < 0.05) were signiﬁcantly down-regulated in the colon of piglets infected with STM1znuABC (group B; Supplementary Figures 2E–G), and IL1-α (p < 0.05) also in the lymph nodes of group C (STMwt; Supplementary Figures 3E–G). members, we used quantitative real time PCR (q-PCR). As depicted in Figure 6, consistent changes in the microbiota were present primarily in the cecal contents at 1 day post-Salmonella infection, with a signiﬁcant increase of total 16S rRNA gene copies (representative of total bacterial numbers) in piglets infected with STMwt (group C; p < 0.05) compared to naïve animals (group A) and piglets infected with STM1znuABC (group B; p < 0.05). Diﬀerences in the Lactobacillus/Lactococcus group were statistically signiﬁcant between groups B and C (p < 0.05) and very close to signiﬁcance between groups A (naïve) and C (STMwt) in the cecum (Figure 6A). In the feces (Supplementary Figure 4), the Lactobacillus/Lactococcus group showed signiﬁcant diﬀerences at 1, 2, 7, and 12 dpi (p < 0.05) between groups A and C, and only at 12 dpi between groups B and C (p < 0.05; Supplementary Figure 4). A decrease in the Eubacterium rectale/Clostridium coccoides group was evident in group C (p < 0.05) at 12 dpi in the cecum and at 2 dpi in the feces (p < 0.01; Figure 6B; Supplementary Figure 4C). No diﬀerences among the three experimental groups were observed for Bacteroides in any of the samples analyzed. Conversely, at 1 dpi an evident increase in the Biﬁdobacterium group was observed in all the districts investigated between groups A and C (p < 0.01 for cecal content and p < 0.001 for colon and feces) and between groups B and C (p < 0.01; Figure 6A; Supplementary Figures 4B, 5A). At 12 dpi, the Biﬁdobacterium group showed a sharp reduction in groups S. Typhimurium Alters Composition of the Microbiota in the Post-Weaned Piglets Model Aiming to more speciﬁcally analyze the impact of STMwt and STM1znuABC on some of the most representative bacterial January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 5 Salmonella Alters Gut Microbiota in Piglets Drumo et al. FIGURE 3 \| STMwt induces a higher organs colonization than STM1znuABC at 1 dpi. Piglets were orally infected with 2 × 109 CFU of STM1znuABC (group B) or STMwt (group C), and bacterial burdens were determined at 1 dpi. Differences between groups B and C were estimated using non-parametric Mann–Whitney test and were considered signiﬁcant when P ≤0.05. Organ samples taken from naïve animals (group A) were negative. Error bars represent one SD from the mean. FIGURE 3 \| STMwt induces a higher organs colonization than STM1znuABC at 1 dpi. Piglets were orally infected with 2 × 109 CFU of STM1znuABC (group B) or STMwt (group C), and bacterial burdens were determined at 1 dpi. Differences between groups B and C were estimated using non-parametric Mann–Whitney test and were considered signiﬁcant when P ≤0.05. Organ samples taken from naïve animals (group A) were negative. Error bars represent one SD from the mean. FIGURE 4 \| (A–C) Photomicrographies showing histological changes of the cecum. (A) Naïve control piglets; (B) piglets infected with STM 1znuABC: multifocal and moderate neutrophilic inﬁltrate (arrows), crypt abscess formation; (C) piglets infected with STMwt: marked and diffuse neutrophilic inﬁltration. FIGURE 4 \| (A–C) Photomicrographies showing histological changes of the cecum. (A) Naïve control piglets; (B) piglets infected with STM 1znuABC: multifocal and moderate neutrophilic inﬁltrate (arrows), crypt abscess formation; (C) piglets infected with STMwt: marked and diffuse neutrophilic inﬁltration. Bacterial Diversity of the Fecal Microbiota after Salmonella Infection Gray and gray-black bars represent STM1znuABC- (group B) and STMwt- (group C) infected piglets, respectively. P-values were calculated using One-way ANOVA with Bonferroni’s post-test. Signiﬁcant differences between groups are indicated by P ≤0.05, P ≤0.01, and P ≤0.001. Eub, all bacteria; Lacto, Lactobacillus/Lactococcus group; Clost, Eubacterium rectale/Clostridium coccoides; Bact, Bacteroides sp.; Biﬁdo, Biﬁdobacterium; Prev, Prevotellaceae; Ent, Enterobacteriaceae other than Salmonella; STM, S. Typhimurium. FIGURE 6 \| STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacriﬁced at 1 and 12 dp FIGURE 6 \| STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacriﬁced at 1 and 12 dpi (A,B). Bacterial genomic DNA was isolated from cecal content and qPCR analysis measured the abundance of speciﬁc commensal bacterial groups. White bars represent uninfected controls (group A). Gray and gray-black bars represent STM1znuABC- (group B) and STMwt- (group C) infected piglets, respectively. P-values were calculated using One-way ANOVA with Bonferroni’s post-test. Signiﬁcant differences between groups are indicated by P ≤0.05, P ≤0.01, and P ≤0.001. Eub, all bacteria; Lacto, Lactobacillus/Lactococcus group; Clost, Eubacterium rectale/Clostridium coccoides; Bact, Bacteroides sp.; Biﬁdo, Biﬁdobacterium; Prev, Prevotellaceae; Ent, Enterobacteriaceae other than Salmonella; STM, S. Typhimurium. FIGURE 6 \| STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacriﬁced at 1 and 12 dpi (A,B). Bacterial genomic DNA was isolated from cecal content and qPCR analysis measured the abundance of speciﬁc commensal bacterial groups. White bars represent uninfected controls (group A). Gray and gray-black bars represent STM1znuABC- (group B) and STMwt- (group C) infected piglets, respectively. P-values were calculated using One-way ANOVA with Bonferroni’s post-test. Signiﬁcant differences between groups are indicated by P ≤0.05, P ≤0.01, and P ≤0.001. Eub, all bacteria; Lacto, Lactobacillus/Lactococcus group; Clost, Eubacterium rectale/Clostridium coccoides; Bact, Bacteroides sp.; Biﬁdo, Biﬁdobacterium; Prev, Prevotellaceae; Ent, Enterobacteriaceae other than Salmonella; STM, S. Typhimurium. group B at 2 dpi (p < 0.05). At the same time, Fisher’s alpha conﬁrmed the signiﬁcant lower alfa diversity in group C at 12 dpi compared to group A (p < 0.05; Figure 7B). STMwt (group C) was signiﬁcantly higher than the naïve animals (group A), at 0 and at 2 dpi respectively (p < 0.01). However, group C showed a signiﬁcant lower alfa diversity at 12 dpi than group A (Figure 7A). Bacterial Diversity of the Fecal Microbiota after Salmonella Infection TNF-α, IL1-α, IL1-β, and INF-γ expression was measured in the cecum at 1 and 12 dpi by real time RT-PCR. Gray bars and black bars represent STM1znuABC-infected (group B) and STMwt-infected piglets (group C), respectively. The asterisk indicates statistical signiﬁcance P ≤0.05, Mann–Whitney test. FIGURE 5 \| (A–H) Cytokines expression reveals that unlike STMwt, STM1znuABC strain is not able to induce a strong host immune response. TNF-α, IL1-α, IL1-β, and INF-γ expression was measured in the cecum at 1 and 12 dpi by real time RT-PCR. Gray bars and black bars represent STM1znuABC-infected (group B) and STMwt-infected piglets (group C), respectively. The asterisk indicates statistical signiﬁcance P ≤0.05, Mann–Whitney test. FIGURE 6 \| STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacriﬁced at 1 and 12 dpi (A,B). Bacterial genomic DNA was isolated from cecal content and qPCR analysis measured the abundance of speciﬁc commensal bacterial groups. White bars represent uninfected controls (group A). Gray and gray-black bars represent STM1znuABC- (group B) and STMwt- (group C) infected piglets, respectively. P-values were calculated using One-way ANOVA with Bonferroni’s post-test. Signiﬁcant differences between groups are indicated by P ≤0.05, P ≤0.01, and P ≤0.001. Eub, all bacteria; Lacto, Lactobacillus/Lactococcus group; Clost, Eubacterium rectale/Clostridium coccoides; Bact, Bacteroides sp.; Biﬁdo, Biﬁdobacterium; Prev, Prevotellaceae; Ent, Enterobacteriaceae other than Salmonella; STM, S. Typhimurium. FIGURE 5 \| (A–H) Cytokines expression reveals that unlike STMwt, STM1znuABC strain is not able to induce a strong host immune response. TNF-α, IL1-α, IL1-β, and INF-γ expression was measured in the cecum at 1 and 12 dpi by real time RT-PCR. Gray bars and black bars represent STM1znuABC-infected (group B) and STMwt-infected piglets (group C), respectively. The asterisk indicates statistical signiﬁcance P ≤0.05, Mann–Whitney test. FIGURE 5 \| (A–H) Cytokines expression reveals that unlike STMwt, STM1znuABC strain is not able to induce a strong host immune response. TNF-α, IL1-α, IL1-β, and INF-γ expression was measured in the cecum at 1 and 12 dpi by real time RT-PCR. Gray bars and black bars represent STM1znuABC-infected (group B) and STMwt-infected piglets (group C), respectively. The asterisk indicates statistical signiﬁcance P ≤0.05, Mann–Whitney test. FIGURE 6 \| STMwt and STM1znuABC differently modify cecal microbiota of piglets. Piglets were sacriﬁced at 1 and 12 dpi (A,B). Bacterial genomic DNA was isolated from cecal content and qPCR analysis measured the abundance of speciﬁc commensal bacterial groups. White bars represent uninfected controls (group A). Bacterial Diversity of the Fecal Microbiota after Salmonella Infection B (p < 0.001) and C (p < 0.001) in the cecal content and in group B (p < 0.05) in the colonic one when compared to group A (naïve; Figure 6B; Supplementary Figure 5B). The levels of the Enterobacteriaceae other than Salmonella decreased signiﬁcantly in both groups of animals infected with Salmonella strains in the cecal and colonic contents at 12 dpi (Figure 6B; Supplementary Figure 5B). A higher level of Salmonella, consistent with the microbiological ﬁndings, was observed in group C (STMwt) compared to group B (STM1znuABC) in all the intestinal samples, while Salmonella was never detected in group A (naïve; Figure 6; Supplementary Figures 4, 5). B (p < 0.001) and C (p < 0.001) in the cecal content and in group B (p < 0.05) in the colonic one when compared to group A (naïve; Figure 6B; Supplementary Figure 5B). The levels of the Enterobacteriaceae other than Salmonella decreased signiﬁcantly in both groups of animals infected with Salmonella strains in the cecal and colonic contents at 12 dpi (Figure 6B; Supplementary Figure 5B). A higher level of Salmonella, consistent with the microbiological ﬁndings, was observed in group C (STMwt) compared to group B (STM1znuABC) in all the intestinal samples, while Salmonella was never detected in group A (naïve; Figure 6; Supplementary Figures 4, 5). Massive parallel sequencing of the 16S rDNA hypervariable V3-V4 region was performed on fecal samples available from the three experimental groups A, B, and C. The sequencing yielded a total of 177198 reads passing quality control (median reads per sample 11030). OTU classiﬁcation yielded a median of 5742 OTUs per sample. Sequencing reads are available at http://www.ncbi.nlm.nih.gov/bioproject/ PRJNA302126 (BioProject accession ID: PRJNA302126). We evaluated the bacterial diversity of the fecal microbiota associated with Salmonella strains by estimating alpha- and beta- diversity. Shannon index demonstrated that the fecal microbiota diversity in piglets infected with STM1znuABC (group B) and These results show that S. Thyphimurium is able to alter intestinal microbiota in pigs inducing modiﬁcations correlated to its virulence. January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 6 Drumo et al. Salmonella Alters Gut Microbiota in Piglets FIGURE 5 \| (A–H) Cytokines expression reveals that unlike STMwt, STM1znuABC strain is not able to induce a strong host immune response. Bacterial Diversity of the Fecal Microbiota after Salmonella Infection Using Fisher’s alpha, an index not inﬂuenced by the sample size and less aﬀected by the abundance of the most common species than Shannon’s index, we found a higher diversity in piglets belonging to group C compared to The beta diversity was calculated using both unweighted Unifrac and Bray-Curtis dissimilarity; principal component analysis (PCoA) was performed. As shown in Figure 8A, using Unifrac, four out of ﬁve samples belonging to group C (STMwt) January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 7 Salmonella Alters Gut Microbiota in Piglets Drumo et al. FIGURE 7 \| Structural comparison of fecal microbiota among groups A, B, and C. The Shannon index (A) and Fisher’s alpha (B) were used to estimate diversity of the fecal microbiota in naïve animals (group A) and in STM1znuABC- (group B) and STMwt- (group C) infected piglets. Boxes represent median, and ﬁrst and third quartiles; whiskers indicate minimum and maximum values. The asterisks indicate statistical signiﬁcance P ≤0.05 and *P ≤0.01, Dunn’s test. FIGURE 7 \| Structural comparison of fecal microbiota among groups A, B, and C. The Shannon index (A) and Fisher’s alpha (B) were used to estimate diversity of the fecal microbiota in naïve animals (group A) and in STM1znuABC- (group B) and STMwt- (group C) infected piglets. Boxes represent median, and ﬁrst and third quartiles; whiskers indicate minimum and maximum values. The asterisks indicate statistical signiﬁcance P ≤0.05 and **P ≤0.01, Dunn’s test. with naïve (group A) is that they showed an abundant presence of lactic acid-producing bacteria and a reduction of short chain fatty acids (SCFAs)-producing bacteria (Figures 9A,B). Analysis of data also revealed that piglets infected with STMwt (group C) initially showed a decrease in Prevotella at 2 dpi compared to the naïve (group A). In addition, at 12 dpi, a more abundant presence of Prevotella, Phascolarcobacterium, and Eubacterium was evident in group C (STMwt) rather than in groups A and B. clustered separately along the principal coordinate 1 (PCA1) at 12 dpi. In addition, a clear separation of group B (STM1znuABC) from the rest of the samples is noticeable along the principal coordinate 2 (PCA2). The PCoA using Bray-Curtis dissimilarity did not allow any clear separation of the groups, although all the ﬁve samples belonging to group C (STMwt), at 2 dpi, clustered at the extreme right along the principal coordinate 1 (PCA1; Figure 8B). Bacterial Diversity of the Fecal Microbiota after Salmonella Infection On the light of these data, it can inferred that Shannon and Unifrac results, in which it seem to be diﬀerences among groups at time 0, could be biased by small sample size. Therefore, the microbiota composition of the diﬀerent groups could be considered similar at time 0. Moreover, clustering analysis highlighted the diﬀerences in the sample distributions according to the treatment type. At 2 dpi, the most represented genera displayed a perfect clusterization of each single sample into its belonging study group (Figure 9A). Similarly, at 12 dpi, each piglet grouped into its belonging treatment group, except 2 samples (5 and 12) clustered in a diﬀerent study group (Figure 9B). Moreover, at 12 dpi, groups A (naïve) and B (STM1znuABC) are more similar to each other, while group C (STMwt) featured more relevant eﬀects (Figure 9B). No signiﬁcant diﬀerences were detected when each single group was analyzed longitudinally according to the three collection times. These data show that infection with diﬀerent strains of S. Typhimurium is associated with diﬀerent alterations of fecal microbiota. Salmonella Strains-Associated Alterations in Fecal Microbiota by NGS In order to compare how the composition of the fecal bacteria diﬀered among treatment groups, the Kruskal–Wallis test and the Benjamini-Hochberg FDR method were used to identify diﬀerentially abundant taxa. Genus-level normalized data are available in Supplementary Table 3. The distribution of reads into phyla revealed that the bacterial communities of all samples consisted primarily of Firmicutes and Bacteroidetes. Microbiota analysis showed that 7 phyla, 112 families, 404 genera, and 15 phyla, 143 families, and 719 genera diﬀered across groups A, B, and C, respectively at 2 and 12 dpi (see Supplementary Table 4). Figures 9A,B and Supplementary Figure 6 represent heatmaps showing the genus-level clustering according to frequency within each sample (abundance ratio greater than 0.1) at times 0, 2, and 12 dpi; abundant genera were color coded red, and white color coding indicated missing genera. The most remarkable diﬀerence reported in the piglets infected with STMwt (group C) compared January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org DISCUSSION The importance of pigs as a source of Salmonella in the food chain is well-known. However, while Salmonella pathogenicity has been widely studied in mice, our knowledge on the modality of interaction of this pathogen with pigs is still limited. It has been known that diﬀerent and multiple factors can inﬂuence the dynamics of Salmonella colonization in swine, including January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 8 Salmonella Alters Gut Microbiota in Piglets Drumo et al. GURE 8 \| Principal Coordinate analysis plot (PCoA) of unweighted UniFrac distances (A) and Bray-Curtis dissimilarity (B) for the fecal microbiota ross the three study groups. PCA, principal coordinate. hogen features (virulence mechanisms, exposure dosage), immune responses and the complex interplay between the hogen and the intestinal microbiota (Bearson et al., 2013). this study, we used a post-weaned piglet model to compare erences in the host colonization, inﬂammatory response, and modiﬁcation of the intestinal microbiota induced by STMwt and STM1znuABC in order to elucidate the interplay among host, pathogen and gut microbiota. STM1znuABC was chosen due to the fact that our previous studies have revealed that this strain is strongly attenuated either in mice or in pigs (Ammendola et al., Principal Coordinate analysis plot (PCoA) of unweighted UniFrac distances (A) and Bray-Curtis dissimilarity (B) for the fecal microbiota three study groups. PCA, principal coordinate. FIGURE 8 \| Principal Coordinate analysis plot (PCoA) of unweighted UniFrac distances (A) and Bray-Curtis dissimilarity (B) for the fecal microbiota across the three study groups. PCA, principal coordinate. pathogen features (virulence mechanisms, exposure dosage), pig immune responses and the complex interplay between the pathogen and the intestinal microbiota (Bearson et al., 2013). In this study, we used a post-weaned piglet model to compare diﬀerences in the host colonization, inﬂammatory response, and modiﬁcation of the intestinal microbiota induced by STMwt and STM1znuABC in order to elucidate the interplay among host, pathogen and gut microbiota. STM1znuABC was chosen due to the fact that our previous studies have revealed that this strain is strongly attenuated either in mice or in pigs (Ammendola et al., Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org January 2016 \| Volume 5 \| Article 106 9 Salmonella Alters Gut Microbiota in Piglets Drumo et al. DISCUSSION GURE 9 \| (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B) with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red gh abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5: oup A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. FIGURE 9 \| (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B) or with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red (high abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5: group A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. GURE 9 \| (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B) with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red gh abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5: oup A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. FIGURE 9 \| (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B) or with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red (high abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5: group A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. DISCUSSION Hence, the reduced abundance of SCFA-producing bacteria induced by STMwt could explain the enhanced inﬂammatory status observed in the gastrointestinal tract of piglets treated with this Salmonella strain; and it could be of interest to investigate the mechanisms leading to a reduction of this potentially protective component of the intestinal microbiota. Moreover, upon infection with Salmonella strains, microbiota composition also showed changes in Prevotella, Phascolarcobacterium and Eubacterium. Similarly to what elsewhere reported (Bearson et al., 2013), we observed a decrease of Prevotella in piglets infected with STMwt at 2 dpi. However the limitation of available information about the biological function of such genera makes diﬃcult to extrapolate any signiﬁcant meaning to our ﬁndings. At the same time it should be acknowledged that the alpha and beta diversity patterns across the three groups within the three time points analyzed presented several discrepancies that can be attributable to the sensitivity of the next-generation sequencing technology and to the relative small sample size. However, both alpha-diversity indices converge on a signiﬁcant lower alpha-diversity in group C compared to group A at dpi 12. At the same time, the signiﬁcant diﬀerence found in the whole microbiome composition at time 0 between group A and group B, highlighted by Shannon alpha index and Unifrac beta diversity PCoA may raise the possibility 2007; Pasquali et al., 2008; Pesciaroli et al., 2013). Moreover, studies carried out in a mouse model showed that ZnuABC- mediated zinc uptake confers resistance to the antimicrobial protein calprotectin and promotes Salmonella growth over the competing intestinal microbiota (Liu et al., 2012). Here, we demonstrate that a diﬀerent organs colonization, intestinal inﬂammation and modiﬁcation of porcine microbiota are correlated with the diﬀerent virulence of Salmonella strains. The inﬂammatory response evaluated through the expression of the immune mediators, and corroborated by histological ﬁndings, has shown that STMwt induces a prompt increase of serum markers of inﬂammation during the early stage of infection (1 dpi). Moreover, at the same time point, the expression of tissue-associated markers showed a tendency to increase even if only IL1-β in cecum and colon (p < 0.01) and IL1-α in ileocecal lymph nodes (p < 0.01) reach statistical signiﬁcance. The prompt induction of host response could be due to the rapid and high-level replication of STMwt as showed by our microbiological data. DISCUSSION FIGURE 9 \| (A,B) Heatmap indicating genus-level changes in the microbiota composition of piglets Naïve (group A), and piglets infected with STM1znuABC (group B) or with STMwt (group C) at 2 and 12 dpi. The relative abundance of the most represented genera is indicated by a gradient of color from white (low abundance) to red (high abundance). The hierarchical clustering analysis of the samples, based on the similarity of the microbiota composition, are displayed on the left. Animals 1–5: group A (Naïve), green; animals 6–10: group B (STM1znuABC), blue; piglets 11–15: group C (STMwt), orange. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org January 2016 \| Volume 5 \| Article 106 10 Salmonella Alters Gut Microbiota in Piglets Drumo et al. to survive under conditions of increased redox potential due to the production of reactive oxygen species by granulocytes inﬁltrating the site of inﬂammation as occurs in a highly inﬂamed gut (Videnska et al., 2013). Indeed, there is evidence that lactic acid accumulation could contribute to impair the intestinal defense barrier and increase the osmotic load in the intestinal lumen (Ling et al., 2014). The utilization of next-generation high-throughput sequencing allowed a wider description of the intestinal microbiota. In our study, clustering analysis shows that the microbiota composition changed after infection with Salmonella strains and the characteristics of the modiﬁcations were correlated with the virulence of the strain used. Our analysis reveals a diﬀerent abundance of the most represented genera in piglets infected with STMwt when compared with STM1znuABC and naïve piglets. In fact, microbiota of piglets infected with STMwt was characterized by an overall reduction of SCFA-producing bacteria (Ruminococcaceae including Faecalibacterium, Roseburia, Butyrivibrio, and Clostridium genera). SCFAs such as acetate and butyric acid are produced by microbial fermentation of carbohydrates and provide beneﬁcial immunomodulatory and anti-inﬂammatory properties (Ling et al., 2014). In particular, butyric acid contributes to the inhibition of Salmonella in an acidic environment (Bearson et al., 2006), decreases invasion of intestinal cells by down-regulating expression of Pathogenicity island 1 (Gantois et al., 2006) and reduces the Salmonella-induced proinﬂammatory response of enterocytic cells in vitro (Malago et al., 2005). In line with these observations, previous studies showed that Faecalibacterium, which is correlated with butyrate production, also exhibits anti-inﬂammatory eﬀects counterbalancing intestinal microbiota dysbiosis (Sokol et al., 2008). Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org January 2016 \| Volume 5 \| Article 106 REFERENCES Chirullo, B., Pesciaroli, M., Drumo, R., Ruggeri, J., Razzuoli, E., Pistoia, C., et al. (2015). Salmonella typhimurium exploits inﬂammation to its own advantage in a porcine enteritis model. Front. Microbiol. 6:985. doi: 10.3389/fmicb.2015.00985 Ahmer, B. M. M., and Gunn, J. S. (2011). Interaction of Salmonella Spp. with the intestinal microbiota. Front. Microbiol. 2:101. doi: 10.3389/fmicb.2011.00101 Ammendola, S., Pasquali, P., Pistoia, C., Petrucci, P., Petrarca, P., Rotilio, G., et al. (2007). 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Genet Evol. 16, 330–340. doi: 10.1016/j.meegid.2013.03.022 Hohmann, E. L. (2001). Nontyphoidal salmonellosis. Clin. Infect. Dis. 32, 263–269. doi: 10.1086/318457 Juricova, H., Videnska, P., Lukac, M., Faldynova, M., Babak, V., Havlickova, H., et al. (2013). Inﬂuence of Salmonella enterica serovar enteritidis infection on the development of the cecum microbiota in newly hatched chicks. Appl. Environ. Microbiol. 79, 745–747. doi: 10.1128/AEM.02628-12 Bearson, S. M., Bearson, B. L., and Rasmussen, M. A. (2006). FUNDING This work was supported by ISS intramural research funds and by Transnational Research Project EMIDA ERA-NET “HealthyGut–Multi-focal strategies to improve gut health and reduce enteritis in poultry and pigs”(MIPAF–DM 27373/7303/2010). This work was supported by ISS intramural research funds and by Transnational Research Project EMIDA ERA-NET “HealthyGut–Multi-focal strategies to improve gut health and reduce enteritis in poultry and pigs”(MIPAF–DM 27373/7303/2010). AUTHOR CONTRIBUTIONS RD planned and performed the research, analyzed data and wrote the manuscript. PP designed and planned the research, participated to the interpretation and discussion of the results, and revised the paper; CM planned and performed the research, analyzed data and revised the manuscript. MP, BC, and MT performed part of the research and revised the manuscript. GA, JR, and NM performed part of the research. SA and AB revised the manuscript. GG revised the manuscript. GP and LM performed part of the research and contributed to the analysis of the data. EM and SP performed histological analysis. VN and SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fcimb. 2015.00106 DISCUSSION On the contrary, at 1 dpi, the histological and immunological analysis revealed a mild intestinal inﬂammation and a poor systemic response induced by STM1znuABC conﬁrming characteristics of attenuation in growth and virulence of this strain. As a whole, these observations indicate that the host is able to mount a rapid innate immune response following Salmonella infection within a few hours after gut colonization. The magnitude of the response and the severity of the clinical manifestations provide evidence that the host response and lesions are correlated and dependent to S. Typhimurium virulence. It is known that, similarly to what happens in vitro and in murine models of infection (Barthel et al., 2003; Stecher et al., 2007; Barman et al., 2008), S. Typhimurium strains induce an acute inﬂammatory response in the intestinal mucosa also in piglets (Bearson et al., 2013). Several studies have proved how S. Typhimurium takes advantages of inﬂammation to compete with the resident microbiota and to colonize the inﬂamed gut in mice (Lupp et al., 2007; Stecher et al., 2007; Barman et al., 2008; Winter et al., 2010) and piglets (Chirullo et al., 2015). In our study, we investigated the impact of S. Typhimurium on the porcine intestinal microbial communities. We found that S. Typhimurium infection modiﬁes either the number or the composition of gut resident bacteria. In particular, these changes were associated with STMwt, while the attenuated STM1znuABC seemed to be less ﬁt to sustain competition with the microbiota. These observations are in agreement with the studies performed in mice, where attenuated Salmonella mutants do not colonize intestine as well as wild-type strains as they are not able to trigger an eﬃcient inﬂammatory response (Stecher et al., 2007; Lawley et al., 2008; Raﬀatellu et al., 2009; Winter et al., 2010). The major changes in the microbiota composition are mainly related to the signiﬁcantly more abundant presence of Lactobacillus/Lactococcus group after STMwt infection. This observation is in agreement with the results obtained by Videnska et al. (2013), which showed a signiﬁcant increase of Lactobacillaceae in chicken cecal microbiota after S. Enteritidis infection. A possible explanation could be attributable to the microaerophilic growth of Lactobacilli, which may allow them January 2016 \| Volume 5 \| Article 106 January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 11 Salmonella Alters Gut Microbiota in Piglets Drumo et al. MP performed next-generation sequencing and analyzed data. DISCUSSION PP and CP contributed to perform technical experiments. allow any speculation and further studies using a larger sample size and, possibly, a more detailed time-course is warranted. Overall, our data show that the results of the interaction among Salmonella, the intestinal microbiota and the gut response are inﬂuenced by the speciﬁc characteristics of the three factors. The virulence of Salmonella and the alteration of microbiota composition is crucial in determining the outcome of the infection. ACKNOWLEDGMENTS We thank Massimiliano Francia for technical assistance. 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REFERENCES 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. Santos, R. L., Raﬀatellu, M., Bevins, C. L., Adams, L. G., Tükel, C., Tsolis, R. M., et al. (2009). Life in the inﬂamed intestine, Salmonella Style. Trends Microbiol. 17, 498–506. doi: 10.1016/j.tim.2009.08.008 Copyright © 2016 Drumo, Pesciaroli, Ruggeri, Tarantino, Chirullo, Pistoia, Petrucci, Martinelli, Moscati, Manuali, Pavone, Picciolini, Ammendola, Gabai, Battistoni, Pezzotti, Alborali, Napolioni, Pasquali and Magistrali. 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. Sassone-Corsi, M., and Raﬀatellu, M. (2015). No Vacancy: How beneﬁcial microbes cooperate with immunity to provide colonization resistance to pathogens. J Immunol. 194, 4081–4087. doi: 10.4049/jimmunol.1403169 Sokol, H., Pigneur, B., Watterlot, L., Lakhdari, O., Bermúdez-Humarán, L. G., Gratadoux, J. J., et al. (2008). Faecalibacterium prausnitzii is an anti- inﬂammatory commensal bacterium identiﬁed by gut microbiota analysis of January 2016 \| Volume 5 \| Article 106 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 13
https://openalex.org/W2102652793	https://europepmc.org/articles/pmc3695513?pdf=render	English	null	Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions	Nucleic acids research	2,013	cc-by	8,519	Benchmarking HMMER3 E-values estimates using Pfam Here, we test the ability of the proﬁle hidden Markov model (proﬁle-HMM) method HMMER3 [http://hmmer. janelia.org/) to correctly infer homology. To this end, we use a library of >13 000 proﬁle-HMMs taken from the Pfam database of protein families (15). Pfam’s compre- hensiveness (the fact that Pfam models annotate a large proportion of all residues in UniProt Knowledgebase, UniProtKB (16)] allows us in many cases to identify sources of discrepancies in HMMER3 E-value estimation without using a benchmark. For example, we can look at cases where Pfam annotation, at a given signiﬁcance threshold, would classify the same region of a sequence into two or more families thought to be evolutionarily unrelated. We call this ‘overlap analysis’. Benchmarks are typically derived from protein structure database clas- siﬁcations, but solved protein structures are depleted for some of the most challenging issues in sequence analysis, such as disordered and/or highly biased sequence compos- ition. Overlap analysis in the entire sequence database ABSTRACT Several methods based on sequence–sequence, sequence– proﬁle or proﬁle–proﬁle alignments have been developed to identify evolutionary-related protein sequences (5–10). Current challenges in homology detection via sequence similarity include problems with homologous overexten- sion of alignments (11) and regions of amino acid com- positional bias under convergent evolution (12). Note that the expression ‘compositional bias’ is often used to indicate a bias in amino acid frequencies within a protein sequence region, that is, a deviation from the frequencies typically observed in globular soluble proteins. An example of this bias is the highly polar, highly charged intrinsically disordered regions (13). Convergent evolution, however, may also lead to a differ- ent type of bias, which is reﬂected not in amino acid frequencies but rather in the way amino acids are distributed along the sequence. The heptad repeats that characterize canonical coiled-coils represent a typical example of this bias (14). Detection of protein homology via sequence similar- ity has important applications in biology, from protein structure and function prediction to recon- struction of phylogenies. Although current methods for aligning protein sequences are powerful, chal- lenges remain, including problems with homologous overextension of alignments and with regions under convergent evolution. Here, we test the ability of the profile hidden Markov model method HMMER3 to correctly assign homologous sequences to >13 000 manually curated families from the Pfam database. We identify problem families using protein regions that match two or more Pfam families not currently annotated as related in Pfam. We find that HMMER3 E-value estimates seem to be less accurate for families that feature periodic patterns of compositional bias, such as the ones typically observed in coiled-coils. These results support the continued use of manually curated in- clusion thresholds in the Pfam database, especially on the subset of families that have been identified as problematic in experiments such as these. They also highlight the need for developing new methods that can correct for this particular type of compositional bias. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions Jaina Mistry1,2,, Robert D. Finn3, Sean R. Eddy3, Alex Bateman1,2 and Marco Punta1,2, 1EMBL-European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK, 2Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK and 3HHMI Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, VA 20147, USA Received October 31, 2012; Revised and Accepted March 22, 2013 To whom correspondence should be addressed. Tel: +44 1223 492541; Fax: +44 1223 494468; Email: jaina@ebi.ac.uk Correspondence may also be addressed to Marco Punta. Tel: +44 1223 492541; Fax: +44 1223 494468; Email: mpunta@ebi.ac.uk Nucleic Acids Research, 2013, Vol. 41, No. 12 e121 doi:10.1093/nar/gkt263 Nucleic Acids Research, 2013, Vol. 41, No. 12 e121 doi:10.1093/nar/gkt263 Published online 17 April 2013 ( ) y y This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. *To whom correspondence should be addressed. Tel: +44 1223 492541; Fax: +44 1223 494468; Email: jaina@ebi.ac.uk Correspondence may also be addressed to Marco Punta. Tel: +44 1223 492541; Fax: +44 1223 494468; Email: mpunta@ebi.ac.uk The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. The Author(s) 2013. Published by Oxford University Press. Pfam proﬁle-HMM database Pfam (15) is a database that uses HMMER3 (http:// hmmer.janelia.org/) to group homologous protein regions into families. Pfam 26.0 comprises 13 672 families. Each family is represented by a proﬁle-HMM generated with HMMER3 using a manually curated ‘seed’ alignment. The proﬁle-HMM is searched against UniProtKB (16), (the Pfam protein sequence database of choice), to ﬁnd additional homologous sequences. Pfam curators deﬁne two separate family-speciﬁc signiﬁcance thresholds for including matches into families, one for domains and one for sequences [a domain can match the same sequence multiple times; see (15) for a detailed ex- planation of what the different thresholds represent]. Each family is annotated with functional and/or structural in- formation where available. In this study, to simplify our overlap analysis that is based on family-independent sig- niﬁcance thresholds, we exclude the 316 families where sequence and domain thresholds differ. These families mostly represent repeats in which the domain threshold is set to be lower than the sequence threshold to capture the highest number of occurrences along individual sequences. Known problems in detecting homology via sequence similarity Homology, or evolutionary descent from a common ancestor, is widely used as a basis to transfer structural and functional annotation between proteins (1–3) and to study protein evolution (4). Homology is most often inferred by establishing statistical signiﬁcance of observed similarity in protein sequence alignments. e121 Nucleic Acids Research, 2013, Vol. 41, No. 12 PAGE 2 OF 10 PAGE 2 OF 10 e121 Deﬁnition of overlap reduces this important bias, as Pfam has models represent- ing many such regions, although it has the disadvantage that an overlap does not necessarily imply a false positive (it could be an as-yet unannotated true homology). Nonetheless, manual examination of particular Pfam families that cause an unusual number of overlaps can be used to identify systematic failure modes in HMMER3’s inference of homology. As outlined in the ‘Introduction’ section, overlaps are sequence regions that match two or more Pfam families that belong to different clans, with families in different clans not annotated as related in the Pfam database. To exclude cases that could be resolved by small changes in family boundaries, we consider only overlapping regions that cover 50% of the alignment co-ordinates of at least one of the two families involved. Note: alignment coord- inates represent the region of the sequence for which HMMER3 can produce a reliable alignment in contrast to envelope coordinates that deﬁne the region for which there is substantial probability mass to support a homolo- gous match. Thus, the envelope coordinates span a region that is equal or wider than the alignment coordinates. Pfam overlaps We deﬁne overlaps as sequence regions that at a given signiﬁcance threshold simultaneously match two or more Pfam families found in different clans (i.e. families that are not annotated as evolutionary related in the Pfam database). Additionally, we require that 50% of the region that matches the proﬁle-HMM of one family also matches the proﬁle-HMM of the other (for at least one of the two families). Overlaps are likely to point to some form of erroneous or incomplete annotation. In particu- lar: (i) the proﬁle-HMMs of one or both of the two families might have been built from a set of non-homolo- gous sequences (incorrect Pfam model); (ii) the overlapping families might be evolutionary related and the relationship not yet annotated in Pfam (incomplete Pfam annotation); and (iii) the proﬁle-HMMs might have incorrectly assigned the sequence to one or both of the overlapping families (false positive). We calculate overlaps between families at E-values that are typically used to suggest homology. Although a number of these overlaps will fall into cases (i) to (ii), we expect this set to be enriched in false positives (iii) with respect to all Pfam matches. Calculating Pfam family overlaps A subset of Pfam 26.0 proﬁle-HMMs (13 356, see earlier) was searched against UniProtKB release 2011_06 using HMMER3.0 hmmsearch with default parameters. E- values are as reported by HMMER3 hmmsearch, with no additional correction for having performed multiple proﬁle-HMM searches. We deﬁne a match as a domain alignment that has an E-value less than or equal to a speciﬁed signiﬁcance threshold. We calculate overlaps between different Pfam families’ matches, as deﬁned earlier in the text, for three different E-value thresholds: 0.001, 0.01 and 0.1. Calculating expected number of false positives The expected number of false positives (FP) can be calculated using the equation: ð1Þ FP ¼ N Eseq+N Eseq Edom ð1Þ where N is the number of Pfam families, Eseq is the per sequence E-value threshold and Edom is the per domain E-value threshold. As we excluded families that in Pfam had different sequence to domain signiﬁcance thresholds, in this study sequence and domain E-value thresholds were always the same. The total number of Pfam families N that we considered here was 13 356. It follows that, for example, the expected number of false positives at an E-value 0.01 was 13 356 0.01+13 356 0.01 0.01=135. where N is the number of Pfam families, Eseq is the per sequence E-value threshold and Edom is the per domain E-value threshold. As we excluded families that in Pfam had different sequence to domain signiﬁcance thresholds, in this study sequence and domain E-value thresholds were always the same. The total number of Pfam families N that we considered here was 13 356. It follows that, for example, the expected number of false positives at an E-value 0.01 was 13 356 0.01+13 356 0.01 0.01=135. MATERIALS AND METHODS Pfam proﬁle-HMM database Pfam proﬁle-HMM database Per family count of overlapping clans In Figure 1, on a logarithmic scale, we report the total number of observed domains with overlaps and the number of expected false positives based on the E-value thresholds used for signiﬁcance [see Equation (1) in ‘Materials and Methods’ section]. We see that domains with overlaps largely exceed expected false positives at all reported thresholds. Note, however, that because not all such domains will correspond to false positives (‘Introduction’ section), a direct comparison between these numbers cannot be drawn. When considering the number of overlapping clans in a family, we counted clan overlaps for each family domain after the greedy algorithm was applied. Where a single domain overlapped with multiple clans, all clans were counted. Note that if a family was not a member of a clan, it was treated as being in a clan with only one member family (i.e. itself). Few families contain most domains with overlaps We take 13 356 families from Pfam release 26.0 (15) (see ‘Materials and Methods’ section for the criterion used to exclude some families), and run their corresponding proﬁle-HMMs against UniProtKB (16). We count the number of domains with overlaps generated when using three different E-value thresholds for establishing signiﬁ- cance: 0.001, 0.01 and 0.1. A winner-takes-all greedy al- gorithm is used for assigning domains with overlaps to families so that each such domain belongs to only one family (‘Materials and Methods’ section). Coiled-coil regions and, in part, transmembrane regions are overrepresented in families overlapping with many clans We now focus on families that overlap with multiple clans (‘Materials and Methods’ section). As, from a Pfam Figure 1. Observed number of overlapping domains (dark grey) and expected number of false positives (light grey) at three different E-value signiﬁcance thresholds for the 13 356 Pfam families considered here. Winner-takes-all greedy algorithm for assigning overlapping domains to families We compiled a list that contained all overlapping domains, and a list of overlaps these domains were involved in (note: a domain could be involved in overlaps with multiple other domains; therefore, in a family, the number of overlapping domains was lower than or equal to the number of overlaps). We selected the family with the highest number of overlapping domains and assigned these overlapping domains to it (at E-value 0.01, this would be PF04156 IncA having 308 overlapping domains, see ‘Results’ section). We recalculated the list of overlapping domains in each re- maining family after removing all overlaps that involved Nucleic Acids Research, 2013, Vol. 41, No. 12 e121 PAGE 3 OF 10 RESULTS the ﬁrst family. In particular, domains that overlapped only with domains in the ﬁrst family would disappear from the list. Families left with no overlapping domains would also be removed. Among the remaining families, we again selected the one with the highest number of overlapping domains and repeated all previous steps. We iterated this procedure until no more overlapping domains (and families) were left. We also experimented with assign- ing the overlap to the domain with the highest E-value instead of to the family with the highest number of overlapping domains. Assigning transmembrane, coiled-coil and disordered region labels to Pfam families Next, we rank families according to the number of member domains with overlaps, from highest to lowest, and plot the cumulative proportion of overlaps (Figure 2A). We see that although overlapping families may be numerous, the majority of member domains with overlaps are found in a small number of families. For E- values of 0.001, 0.01 and 0.1, we ﬁnd that 50% of domains with overlaps are found in 22, 30 and 59 families (of 13 356), respectively. At the same signiﬁcance thresholds, 75% of domains with overlaps are found in 87, 124 and 214 families. The presence of transmembrane helices, coiled-coil and disordered regions was predicted in the seed alignments of all families. Transmembrane helices were predicted using Phobius with default parameters (17), coiled-coil regions were predicted using ncoils with default param- eters (http://www.russelllab.org/cgi-bin/coils/coils-svr.pl) and disordered regions using IUPred with the long option (18). For each seed alignment, we determined whether 50% of seed member regions had 20 consecutive residues predicted to be coiled-coil, 20 consecutive residues predicted to be disordered and 2 predicted transmembrane helices. Families that satisﬁed these con- straints were labelled as coiled-coil, transmembrane (order is important) and disordered. Additionally, we also identiﬁed seed alignments that had 50 consecutive pre- dicted coiled-coil regions in 50% of seed members. Note that in these calculations, predicted coiled-coil, transmembrane or disordered residues falling outside of the family seed regions were not taken into consideration. Hence, for example, a family covering a soluble domain of an a-helical integral membrane protein would not be labelled as transmembrane. Coiled-coil regions and, in part, transmembrane regions are overrepresented in families overlapping with many clans Null2 model corrections The HMMER3 hmmsearch output gives a bias compos- ition correction score for each sequence and domain, which is the bit score difference contributed by the null2 model (see ftp://selab.janelia.org/pub/software/hmmer3/3. 0/Userguide.pdf). The sequence and domain scores include this bias composition correction. A high bias score may indicate a false positive, especially if the bias score is larger than the overall bit score. We took the domains that had E-value 0.01 and calculated the pro- portion of families that had >50% of domains with a bias score/bit score ratio >0.1. Figure 1. Observed number of overlapping domains (dark grey) and expected number of false positives (light grey) at three different E-value signiﬁcance thresholds for the 13 356 Pfam families considered here. . (A) Cumulative proportion of overlapping domains in Pfam families. Families are ranked according to the number of their domains that (in descending order) after applying a winner-takes-all greedy algorithm that assigns overlapping domains to families (see ‘Materials and ’ section). Data shown for three E-value signiﬁcance thresholds. (B) Same as 2A red line, with additional plots for families that overlap with more, and three or more clans only (E-value = 0.01). Nucleic Acids Research, 2013, Vol. 41, No. 12 PAGE 4 OF 10 e121 Nucleic Acids Research, 2013, Vol. 41, No. 12 PAGE 4 OF 10 Figure 2. (A) Cumulative proportion of overlapping domains in Pfam families. Families are ranked according to the number of their domains that overlap (in descending order) after applying a winner-takes-all greedy algorithm that assigns overlapping domains to families (see ‘Materials and Methods’ section). Data shown for three E-value signiﬁcance thresholds. (B) Same as 2A red line, with additional plots for families that overlap with two or more, and three or more clans only (E-value = 0.01). Figure 2. (A) Cumulative proportion of overlapping domains in Pfam families. Families are ranked according to the number of their domains that overlap (in descending order) after applying a winner-takes-all greedy algorithm that assigns overlapping domains to families (see ‘Materials and Methods’ section). Data shown for three E-value signiﬁcance thresholds. (B) Same as 2A red line, with additional plots for families that overlap with two or more, and three or more clans only (E-value = 0.01). Null2 model corrections (A) Venn diagram with overlap between families predicted to be coiled-coil, disordered and transmembrane (see ‘Materials and Methods’ section) as observed in 13 356 total families. Coiled-coil: consecutive coiled-coil regions of 20 residues predicted in 50% of seed member regions. Disordered: consecutive intrinsic disordered regions of 20 residues predicted in 50% of seed member regions. Transmembrane helices: 2 trans- membrane helices predicted in 50% of seed members regions. (B) Overrepresentation of predicted coiled-coil, transmembrane helices and instrinsic disorder when considering Pfam families with overlapping domains versus all Pfam families. Overlaps are calculated with respect to an E-value signiﬁcance threshold of 0.01. Families are sorted by the number of clans they overlap with (descending) after a winner-takes-all greedy algorithm for assigning overlapping domains to families is applied. Note: two/three or more means two/three or more clans other than the one the family belongs to. Overrepresentation at each point x in the x axis is obtained by calculating the proportion of families with a given label (e.g. coiled-coil) among the ﬁrst x families and dividing by the proportion of all families (n = 13 356) with that label. Note that for the sake of simplicity, we truncated the x- axis at 400 families. Labels assigned to families as described in 3A. two or more clans (crossing point between the solid red line and the dashed grey vertical line) and almost 7-fold in families that overlap with three or more clans (crossing point between the solid red line and the solid grey vertical line). Overall, we can assign a coiled-coil label to 42% of families that overlap with three or more clans. Overrepresentation correlates with predicted coiled-coil length: 19% of all families that overlap with three or more clans can be labelled as coiled-coil based on pre- dicted consecutive regions of 50 amino acids (instead of 20 as used elsewhere in this article), accounting for a 45-fold enrichment with respect to all 13 356 families considered here. caveat. Predicted disordered residues in overlap regions seem to be highly enriched in residues that are also pre- dicted to be coiled-coil (59% of cases in overlaps versus 4% observed in the entire UniProtKB, Figure 4). The opposite is not true, with the percentage of predicted coiled-coil residues additionally predicted as disordered being similar in overlap regions and in UniProtKB (33% versus 34%). Null2 model corrections In fact, if we consider only the disordered regions that have no predicted coiled-coil residues, we see no overrepresentation in overlapping families. These data suggest that even in disordered families, it is coiled-coil regions, rather than disordered ones, that correlate with the presence of overlaps. two or more clans (crossing point between the solid red line and the dashed grey vertical line) and almost 7-fold in families that overlap with three or more clans (crossing point between the solid red line and the solid grey vertical line). Overall, we can assign a coiled-coil label to 42% of families that overlap with three or more clans. Overrepresentation correlates with predicted coiled-coil length: 19% of all families that overlap with three or more clans can be labelled as coiled-coil based on pre- dicted consecutive regions of 50 amino acids (instead of 20 as used elsewhere in this article), accounting for a 45-fold enrichment with respect to all 13 356 families considered here. Predicted transmembrane families are also overrepresented but to a much lesser extent than coiled- coil ones (2-fold, Figure 3B). Predicted disordered families are overrepresented but with the following Null2 model corrections if at least 50% of the seed alignment regions of a family are predicted to have two or more transmembrane helices, the family is labelled as transmembrane; if the same per- centage of seed sequence regions are predicted to have 20 consecutive amino acids in a coiled-coil region or in an intrinsically disordered region, the family is labelled as coiled-coil or disordered, respectively (‘Materials and Methods’ section, see also Supplementary Materials, ‘labels for all families’ tab, for list of labels). The same family may receive multiple labels (Figure 3A). Note that the seed sequence regions are the manually curated set of representative sequences that are aligned and used to build Pfam families’ proﬁle-HMMs. In Figure 3B, we rank all overlapping families according to the number of clans they overlap with (from most to least, left to right) and look at overrepresentation of each of the aforemen- tioned regions with respect to what was observed in all 13 356 families considered here. Coiled-coil families are overrepresented >4-fold in families that overlap with perspective, these families seem to be particularly promis- cuous, we hypothesize that this set is more likely to have a high false-positive enrichment rate (although it may also include some of the most interesting cases of as yet un- detected evolutionary relationships). Hereafter, for the sake of simplicity, we report results only for E-value 0.01 (see Supplementary Materials, ‘GreedyByDomain E-value 0.01’ tab, for the complete list of overlapping families). Results for E-value 0.1 and 0.001 follow similar trends (data not shown). In Figure 2B, we plot the cumulative distribution of overlaps for families that overlap with two or more, or three or more clans. For comparison, the cu- mulative distribution for all overlapping families is also shown (dark red, same as in Figure 2A). In total, 235 families overlap with two or more clans and 96 families overlap with three or more clans. We next assign, to each family, labels that correspond to three predicted sequence features, all of which represent different types of amino acid compositional bias. In brief, Nucleic Acids Research, 2013, Vol. 41, No. 12 e121 PAGE 5 OF 10 Figure 3. (A) Venn diagram with overlap between families predicted to be coiled-coil, disordered and transmembrane (see ‘Materials and Methods’ section) as observed in 13 356 total families. Coiled-coil: consecutive coiled-coil regions of 20 residues predicted in 50% of seed member regions. Null2 model corrections Disordered: consecutive intrinsic disordered regions of 20 residues predicted in 50% of seed member regions. Transmembrane helices: 2 trans- membrane helices predicted in 50% of seed members regions. (B) Overrepresentation of predicted coiled-coil, transmembrane helices and instrinsic disorder when considering Pfam families with overlapping domains versus all Pfam families. Overlaps are calculated with respect to an E-value signiﬁcance threshold of 0.01. Families are sorted by the number of clans they overlap with (descending) after a winner-takes-all greedy algorithm for assigning overlapping domains to families is applied. Note: two/three or more means two/three or more clans other than the one the family belongs to. Overrepresentation at each point x in the x axis is obtained by calculating the proportion of families with a given label (e.g. coiled-coil) among the ﬁrst x families and dividing by the proportion of all families (n = 13 356) with that label. Note that for the sake of simplicity, we truncated the x- axis at 400 families. Labels assigned to families as described in 3A. PAGE 5 OF 10 Nucleic Acids Research, 2013, Vol. 41, No. 12 e121 Figure 3. (A) Venn diagram with overlap between families predicted to be coiled-coil, disordered and transmembrane (see ‘Materials and Methods’ section) as observed in 13 356 total families. Coiled-coil: consecutive coiled-coil regions of 20 residues predicted in 50% of seed member regions. Disordered: consecutive intrinsic disordered regions of 20 residues predicted in 50% of seed member regions. Transmembrane helices: 2 trans- membrane helices predicted in 50% of seed members regions. (B) Overrepresentation of predicted coiled-coil, transmembrane helices and instrinsic disorder when considering Pfam families with overlapping domains versus all Pfam families. Overlaps are calculated with respect to an E-value signiﬁcance threshold of 0.01. Families are sorted by the number of clans they overlap with (descending) after a winner-takes-all greedy algorithm for assigning overlapping domains to families is applied. Note: two/three or more means two/three or more clans other than the one the family belongs to. Overrepresentation at each point x in the x axis is obtained by calculating the proportion of families with a given label (e.g. coiled-coil) among the ﬁrst x families and dividing by the proportion of all families (n = 13 356) with that label. Note that for the sake of simplicity, we truncated the x- axis at 400 families. Labels assigned to families as described in 3A. Figure 3. Top 20 overlapping families In Table 1, we report the top 20 families in terms of number of overlapping clans. These families overlap e121 Nucleic Acids Research, 2013, Vol. 41, No. 12 PAGE 6 OF 10 PAGE 6 OF 10 Figure 4. Comparison between the proportion of residues predicted to be in coiled-coil and in disordered regions (dark and light grey, respect- ively) in overlaps versus the proportion in UniProtKB (version 2011_06). (MFS_1) and PF12698 (ABC2_membrane_3), belong to two of the largest helical membrane protein clans in Pfam. PF13166 (AAA_13) is in the P-loop NTPase clan (CL0023), which includes ATPase family PF00004 (AAA). Although the ATPase domain is not a coiled-coil struc- ture, PF13166 members seem to be bacterial condensin- like proteins that contain a central zinc-hook motif and have an AAA domain that is split by a large coiled-coil region. For the remaining families, little is known experi- mentally that may conﬁrm or reject the computationally derived hypothesis of them harbouring coiled-coil regions: PF06160 (Ezra) and PF08317 (Spc7) are families involved in cell division, in Firmicutes and Fungi, respectively, and PF04513 (Baculo_PEP_C) is a Baculovirus polyhedron envelope protein. Of the three domains of unknown function in the list, one (PF05701, DUF827) has been recently annotated as containing proteins with weak chloroplast movement under blue light (31), and the new annotation is in Pfam 27.0. Overall, the top 20 overlapping families generate 31% of all overlapping domains and 59% of those that occur in families that overlap with three or more clans. As we have seen, all of these families feature predicted coiled-coil or transmem- brane regions with the predictions supported in several cases by experimental evidence. Figure 4. Comparison between the proportion of residues predicted to be in coiled-coil and in disordered regions (dark and light grey, respect- ively) in overlaps versus the proportion in UniProtKB (version 2011_06). Outside of the top 20 list PF00001 (7tm_1), the seven transmembrane receptor family is the one with the largest number of overlapping domains (231 overlapping domains with four different clans). Of 231 overlaps (note: in this case the number of overlaps is equal to the number of overlapping domains), 226 occur with PF07264 (EI24), a family of multi-span integral membrane proteins believed to be involved in growth suppression and apop- tosis in eukaryotes (32). with at least 10 different clans. Top 20 overlapping families All can be labelled with one or more of the compositionally biased regions of Figure 3A. In particular, 18 families are labelled as coiled-coil, 4 as transmembrane and 2 as both. Ten families can additionally be labelled as disordered. In most cases, overlap regions are enriched in compositional bias (second to last column in Table 1). ( ) PF04156 (IncA) is the top family both in terms of number of domains with overlaps (308) and of overlapping clans (61). The family is restricted to Chlamydiaceae and is functionally associated with the homotypic fusion of the inclusions of these obligate intra- cellular parasites (19). Sequences in this family are believed to include two N-terminal transmembrane segments and a coiled-coil region (20). Families with the most overlaps to IncA include membrane families PF00664 (ABC_membrane; 49 overlaps) and PF06305 (DUF1049; 33 overlaps), and coiled-coil families PF04582 (Reo_sigmaC; 45 overlaps), PF13166 (AAA_13; 35 overlaps) and PF01576 (Myosin_tail_1; 29 overlaps). Interestingly, PF04156 (IncA) is the family with the longest HMMER3 tested running time (21). Slowness of HMMER3 on PF04156 is related to the compositional bias of its sequence (21). Among the other families, 10 have well known and characterized members featuring coiled-coil regions. These include PF01576 (Myosin_tail_1) (22), PF04582 (Reo_sigmaC) (23), PF00038 (Filament) (24), PF00261 (Tropomyosin) (25), PF12718 (Tropomyosin_1) (25), PF08614 (ATG16) [see coiled-coil structures 3A7P and 3A70 in the Protein Data Bank (26)], PF00015 (MCPsignal) (27), PF00769 (ERM) (28), PF10473 (Cenp-F_leu_zip) (29) and PF07926 (TPR_MLP1_2) (30). Two families, PF07690 Overlapping regions enriched in predicted coiled-coil and transmembrane residues The 20 Pfam-A families (Pfam 26.0) with the highest number of overlapping clans at an E-value threshold of 0.01, after a winner-takes-all greedy algorithm for assigning overlapping domains to families was applied (see ‘Materials and Methods’ section) Pfam-A ID (accession) Description Number of domains in the family Number of domains with overlaps Number of clans the family overlaps with TMH CC DIS Proportion of residues in overlap regions predicted to be TMH, CC or DIS (%) Pfam sequence E-value threshold Table 1. Overlapping regions enriched in predicted coiled-coil and transmembrane residues The 20 Pfam-A families (Pfam 26.0) with the highest number of overlapping clans at an E-value threshold of 0.01, after a winner-takes-all greedy algorithm for assigning overlapping domains to families was applied (see ‘Materials and Methods’ section) Pfam-A ID (accession) Description Number of domains in the family Number of domains with overlaps Number of clans the family overlaps with TMH CC DIS Proportion of residues in overlap regions predicted to be TMH, CC or DIS (%) Pfam sequen E-value threshold IncA (PF04156) Inclusion membrane protein A 1231 308 61 3 3 5 62.6 1.5e-10 Myosin_tail_1 (PF01576) This family consists of the coiled-coil myosin heavy chain tail region 2383 271 49 5 3 3 82.4 3.0e-11 AAA_13 (PF13166) Many of the proteins in this family are conjugative transfer protein 986 170 45 5 3 5 77.6 3.0e-09 Reo_sigmaC (PF04582) A homo trimer, this family features an a-helical triple coiled-coil 456 116 41 5 3 5 61.0 9.8e-08 Baculo_PEP_C (PF04513) Baculovirus polyhedron envelope protein, C-terminus 189 52 35 5 3 5 62.9 9.9e-07 EzrA (PF06160) This family represents proteins involved in bacterial cell division 669 28 25 5 3 5 72.7 1.8e-05 DUF827 (PF05701) Domain of unknown function 474 172 23 5 3 3 43.6 3.3e-05 Filament (PF00038) This family represents the coiled-coil central rod domain of intermediate ﬁlament proteins 2315 35 22 5 3 5 76.3 2.4e-07 DUF869 (PF05911) Domain of unknown function 246 15 20 5 3 3 78.8 9.6e-08 MFS_1 (PF07690) This family belongs to the major facilitator superfamily of membrane transporters 175 494 30 19 3 5 5 62.1 2.6e-05 Tropomyosin (PF00261) Tropomyosin is an a-helical protein that forms a coiled-coil structure of two parallel helices 1389 39 19 5 3 3 80.9 6.5e-06 ATG16 (PF08614) Autophagy proteins 16 have a dimeric coiled- coil structure 298 26 17 5 3 3 74.3 7.6e-06 MCPsignal (PF00015) This family represents the coiled-coil bacterial cytoplasmic domain of chemotaxis receptors 20 808 50 17 5 3 5 41.2 3.0e-4 ERM (PF00769) This family represents the coiled-coil domain of ERM proteins 311 11 15 5 3 3 77.7 3.1e-4 Cenp-F_leu_zip (PF10473) This family represents a microtubule-binding protein consisting of long coiled-coil regions 138 76 14 5 3 5 60.7 3.4e-4 Spc7 (PF08317) This family represents proteins involved in cell division in Fungi 183 24 14 5 3 3 69.3 9.2e-06 Tropomyosin_1 (PF12718) Tropomyosin_1, in the same clan as Tropomyosin aforementioned 964 28 14 5 3 3 92.4 2.9e-4 ABC2_membrane_3 (PF12698) ABC-2 membrane transporter family 17 995 60 10 3 5 5 34.8 6.3e-05 TPR_MLP1_2 (PF07926) Proteins in this family feature coiled-coil regions 193 12 10 5 3 3 58.5 2.8e-4 DUF3584 (PF12128) Domain of unknown function 173 14 10 3 3 3 68.3 1.4e-4 For the purposes of counting the number of clans that a family overlaps with, a family that is not in a clan was counted as being in a clan by itself. Overlapping regions enriched in predicted coiled-coil and transmembrane residues So far, we have analysed the presence of different types of amino acid compositional bias in the seed regions of families with overlapping domains. This allowed us to see that many families featuring overlapping domains, es- pecially when overlaps occur with multiple Pfam clans, are rich in coiled-coil regions. We now want to verify whether the overlap regions themselves are enriched in residues predicted to be part of coiled-coil or other compositionally biased regions. We see that 22% of residues in all overlap regions are predicted to be coiled-coil (Figure 5, red bars); this is 17 times more than what was observed in UniProtKB and 26 times more than observed in all Pfam domains at an E-value threshold of 0.01. The per- centage of residues predicted to be coiled-coil increases for the overlap regions of families that overlap with at least two or three different clans (29 and 36%, respectively). In the top 20 families reported in Table 1, nearly 55% of the residues in the overlapping regions are predicted to be coiled-coil. Overlap regions are also enriched in residues predicted to be in transmembrane helices (Figure 5, blue bars) and in disordered regions (Figure 5, green bars), albeit to a much lower extent. As discussed earlier in the text, overrepresentation of predicted disordered residues PAGE 7 OF 10 Nucleic Acids Research, 2013, Vol. 41, No. 12 e121 ses of counting the number of clans that a family overlaps with, a family that is not in a clan was counted as being in a clan by itself. Note that ‘number of domains in the family’ is er of regions aligned to the family proﬁle HMM with E-value 0.01, before clan competition. Columns ‘TMH’, ‘CC’ and ‘DIS’ show whether the families have 2 transmembrane , or consecutive coiled-coil regions of 20 residues (CC), or consecutive disordered regions of 20 residues (DIS), predicted in 50% of seed member regions. E-values in the last culated from the sequence gathering threshold, i.e. the family-speciﬁc bit score signiﬁcance threshold (15), according to the following formula: E = N exp[·(x t)], where x is athering threshold, and t are parameters derived from the proﬁle-HMM model ( is the slope parameter, t is the location parameter) and N is the database size (in this case the tKB). Table 1. Overlapping regions enriched in predicted coiled-coil and transmembrane residues Note that ‘number of domains in the family’ the total number of regions aligned to the family proﬁle HMM with E-value 0.01, before clan competition. Columns ‘TMH’, ‘CC’ and ‘DIS’ show whether the families have 2 transmembra helices (TMH), or consecutive coiled-coil regions of 20 residues (CC), or consecutive disordered regions of 20 residues (DIS), predicted in 50% of seed member regions. E-values in the la column are calculated from the sequence gathering threshold, i.e. the family-speciﬁc bit score signiﬁcance threshold (15), according to the following formula: E = N exp[·(x t)], where x the bit score gathering threshold, and t are parameters derived from the proﬁle-HMM model ( is the slope parameter, t is the location parameter) and N is the database size (in this case t size of UniProtKB). e121 Nucleic Acids Research, 2013, Vol. 41, No. 12 PAGE 8 OF 10 Figure 5. Proportion of residues in predicted transmembrane helices, coiled-coil regions and intrinsically disordered regions in different sets of sequences: UniProtKB (version 2011_06), all domains in the 13 356 Pfam families that we consider in this study, all overlapping regions, overlapping regions of families that overlap with two or more and three or more clans, overlapping regions of the top 20 families in Table 1. Both Pfam domains and overlapping regions are calculated based on an E-value threshold of 0.01. Figure 5. Proportion of residues in predicted transmembrane helices, coiled-coil regions and intrinsically disordered regions in different sets of sequences: UniProtKB (version 2011_06), all domains in the 13 356 Pfam families that we consider in this study, all overlapping regions, overlapping regions of families that overlap with two or more and three or more clans, overlapping regions of the top 20 families in Table 1. Both Pfam domains and overlapping regions are calculated based on an E-value threshold of 0.01. seems to be related to the fact that many of these residues are also predicted to be part of coiled-coil regions (Figure 4). proﬁle-HMM’s emission states, within the aligned region. The bias correction, i.e. the bit score difference contributed by the null2 model, is reported as part of the HMMER3 output. In practice, bias corrections that compare in magnitude with ﬁnal bit score values should be regarded with suspicion and may be indicative of false positive hits. DISCUSSION Aligning and establishing homology between sequences with amino acid compositional bias is difﬁcult. Sequence comparison methods, including HMMER3, evaluate whether the query and target seem more likely to be hom- ologous (under a model of residue similarity scores), versus random non-homologous sequences. Some low- complexity regions of proteins convergently evolve similar sequences that can score more highly in such sequence comparison than random sequences are expected to. These scores may be ‘statistically signiﬁcant’, rejecting the null hypothesis (i.e. matches are not recognized as random), but only because the sequences do not ﬁt the simple null hypothesis. This is the reason Overlapping regions enriched in predicted coiled-coil and transmembrane residues If, for example, we calculate the number of families for which >50% of the member sequences have a bias correction of >10% of their bit score value (member sequences determined according to an E-value threshold of 0.01), we see that 744 of the 13 356 satisfy these con- straints. Thirty-six of them are found among the families that overlap with three or more clans (96 families overall), accounting for >6-fold enrichment in this set. Similarly, we observe >5-fold enrichment when considering families such that 75% of sequences have a bias correction >10% of their bit score values. These data indicate that the relative magnitude of the bias correction of a sequence match can provide some indication about the likelihood of matches to be false positives. E-value-based strategy for assigning overlapping domains to families gives similar overrepresentation results The data we reported in the last two sections are obtained using an E-value-independent protocol to assign overlapping domains to families, when E-values are the quantity that is being tested. We have, however, also ex- perimented with an assignment strategy where the overlapping domain between two families is ascribed to the family that matches with the higher E-value (in other words, the family with the lower E-value is instead awarded the genuine match). This alternative way of as- signing overlaps produces overrepresentation curves (data not shown) that show similar trends with respect to the ones in Figure 3B. Predicted coiled-coil regions, for example, are overrepresented 6- and 10-fold in families that overlap with two or more or three or more clans, respectively. In the same way, when compiling the list of top 20 overlapping families, 15 of the 20 families are shared with the list in Table 1. Nineteen families in this alternative top 20 list are labelled as coiled-coil and three are labelled as transmembrane (with two having both labels). PAGE 9 OF 10 for so many ad hoc methods for low-complexity sequence masking and biased composition score corrections (33–35). HMMER3 has two unpublished biased compos- ition methods. As both are based on an independent and identically distributed random null hypothesis, however, they only model biased sequence composition (i.e. zeroth order effects), not higher order correlations, such as short-period tandem repetitions. Coiled-coil regions, with their characteristic heptad repeat pattern (14), are an example of non-zeroth order correlations in protein amino acid sequences. Patterns of alternating hydropho- bic membrane-embedded and polar soluble regions may produce a similar effect in some multi-span membrane proteins. This can explain why families that appear prob- lematic from our analysis often feature coiled-coil or transmembrane regions. program HMMER3 assigns protein sequences to homolo- gous families. We identify a small set of families (repre- senting 1–2% of the total) that, according to our analysis, are likely to be enriched in false positives at E-value sig- niﬁcance thresholds generally used to assign homology. We ﬁnd that sequences in these families often harbour regions that exhibit periodic patterns of compositional bias. These include coiled-coiled and, to a lesser extent, multi-span helical transmembrane domains, both examples of regions that are believed to have arisen inde- pendently in non-homologous proteins following conver- gent evolution. Manual curation of homology thresholds, as implemented in the Pfam database, can help minimize the number of false positives in families with this type of amino acid bias, but development of better bias correction methods is needed to prevent this problem from occurring in automatic database searches. for so many ad hoc methods for low-complexity sequence masking and biased composition score corrections (33–35). HMMER3 has two unpublished biased compos- ition methods. As both are based on an independent and identically distributed random null hypothesis, however, they only model biased sequence composition (i.e. zeroth order effects), not higher order correlations, such as short-period tandem repetitions. Coiled-coil regions, with their characteristic heptad repeat pattern (14), are an example of non-zeroth order correlations in protein amino acid sequences. Patterns of alternating hydropho- bic membrane-embedded and polar soluble regions may produce a similar effect in some multi-span membrane proteins. This can explain why families that appear prob- lematic from our analysis often feature coiled-coil or transmembrane regions. Conﬂict of interest statement. None declared. Conﬂict of interest statement. None declared. Conﬂict of interest statement. None declared. Supplementary Data are available at NAR Online. Supplementary Data are available at NAR Online. Supplementary Data are available at NAR Online. REFERENCES 1. Friedberg,I. (2006) Automated protein function prediction—the genomic challenge. Brief. Bioinform., 7, 225–242. 2. Punta,M. and Ofran,Y. (2008) The rough guide to in silico function prediction, or how to use sequence and structure information to predict protein function. PLoS Comput. Biol., 4, e1000160. 3. Loewenstein,Y., Raimondo,D., Redfern,O.C., Watson,J., Frishman,D., Linial,M., Orengo,C., Thornton,J. and Tramontano,A. (2009) Protein function annotation by homology- based inference. Genome Biol., 10, 207. PAGE 9 OF 10 g It should be emphasized, however, that our overlap analysis suggests false positive enrichment for only a small fraction of all Pfam families that feature coiled- coil and transmembrane regions. Of 13 356 families con- sidered here, we have classiﬁed 806 and 1863 as coiled-coil and transmembrane, respectively. For 89% of coiled-coil and 91% of transmembrane families, we do not observe any overlap when considering an E-value signiﬁcance threshold of 0.01, leaving us with no evidence of false positives occurring in these families. On the other hand, among the 56 families featuring coiled-coil regions of 50 consecutive amino acids, 41% produce overlaps and 32% produce overlaps with three or more clans. This suggests that the longer the coiled-coil region, the more problem- atic it is for HMMER3 to discriminate between homo- logues and non-homologues. ACKNOWLEDGEMENTS The authors are grateful for the infrastructure support provided by the Systems, Web and Database administra- tion teams at Wellcome Trust Sanger Institute (WTSI). M.P. would like to thank Johannes So¨ ding of the Ludwig Maximilian University in Munich (Germany) and Lars Barquist, Penny Coggill and Ruth Eberhardt of WTSI for useful discussions. Overall, our results suggest that the ‘random sequence’ null hypothesis in the denominator of every method’s log- odds score test [including HMMER3, BLAST (7) and FASTA (5)] is oversimpliﬁed. In fact, we expect non-hom- ologous sequences to contain other higher-order features ‘by chance’, such as coiled-coil; hence, perhaps such common convergent features should be included in the null model. Monitoring HMMER3 bias correction can help identifying problematic families One of the two methods that HMMER3 uses to handle sequences that have a biased composition is an unpub- lished method called null2. The null2 method retests a local sequence comparison under the following null hy- pothesis: that it was generated from an independent and identically distributed random sequence composition calculated from the weighted mean composition of the Nucleic Acids Research, 2013, Vol. 41, No. 12 e121 PAGE 9 OF 10 FUNDING Wellcome Trust [WT077044/Z/05/Z]; BBSRC Bioinformatics and Biological Resources Fund [BB/ F010435/1]; Howard Hughes Medical Institute (to S.R.E. and R.D.F.); EMBL-European Bioinformatics Institute (to J.M. and A.B.). Funding for open access charge: Wellcome Trust Sanger Institute. Finally, we note that Pfam relies on manually curated family-speciﬁc bit score thresholds for deﬁning signiﬁ- cance. One of the leading criteria in deﬁning such thresh- olds is to avoid overlaps with other, supposedly unrelated families. As a consequence, we can see that thresholds in the top 20 overlapping families (Table 1) tend to be on the conservative side (i.e. correspond to low E-values). 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https://openalex.org/W2043267365	https://zenodo.org/records/2484987/files/article.pdf	Latin	null	LXVI. Neue Formen von <i>Glaucium</i> aus Nord‐ und Westpersien	Feddes repertorium specierum novarum regni vegetabilis	1,906	public-domain	436	1) Nomen omiditandurn ob GI, leiomrpum Boiss., quocum facile confundatur. F. Fed,de: Neue Formen von Glaucium aus Nord- und Westpersien. F. Fed,de: Neue Formen von Glaucium aus Nord- und Westpersien. 215 (Origineldiagnosen.) I . Glaudmm grdifEorzcm Boiss. var. malaoooarpum (Hausskn.) Fedde apud Bornmtiller in Beih. Bot. Centrbl., XIX, ser. 2 (1905), p. 203 (nomen nudum); diagn. nov. ) g G1. malacocarpum Hausskn. in sched.! Herba mod0 20-25 cm alta. Folia minus profunde incisa. Siliqua quasi flacoida, non tam robusta quam formae typicae, valde elongata, fere longior quam oetera herba, usque ad 15 cm longa, valde angusta, setis brevioribus minus adpressis obsita. p West-Persien: Prov. Irakadjmi, Sultanabad bei Latetar (Strauss 1 89 7 !) . ) Nota: J. Bornmiiller suspicatw hanc varietatem formam apricam. 2. Glawim Haussheohtii Bornmuller et Fedde apud Bornmiiller, 1. c., p. 203 (nomen nudum); diagn. nov. Herba dense virgulta, foliis basalibus junioribus caulibusque fere aequilongis, circiter 15 cm alta, tomentella perennis (?) radice robusto perpendiculari. Caules multi e radicis collo dense rosulatim enascentes, sparsim dichotome ramosi, foliosi. Folia basalia petiolata rosulata, primaria circiter 7--8 cm longa, sub aestivatione mod0 submarcida incumbentia, juniora 10-15 om ionga erecta, subpinnatipartita, segmentis ala plerum- que angustissima inter se conjunctis, mboppositis basi lata sessilibus et cum oppositis confluentibus, inferiori bus triangularibus, superioribus am- bitu late obovatis pinnatim lohatis, lobis late oblongis ad apicem late cuneato -rotundatis setula terminatis, lob0 terminali segmentorum sub- quadrato-cruciformi, supra sparsim, subtus densius tomentella. caulina basalibus similia, sed minora. Alabastra oblongo-fusiformia ad apicem paulatim acutata, tomentella, 2-3 cm longa. Petala magna violacea (?), 2-3 cm longa. Siliqua recta setis longis adpressis hirsuta (matura non visa!), stigmate subhastato, lobis deflexis, capsula vix latiore. g p West -Persien: Prov. Irakadjmi, Chanekin bei Sultanabad (S trauss 1894 !). West -Persien: Prov. Irakadjmi, Chanekin bei Sultanabad (S trauss 1894 !). 1894 !). Nota: Affinis .G2. gra'andifloro, a quo differt habitu, foliorum forma et (?) florum colore. Nota: Affinis .G2. gra'andifloro, a quo differt habitu, foliorum forma et (?) florum colore. 3. GZazsCizLnt degans Fischer et hleyer var. Bornmiilleri Fedde, nov. var. GI. eleguns f. Zeiocaqa') Bornm., Iter Pers. alt. (1902), no. 6087b! Herba fere omnino glabrescens. Siliquae fere glabrae, in terdum mod0 setis conicis adpressis perpaucis instructae. 3. GZazsCizLnt degans Fischer et hleyer var. Bornmiilleri Fedde, nov. var. GI. eleguns f. Zeiocaqa') Bornm., Iter Pers. alt. (1902), no. 6087b! 3. GZazsCizLnt degans Fischer et hleyer var. Bornmiilleri Fedde, nov. var. GI. eleguns f. Zeiocaqa') Bornm., Iter Pers. alt. (1902), no. 6087b! Herba fere omnino glabrescens. Siliquae fere glabrae, in terdum mod0 setis conicis adpressis perpaucis instructae. Nord-Persien: Scheheristanek im Elburs (Bornm., 1. c., no. 6087b!). p p p
https://openalex.org/W4211016647	https://www.pure.ed.ac.uk/ws/files/301141044/cells_11_00618.pdf	English	null	Diagnosis, Prognosis and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors	Cells	2,022	cc-by	29,122	Edinburgh Research Explorer Published In: C ll General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Diagnosis, Prognosis and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors Citation for published version: Nardi, ABD, Horta, RDS, Fonseca-Alves, CE, Paiva, FND, Linhares, LCM, Firmo, BF, Sueiro, FAR, Oliveira, KDD, Lourenço, SV, Strefezzi, RDF, Brunner, CHM, Rangel, MMM, Jark, PC, Castro, JLC, Ubukata, R, Batschinski, K, Sobral, RA, Cruz, NOD, Nishiya, AT, Fernandes, SC, Cunha, SCDS, Gerardi, DG, Challoub, GSG, Biondi, LR, Laufer-Amorim, R, Paes, PRDO, Lavalle, GE, Huppes, RR, Grandi, F, Vasconcellos, CHDC, Anjos, DSD, Luzo, ÂCM, Matera, JM, Vozdova, M & Dagli, MLZ 2022, 'Diagnosis, Prognosis and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors', Cells, vol. 11, no. 4, 618, pp. 1-37. https://doi.org/10.3390/cells11040618 Citation for published version: Digital Object Identifier (DOI): 10.3390/cells11040618 Link: Link to publication record in Edinburgh Research Explorer Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record cells cells Review Diagnosis, Prognosis and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors Andrigo Barboza de Nardi 1 , Rodrigo dos Santos Horta 2, Carlos Eduardo Fonseca-Alves 3,4 , Felipe Noleto de Paiva 1 , Laís Calazans Menescal Linhares 1 , Bruna Fernanda Firmo 5, Felipe Augusto Ruiz Sueiro 6 , Krishna Duro de Oliveira 7, Silvia Vanessa Lourenço 8 , Ricardo De Francisco Strefezzi 9 , Carlos Henrique Maciel Brunner 10, Marcelo Monte Mor Rangel 11, Paulo Cesar Jark 12, Jorge Luiz Costa Castro 13, Rodrigo Ubukata 14, Karen Batschinski 14, Renata Afonso Sobral 15, Natália Oyafuso da Cruz 16, Adriana Tomoko Nishiya 17, Simone Crestoni Fernandes 18 , Simone Carvalho dos Santos Cunha 19, Daniel Guimarães Gerardi 20, Guilherme Sellera Godoy Challoub 21, Luiz Roberto Biondi 22 , Renee Laufer-Amorim 23 , Paulo Ricardo de Oliveira Paes 2, Gleidice Eunice Lavalle 24, Rafael Ricardo Huppes 25, Fabrizio Grandi 26, Carmen Helena de Carvalho Vasconcellos 27, Denner Santos dos Anjos 1,28 , Ângela Cristina Malheiros Luzo 29 , Julia Maria Matera 30 , Miluse Vozdova 31 and Maria Lucia Zaidan Dagli 32,* 1 Department of Veterinary Clinic and Surgery, Universidade Estadual Paulista (UNESP), 1 Department of Veterinary Clinic and Surgery, Universidade Estadual Paulista (UNESP), Jaboticabal 14884-900, Brazil; andrigobarboza@yahoo.com.br (A.B.d.N.); n-paiva@hotmail.com (F.N.d.P.); laiscmlinhares@gmail.com (L.C.M.L.); denner.anjosoncology@gmail.com (D.S.d.A.) Jaboticabal 14884-900, Brazil; andrigobarboza@yahoo.com.br (A.B.d.N.); n-paiva@hotmail.com (F.N.d.P.); l i li h @ il (L C M L ) d j l @ il (D S d A ) Jaboticabal 14884-900, Brazil; andrigobarboza@yahoo.com.br (A.B.d.N.); n-paiva@hotmail.com (F.N.d.P.); laiscmlinhares@gmail.com (L.C.M.L.); denner.anjosoncology@gmail.com (D.S.d.A.) Citation: de Nardi, A.B.; dos Santos Horta, R.; Fonseca-Alves, C.E.; de Paiva, F.N.; Linhares, L.C.M.; Firmo, B.F.; Ruiz Sueiro, F.A.; de Oliveira, K.D.; Lourenço, S.V.; De Francisco Strefezzi, R.; et al. Diagnosis, Prognosis and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors. Cells 2022, 11, 618. https://doi.org/ 10.3390/cells11040618 Academic Editor: Alexander E. Kalyuzhny Received: 10 December 2021 Accepted: 3 February 2022 Published: 10 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 2 Department of Veterinary Medicine and Surgery, Veterinary School, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil; rodrigohorta@ufmg.br (R.d.S.H.); paulopaes@vet.ufmg.br (P.R.d.O.P.) Take down policy p y The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 3 Institute of Health Sciences, Universidade Paulista (UNIP), Bauru 17048-290, Brazil; carlos.e.alves@unesp.br 4 Department of Veterinary Surgery and Animal Reproduction, Universidade Estadual Paulista (UNESP), Botucatu 18618-681, Brazil Citation: de Nardi, A.B.; dos Santos Horta, R.; Fonseca-Alves, C.E.; de Paiva, F.N.; Linhares, L.C.M.; Firmo, B.F.; Ruiz Sueiro, F.A.; de Oliveira, K.D.; Lourenço, S.V.; De Francisco Strefezzi, R.; et al. Diagnosis, Prognosis and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors. Cells 2022, 11, 618. https://doi.org/ 10.3390/cells11040618 Academic Editor: Alexander E. Kalyuzhny Received: 10 December 2021 Accepted: 3 February 2022 Published: 10 February 2022 C.M.; 5 Department of Veterinary Medicine, Universidade Federal do Paraná, Curitiba 80035-050, Brazil; brunaf_vetxlv@hotmail.com 6 Histopathological Diagnosis Department, VETPAT—Animal Pathology & Molecular Biology, 7 Surgical Pathology, Vet Câncer LTDA-ME, São Paulo 04523-013, Brazil; vclabpathology@gmail.com 8 7 Surgical Pathology, Vet Câncer LTDA-ME, São Paulo 04523-013, Brazil; vclabpathology@gmail.com 8 General Pathology Department, Dental School, Universidade de São Paulo (USP), São Paulo 05508-000, Brazil; il i l @ b g gy, , , ; p gy g 8 General Pathology Department, Dental School, Universidade de São Paulo (USP), São Paulo 05508-000, Brazil; silvialourenco@usp.br 9 Laboratory of Comparative and Translational Oncology (LOCT), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, Universidade de São Paulo (USP), Pirassununga 13635-900, Brazil; rstrefezzi@usp.br 10 Institute of Health Sciences, Universidade Paulista (UNIP), São Paulo 01533-000, Brazil; carlosbrunner@hotmail.com 11 Clinical and Surgical Oncology, Vet Cancer Animal Oncology and Pathology, São Paulo 04523-013, Brazil; diretoriavetcancer@gmail.com 11 Clinical and Surgical Oncology, Vet Cancer Animal Oncology and Pathology, São Paulo 04523-013, Brazil; diretoriavetcancer@gmail.com 12 Clinical Oncology, Onccarevet e Onconnectionvet, Ribeirão Preto 14026-587, Brazil; paulocjark@hotmail.com 13 13 Técnica Cirúrgica da Pontifícia, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba 80215-901 Brazil; castrojlc@jorgecastrovet com 13 Técnica Cirúrgica da Pontifícia, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba 80215-901, Brazil; castrojlc@jorgecastrovet.com 13 Técnica Cirúrgica da Pontifícia, Pontifícia Universidade Católica do Paraná (PUCPR), C i ib 80215 901 B il jl @j 13 Técnica Cirúrgica da Pontifícia, Pontifícia Universidade Católica do Paraná (PUC 14 Clinical and Surgical Oncology, E+ Veterinary Specialties, São Pau ubukata@gmail.com (R.U.); karen.batschinski@gmail.com (K.B.) 14 Clinical and Surgical Oncology, E+ Veterinary Specialties, São Paulo 04078-012, Brazil; Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. https://doi.org/10.3390/cells11040618 https://www.mdpi.com/journal/cells Cells 2022, 11, 618 2 of 37 23 Department of Veterinary Clinic, School of Veterinary Science and Animal Health, Universidade Estadual Paulista (UNESP), Botucatu 18618-681, Brazil; Renee.laufer-amorim@unesp.br 23 Department of Veterinary Clinic, School of Veterinary Science and Animal Health, Universidade Estadual Paulista (UNESP), Botucatu 18618-681, Brazil; Renee.laufer-amorim@unesp.br ( ) p 24 School of Veterinary, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil; gleidicel@yahoo.com.br 24 School of Veterinary, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil; gleidicel@yahoo.com.br g y 25 Surgery Department, Univet Veterinary Clinic—São José Do Rio Preto, São José do Rio Preto 15085-420, Brazil; rafaelhuppes@hotmail.com 25 Surgery Department, Univet Veterinary Clinic—São José Do Rio Preto, São José do Rio Preto 15085-420, Brazil; rafaelhuppes@hotmail.com 26 Vetschool São Paulo: Veterinária, Universidade Estadual Paulista (UNESP), São Paulo 03308-010, Brazil; fgrandivet@gmail.com g g 27 Surgical and Clinical Oncology, Bota Fogo Veterinary Hospital, Rio de Janeiro 22281-180, Brazil; carmenvasconcellos@hotmail.com 28 Eletro-Onkovet Service, Franca 14406-005, Brazil 28 Eletro-Onkovet Service, Franca 14406-005, Brazil 29 Department of Surgery, Medical Sciences College, Universidade Estadual de Campinas (UNICAMP), 29 Department of Surgery, Medical Sciences College, Universidade Estadual de Campinas (UNICAMP), Campinas 13083-970, Brazil; angela.luzo@gmail.com 30 Department of Surgery, School of Veterinary Medicine and Animal Science, Universidade de São Paulo (USP), 30 Department of Surgery, School of Veterinary Medicine and Animal Science, Universidade de São Paulo (U São Paulo 05508-270 Brazil; materajm@usp br j p 31 Veterinary Research Institute (VRI), 621 00 Brno, Czech Republic; vozdova@vri.cz y p 32 Department of Pathology, School of Veterinary Medicine and Animal Science, Universidade de São 32 Department of Pathology, School of Veterinary Medicine and Animal Science, Univers Paulo (USP), São Paulo 05508-900, Brazil p gy, y , Paulo (USP), São Paulo 05508-900, Brazil Paulo (USP), São Paulo 05508-900, Brazil * Correspondence: mlzdagli@usp.br; Tel.: +55-11-3091-7712 Abstract: Mast cell tumors (MCTs) are hematopoietic neoplasms composed of mast cells. It is highly common in dogs and is extremely important in the veterinary oncology ﬁeld. It represents the third most common tumor subtype, and is the most common malignant skin tumor in dogs, corresponding to 11% of skin cancer cases. The objective of this critical review was to present the report of the 2nd Consensus meeting on the Diagnosis, Prognosis, and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors, which was organized by the Brazilian Association of Veterinary Oncology (ABROVET) in August 2021. 1. Introduction Several epidemiological studies from many countries point out that mast cell tumors (MCTs) have a high frequency in dogs, and is thus important in veterinary oncology. In dogs, it is the third most common tumor subtype, and is the most common malignant skin tumor, accounting for 11% of skin cancer cases [1–14]. Access to up-to-date information about diagnostic techniques, treatment, and prognosis is extremely important to ensure an optimized approach and improve the outcomes for the patient. The 1st Brazilian consensus meeting on canine MCTs occurred in 2012, and the results were reported by Nardi et al. in 2018 [14]. The present review is the result of the 2nd Consensus Meeting on the Diagnosis, Prognosis, and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors organized by the Brazilian Association of Veterinary Oncology (ABROVET) in August 2021. The present review presents the most recent information on cutaneous and subcutaneous mast cell tumors in dogs. The work was prepared by veterinarians working in the ﬁeld of veterinary oncology from across Brazil and from the Czech Republic (Miluse Vozdova). This review aims to present a complete report to all scientists interested in mast cell diseases. The most recent information on cutaneous and subcutaneous mast cell tumors in dogs is presented and discussed. Keywords: cutaneous; dog; mast cell tumor; subcutaneous 15 Clinical, Surgical and Palliative Care Oncology, Onco Cane Veterinary, São Paulo 04084-002, Brazil; renatasobral@oncocane.com 15 Clinical, Surgical and Palliative Care Oncology, Onco Cane Veterinary, São Paulo 04084-002, Brazil; renatasobral@oncocane.com 16 Radiation Oncology Service, Pet Care Oncologic Center, São Paulo 05659-010, Brazil; nataliacruz.vet@gmail.com 16 Radiation Oncology Service, Pet Care Oncologic Center, São Paulo 05659-010, Brazil; nataliacruz.vet@gmail.com 17 Naya Specialties, Campo Belo 04608-003, Brazil; adriananishiya@hotmail.com 17 Naya Specialties, Campo Belo 04608-003, Brazil; adriananishiya@hotmail.com y p p y 18 SEOVET—Specialized Service in Veterinary Oncology, Clinical and Surgical Oncology, São Paulo 05016-000, Brazil; sicrestoni@gmail.com 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/). 19 Clinical Oncology and Radiation Therapy, Oncopet Veterinary, Rio de Janeiro 22631-000, Brazil; Simonecsc@gmail.com 19 Clinical Oncology and Radiation Therapy, Oncopet Veterinary, Rio de Janeiro 22631-000, Brazil; Simonecsc@gmail.com 20 Department of Animal Medicine, Veterinary School, Universidade Federal do Rio Grande do Sul, Porto Alegre 91540-000, Brazil; daniel.gerardi@ufrgs.br 20 Department of Animal Medicine, Veterinary School, Universidade Federal do Rio Grande do Sul, Porto Alegre 91540-000, Brazil; daniel.gerardi@ufrgs.br 21 ONE Patologia Veterinária, Santos 11055-051, Brazil; onepatovet@gmail.com 21 ONE Patologia Veterinária, Santos 11055-051, Brazil; onepatovet@gmail.com g p g 22 Small Animal Internal Medicine Department, School of Veterinary Medicine, Universidade Metropolitana de Santos (UNIMES), Santos 11045-002, Brazil; lrbiondi@gmail.com 22 Small Animal Internal Medicine Department, School of Veterinary Medicine, Universidade Metropolitana de Santos (UNIMES), Santos 11045-002, Brazil; lrbiondi@gmail.com Cells 2022, 11, 618. 1.2. Genetics of Canine MCTs Although mast cell tumors are among the most common cancers in dogs, the ge- netic basis of their origin and development has not been fully elucidated. Mutations in KIT, which encodes the c-KIT tyrosine kinase receptor, have been found in 10–45% of cutaneous MCTs in dogs. The most common type of mutation is an internal tandem du- plication (ITD), typically in exons 8 or 11 of KIT, followed by point mutations in exons 8, 9, 11, and 17 [28,30,33–38]. The KIT transmembrane receptor is involved in cell differenti- ation and proliferation. Most known KIT mutations cause constitutive activation of the KIT receptor in the absence of its ligand, i.e., SCF. Mutations in KIT, other than those in exon 8, are associated with aggressive behavior of MCTs, reduced survival time, and increased incidence of tumor-related recurrence and death [11,30,32,39–42]. In addition, mutations in the GNB1 (17.3% of MCTs), TP53 (14.6% of MCTs), and several other genes have been identiﬁed at low frequency in canine cutaneous MCTs [41,43,44]. TP53 is a known tumor suppressor gene mutated in a number of human and animal tumors, although TP53 mutations have not proven to be reliable prognostic markers in canine MCTs [41]. GNB1 encodes a subunit of the guanine nucleotide-binding protein. Mutations in GNB1 have been found to be associated with MAPK and anti-apoptotic pathways in human leukemia [45]. In dogs, GNB1 mutations have been detected in cutaneous and subcutaneous MCTs, with a trend towards a positive prognosis [41]. p p g Regarding cytogenetic abnormalities, copy number variations in several chromosomes and chromosomal translocations have been detected in canine MCTs by Array CGH and ﬂuorescence in situ hybridization. Speciﬁcally, loss of chromosome 5, gain of chromosome 31, and partial gain or loss of chromosome 20 were found to be associated with aggressive MCTs [46]. Interestingly, copy number loss of PTEN and FAS, as well copy number gains of MAPK3, WNT5B, FGF, FOXM1, and RAD51 have previously been associated with poor outcomes in MCT-affected dogs [47]. In summary, KIT mutations (excluding mutations in exon 8), multiple aneuploidy, polyploidy, and accumulation of gene mutations and chromosomal and subchromosomal copy number variants are associated with aggressive, higher-grade MCTs and a negative prognosis. However, further research is needed to elucidate the etiology of MCTs and to discover new reliable prognostic markers and targets for therapy. 1.1. Incidence and Etiology MCT is recognized as one of the most common skin tumors in dogs [15]. Studies perforMed. in Brazil have described an incidence ranging between 20.9% and 22.4%, revealing MCT as the second-most frequent malignant neoplasm in dogs, after mammary gland tumors [16–18]. Some breeds are predisposed to MCT development, including Boxer; Bull Terrier; French Bulldog; Golden Retriever; Labrador Retriever; Shar-pei; and Dachshund [16,19–27]. Cells 2022, 11, 618 3 of 37 3 of 37 On the other hand, some breeds present a lower risk of MCT development, including the German Shepherd, Chihuahua, Poodle, Yorkshire Terrier, and Cocker Spaniel [24,25]. Recent studies also sought to correlate the breed predisposition to the biological behavior of MCT, and suggested that Pug and Boxer dogs are more prone to tumors with less aggressive behavior, while the shar-pei tends to develop more aggressive tumors [15,24,25]. In terms of sexual predisposition, results have been contradictory. To date, no sexual predisposition has been considered. In terms of age groups, MCT can develop at any age, but it is more common in adult to older animals [15,25]. The etiology of MCT has not been completely elucidated. However, Vail et al. [15] suggested the inﬂuence of chronic inﬂammation in the skin and exposure to irritating compound. Furthermore, the presence of mutations in the c-KIT gene (KIT) has been related to tumor development in MCT cases. This gene encodes a receptor tyrosine kinase that binds stem cell factor (SCF) in canine mast cells. Mutations drive uncontrolled cell survival and proliferation, which is related to MCT development and progression [28–32]. 1.3. Biological Behavior The biological behavior of MCTs in dogs is considered to be extremely variable. In general, according to Patnaik et al. [48], when MCTs are well-differentiated, they present a milder behavior. In contrast, less-differentiated tumors have a more aggressive behavior. The organs most frequently affected by metastases are the lymph nodes, skin, spleen, and liver, and less frequently the lungs [15]. The metastatic potential of MCTs varies according to its histopathological classiﬁcation, occurring in less than 10% of cases of Cells 2022, 11, 618 4 of 37 4 of 37 well-differentiated mast cell tumors, in 5% to 22% of moderately differentiated neoplasms, and in 55% to 95% of poorly differentiated cases. Metastases occur mainly on locoregional lymph nodes, and may later affect the spleen, liver, and other organs. Patients with mast cell tumors of any degree and who have regional lymph node involvement in general have a poorer prognosis [20,21,49–51]. Clinically, canine MCTs can manifest as formations of different sizes and aspects, with widely variable presentation (Figure 1). They can be delimited (Figure 1A), elevated, ﬁrm, soft, pruritic, and can also present erythematous areas (Figure 1B), with invasion of the subcutaneous tissue (Figure 1C), and in up to 30% of cases, ulceration (Figure 1D). Some cases may present with irregular, elevated formations, with a soft consistency, without erythema and ulceration, and may macroscopically appear to be cutaneous lipomas. Cutaneous and subcutaneous manifestations are macroscopically similar and can only be differentiated by histopathological examination. In terms of location, about 50% of canine MCTs develop in the trunk, perineum, and inguino-genital regions, 40% occur in the limbs, and 10% in the head and neck [15,52]. Figure 1. Canine mast cell tumors evidencing different clinical presentations. (A) Solitary, well- delimited and alopecic lesion in a dog. (B) Solitary irregular erythematous and partially alopecic cutaneous lesion. (C) Disseminated lesion with cutaneous/subcutaneous involvement and limb edema. (D) Multiple lesions with ulceration aspect. Figure 1. Canine mast cell tumors evidencing different clinical presentations. (A) Solitary, well- delimited and alopecic lesion in a dog. (B) Solitary irregular erythematous and partially alopecic cutaneous lesion. (C) Disseminated lesion with cutaneous/subcutaneous involvement and limb edema. (D) Multiple lesions with ulceration aspect. MCTs most commonly develop as solitary lesions; however, multiple formations can occur, independently, or with disease progression, making it difﬁcult to differentiate between these situations. 1.3. Biological Behavior Lesions can be considered separately, and prognostic factors should be assessed in relation to each tumor in isolation [20]. However, although O’Connell Cells 2022, 11, 618 5 of 37 5 of 37 and Thompson [53] suggested that the presence of multiple MCTs does not inﬂuence overall survival, Kiupel et al. [19] have described that these cases have worse prognosis. Some cases may prove even more challenging because they have low histopathological grades, but are nevertheless associated with aggressive behavior, with regional or distant metastases already present at the time of diagnosis [54]. In contrast, some high-grade MCTs have less aggressive behavior and a favorable prognosis [55]. Subcutaneous MCTs tend to exhibit less aggressive behavior [2], although some veterinarians have reported contradictory observations. Although a recent publication describes the characteristics of cutaneous and subcutaneous tumors [56], the biological behavior of cutaneous and subcutaneous MCT have not yet been directly compared in a study. In terms of assessing location-related behavior, previous studies have reported that MCTs located in the oral cavity, nail bed, and inguinal, perineal, and preputial regions exhibited more aggressive behavior [57–60]. However, different studies have ruled out the association of this behavior with locations in the oral cavity and in the inguinal and perineal region [61,62]. MCTs located in the scrotum and nasal plane are more prone to higher grades and earlier metastasis [11,25]. In addition to the cutaneous and subcutaneous MCT forms, a visceral form is also considered, characterized by inﬁltration into the ab- dominal lymph nodes, liver, and spleen [15], and disseminated mastocytosis, when there is systemic involvement in the bone marrow [52]. However, these are considered uncommon presentations in dogs. p g Other forms of dissemination related to MCTs are mastocythemia (when mast cells are present in the peripheral blood), and mast cell leukemia (when there is exacerbated and uncontrolled proliferation of mast cells in the bone marrow). Both are considered uncommon and are mainly related to cases of advanced MCTs, although they can also occur in association with other illnesses [15,63–65]. 2. Diagnostic Approach The diagnostic evaluation of dogs suspected of having MCTs aims to reach a deﬁnitive diagnosis through cytopathological, histopathological, and immunohistochemical examina- tion; deﬁne complete clinical staging; identify concomitant paraneoplastic syndromes; and assess associated prognostic factors. 1.4. Clinical Signs and Paraneoplastic Syndromes In addition to the presence of tumor formations, secondary clinical signs of MCT are present in about half of affected dogs, and include signs resulting from mast cell degranulation, and consequent release of histamine, heparin, eosinophil chemotactic factor, and proteolytic enzymes [15,52,66]. The effects of degranulation can be observed during physical examination, when, after mechanical palpation, erythema, edema, and papule formation are observed in the region, in a manifestation called Darier’s sign. Delayed wound healing, coagulation abnormalities, and, less frequently, hypotension and circulatory collapse may also occur [15,52,66,67]. p y Gastrointestinal complications are also seen, with signs of ulceration mainly affecting the stomach and less frequently the duodenum. These lesions are usually multiple and superﬁcial, although, in some situations, more severe ulcerations can occur. Its manifes- tation is attributed to high blood levels of histamine that stimulate the H2 receptor on parietal cells, resulting in excessive production of gastric acid and increased gastric motil- ity. In addition, histamine damages the vascular endothelium of arterioles and venules and releases ﬁbrolysin, leading to intravascular thrombosis and ischemic necrosis of the stomach mucosa. Heparin, in turn, tends to block the effects of histamine, but is present in low concentrations [52,66,67]. In these cases, clinical signs of hematemesis, anorexia, hematochezia, melena, anemia, abdominal pain, and, in severe cases, intestinal perfora- tion, peritonitis, and sepsis can be observed [15,52,66,67]. According to Welle et al. [66], gastrointestinal ulcerations are observed in 35–83% of canines affected by MCTs, based on necropsy examinations. The delay in healing and dehiscence, often observed after surgical resection of MCTs, are related to the release of vasoactive amines and proteolytic enzymes by mast cells, which, once linked to H1 and H2 receptors, can lead to the suppression of ﬁbroblast growth factor, reducing the ﬁbroplasia [52,66]. In addition, local bleeding can also be seen during surgical resection, probably due to coagulation deﬁcits caused by heparin release [66]. Circulatory collapse cases, although rare, can occur in the presence of massive histamine release from Cells 2022, 11, 618 6 of 37 neoplastic cells, particularly in animals with extensive disease [66,67]. The presence of hypereosinophilia is described as a paraneoplastic syndrome associated with MCTs in cases of cutaneous and visceral disease. Its manifestation is associated with the release of eosinophil chemotactic factors [68–70]. 2.1. Cytological Analysis Fine-needle cytology is the basis of MCT diagnostic investigation and is often per- forMed. during the initial consultation [1,53], achieving the correct diagnosis in 92–96% of cases (Figure 2) [67]. Tissue collection is usually perforMed. with a ﬁne needle, with or without aspiration. The use of a 13 × 4.5 mm (26 G) needle is recommended in order to re- duce blood contamination, thus increasing the accuracy of the diagnosis [71]. The technique is considered quick, less costly, and non-invasive, and is very useful for obtaining an early diagnosis [72], which facilitates treatment. In routine Brazilian veterinary pathology, there are two staining kits that are known to be useful, including the May–Grünwald–Giemsa and Instant Prov stains. Both are based on the Romanowski technique and are effective for staining mast cell granules. It should be noted that the Diff-Quik dye, frequently used in the USA and other countries, may not stain or only partially stain mast cell granules, making it difﬁcult to diagnose MCTs [72–75]. In inconclusive cases, in which mast cell staining does not occur, the use of stains, such as Giemsa and toluidine blue, is recommended [72,74]. Figure 2. Cytological analysis of a cutaneous well-differentiated mast cell tumor stained with Giemsa. It is observed a round ell population, showing a high degree of cytoplasmic granulation. Granules are also observed in the slide background. Objective: 40×. Figure 2. Cytological analysis of a cutaneous well-differentiated mast cell tumor stained with Giemsa. It is observed a round ell population, showing a high degree of cytoplasmic granulation. Granules are also observed in the slide background. Objective: 40×. Among round cell neoplasms, MCTs have a characteristic cytomorphology, with the presence of ﬁne to coarse basophilic intracytoplasmic granules. However, high-grade tu- mors may not present abundant cytoplasmic granulation. A possible explanation would be the loss of cell differentiation during the process of malignant transformation. Nevertheless, other potential causes for a reduction in the number of granules, such as spontaneous or palpation-induced degranulation, should also be considered. Furthermore, poorly dif- ferentiated tumors are more prone to degranulation and not necessarily to less granule Cells 2022, 11, 618 7 of 37 7 of 37 production. Detailed studies on this subject are lacking. Strefezzi et al. [73] demonstrated a correlation between the mean nuclear area and survival time in samples submitted to morphometric study. 2.1. Cytological Analysis p y Assessment of the tumor grade in cytological smears does not allow us to follow the grading system proposed by Patnaik et al. [48] strictly, which, in addition to cytomorpho- logical criteria, also considers the level of dermal invasion not determinable by cytological examination. Among the various articles published, the study by Camus et al. [76] has been the most objective and was based on speciﬁc morphological criteria selected after statistical analysis. The selected algorithm that presented the highest correlation with Kiupel’s histological grade classiﬁed MCTs into two groups: high- and low-grade. The selected criteria were: at least one smear with adequate cellularity; granulation; presence or absence of mitotic ﬁgures; nuclear pleomorphism; presence or absence of binucleation or multinucleation; and >50% anisokaryosis. Hergt et al. [77] also proposed criteria for a cytological classiﬁcation system for MCTs, dividing tumors into low- and high-grade, and concluded that the classiﬁcation is useful for the initial assessment, although the reliability of cytology is considered inadequate at this point. The proposed classiﬁcation resulted in a sensitivity of 86.8% and a speciﬁcity of 97.1% for evaluating the agreement between histological and cytological grading. Nevertheless, ﬁve high-grade tumors, according to histology, were classiﬁed as low-grade on cytology. Cytological grading is promising and can provide important pre-operative information, but further validation is still needed. To date, this second consensus meeting recommended using the classiﬁcation system proposed by Camus et al. [76]. The use of cytology to assess lymph nodes for the presence of metastases is rec- ommended as a screening method; however, false negative results may occur, since the punctured area may not comprise, the area affected by neoplastic development [15,52]. Thus, lymphadenectomy and histopathological evaluation of the lymph node in its entirety is always recommended. 2.2. Histopathological Analysis Histopathological grading is considered the main tool to suggest the biological be- havior of cutaneous and subcutaneous MCT, and is fundamental in therapeutic decision- making and prognostic investigation [48,78–81]. A correct histopathological analysis de- pends on the quality of the collected sample, as well as on good packaging, ﬁxation, and transport practices. It is important to bear in mind that incisional biopsies may not faithfully represent the formation, which may interfere both with the identiﬁcation of the cutaneous or subcutaneous location, and with the grading and/or identiﬁcation of criteria and parameters for prognostic evaluation. Instructions for sample collection and handling are as follows: Instructions for sample collection and handling are as follows: 1. When it is necessary to perform cytoreductive therapy with the use of corticosteroids or chemotherapy, it is recommended that material ﬁrst be collected by incisional biopsy, considering that cytoreductive protocols can change the assessment of the cell proliferation index (mitotic count and immunohistochemical analysis for Ki67), and when possible, the excised formation should be reassessed; p 2. When an incisional biopsy is necessary, maximum caution is recommended in han- dling the tumor, in order to avoid degranulation of mast cells and risk to the patient; 3. For a better assessment of the surgical margins, the use of India ink or suture stitches is recommended to identify the lateral and deep margins; 4. When there are multiple nodules, the submission must be completed separately with the appropriate identiﬁcations, allowing individual analysis of each lesion; 5. The sentinel lymph node (considered the ﬁrst lymph node to receive tumor drainage) should be sent for analysis whenever possible, and should be packaged separately to allow for metastasis research [82–84]. In terms of margin analysis, performing a trans-surgical pathological analysis to aid in the complete excision of a formation can be considered for MCTs. There are no well- Cells 2022, 11, 618 8 of 37 8 of 37 deﬁned, proven, and published criteria for deﬁning free, suspicious, or compromised margins. In addition, it is impossible to differentiate normal mast cells from neoplastic mast cells in most cases. Thus, current interpretations are empirical, although supported by evidence-based medicine, and, thus, require further studies. It has been suggested to interpret as ‘inﬁltration’ the presence of mast cells in foci or cords which, when discrete, are considered only if not associated with the cutaneous annex structures and vessels. 2.2. Histopathological Analysis We interpreted these as suspect, since the skin of allergic dogs tends to present numerous mast cells in the superﬁcial dermis, preferentially concentrated in vessels and annexes. When possible, in these cases, a skin biopsy from a distant location is requested for comparison and better interpretation. Some authors have suggested that low-grade MCTs would not relapse, even when margins are compromised [85], while about 40% of high-grade MCTs would relapse, even when margins are free [86]. g g p g The grading system proposed by Patnaik et al. [48] is the most commonly used system for cutaneous histological classiﬁcation of MCTs, and divides tumors into three groups: grades 1, 2, and 3. It must be emphasized that the Patnaik Grading System should not be used for subcutaneous tumors [64]. According to the authors’ criteria, grade 1 MCT is characterized by rows or clusters of neoplastic mast cells that are well-differentiated and monomorphic, with rounded nuclei, small intracytoplasmic granules, with cell pro- liferation conﬁned to the dermis. Grade 1 MCTs have no mitotic ﬁgures and binucleate cells and may have minimal stromal reactions or necrosis. Grade 2 MCT is characterized by moderately pleomorphic neoplastic mast cells, with round and/or pleomorphic nuclei and intracytoplasmic granulation of varying sizes. They extend deep into the dermis, subcutaneous tissue, and occasionally even deeper. Grade 2 MCTs show zero to two mitosis ﬁgures per higher magniﬁcation ﬁeld, discrete areas of edema, necrosis, and collagen hyalinization (Figure 3). Grade 3 MCTs are characterized by neoplastic mast cells with remarkable pleomorphism, rounded pleomorphic vesicular nuclei, containing multiple prominent nucleoli. The cells are arranged in dense layers that replace the subcutaneous tissue and deep planes. Grade 3 MCTs contain three to six mitosis ﬁgures per high-power ﬁeld, and areas of hemorrhage, edema, necrosis, and collagen hyalinization. Figure 3. Photomicrograph of a canine mast cell tumor, revealing a round cell proliferation, with evi- dent nucleolus and moderate cytoplasmic granulation. Hematoxylin and eosin staining, Objective: 40×. Although the Patnaik system is considered the “gold standard” in the prognostic evaluation of canine MCT, the prevalence of cases considered grade 2, and the variability of interpretation between pathologists for the same sample reduce the accuracy of the system. Figure 3. Photomicrograph of a canine mast cell tumor, revealing a round cell proliferation, with evi- dent nucleolus and moderate cytoplasmic granulation. Hematoxylin and eosin staining, Objective: 40×. 2.2. Histopathological Analysis Although the Patnaik system is considered the “gold standard” in the prognostic l i f i MCT h l f id d d 2 d h i bili f Figure 3. Photomicrograph of a canine mast cell tumor, revealing a round cell proliferation, with evi- dent nucleolus and moderate cytoplasmic granulation. Hematoxylin and eosin staining, Objective: 40×. Although the Patnaik system is considered the “gold standard” in the prognostic evaluation of canine MCT, the prevalence of cases considered grade 2, and the variability of interpretation between pathologists for the same sample reduce the accuracy of the system. Cells 2022, 11, 618 9 of 37 Considering Patnaik’s criteria for grade 3 tumors, when evaluating proliferative indices, some cases with low mitotic indices that present with aggressive behavior are excluded or underestimated [73,79,80,87–89]. Shaw et al. [90] evaluated the degree of agreement of the histopathological grading systems in samples collected by incisional techniques (punch, Tru-cut needle and similar devices, and collection with a scalpel) compared to those collected by excisional biopsy, and observed agreement of 96% by the system of Patnaik and 92% by the Kiupel system. They concluded that all techniques were adequate in differentiating between low-grade and high-grade MCTs. Some grade 2/high-grade cases may be underestimated in pre-operative biopsies, and are, thus, not recommended as the only source of therapeutic guidance and tumor staging [54]. To reduce the variability of interpretation and obtain a more reliable histological classiﬁcation, Kiupel et al. [80] proposed a second classiﬁcation system for the canine MCTs, using low- and high-grades. When presenting at least one of the following characteristics, the MCT is considered high-grade: 1. A minimum of seven mitotic ﬁgures counted in 10 ﬁelds of higher magniﬁcation, and evaluated in the areas with the greatest number of mitotic ﬁgures; g g 2. At least three multinucleated cells (three or more nuclei) in 10 high-power ﬁelds; 3. At least three bizarre nuclei or markedly pleomorphic nuclei in 10 ﬁelds of higher magniﬁcation; or 4. Karyomegaly [15,80]. In the study by Kiupel et al. (2011) [80], three samples of MCT classiﬁed as having a high degree of malignancy did not have the seven mitotic ﬁgures counted in 10 ﬁelds of highest magniﬁcation, which indicates that the number of mitotic ﬁgures should not be the only criterion to classify the MCT as being of high degree. 3. Inﬁltrative. 3. Inﬁltrative. In this study, cases with lower survival were associated with a mitotic count > 4 in 10 ﬁelds of high magniﬁcation (40×), an inﬁltrative histological pattern, and the presence of multinucleated neoplastic mast cells. It is also recommended to send a sample, including the associated cutaneous portion, for analysis, for the correct classiﬁcation of MCTs in the subcutaneous tissue. This consensus recommends the classiﬁcation by Thompson et al. [84] for the classiﬁcation of subcutaneous MCTs. For lymph node histopathological evaluation, it is recommended that serial sections of the lymph node be made, transverse to the longest axis, and with each section 2 mm-thick, in order to represent the largest possible area of the subcapsular sinuses and lymph node parenchyma, with special attention to capsular and extra-capsular invasion. Immunohis- tochemical analysis and the toluidine blue staining technique can be added for a more effective diagnosis. For classiﬁcation, Weishaar et al. [98] proposed a model that divides cases into: HN0 (non-metastatic lymph node), characterized by absent or rare mast cells (0–3), isolated in the sinuses (subcapsular, paracortical, or medullary), and/or lymph node parenchyma per 40× ﬁeld. HN1 (pre-metastatic lymph node), characterized by more than three isolated mast cells in the sinuses (subcapsular, paracortical, or medullary), and/or lymph node parenchyma per 40× ﬁeld, in at least four ﬁelds. HN2 (early or early metas- tasis), characterized by aggregates (clusters) of mast cells (more than three cells) in the sinuses (subcapsular, paracortical or medullary) and/or parenchyma, or sinusoidal cords of mast cells. HN3 (obvious or advanced metastasis), characterized by altered lymph node architecture, with foci of mast cells organized in a mantle or cord. However, the authors of this consensus have reservations about the term “pre-metastatic” used in the HN1 classiﬁcation, as it can generate doubts, and should not be confused with cases of already established metastasis. y Histochemical techniques, such as Giemsa, toluidine blue, and Alcian blue-safranin are extremely important to establish a differential diagnosis with other round-cell neoplasms, and to identify intracytoplasmic granulation, particularly in cases where cytoplasmic granules are scarce. However, poorly differentiated MCT cases may require immunohisto- chemistry for diagnostic conﬁrmation [99]. In human medicine, the tissue freezing technique is typically used to determine the presence of tumor cells in surgical margins and metastases in sentinel lymph nodes [100,101]. Its accuracy is similar to that of tissues embedded in parafﬁn [102,103], and this technique can be perforMed. 3. Inﬁltrative. to conﬁrm the diagnosis of MCTs, as well as to analyze the lateral surgical margins and depth of the tumor during surgery. Historically, there are several stains that can be used to detect mast cells in histology; however, in frozen tissues, toluidine blue remains the stain of choice [104]. It is suggested that histopathology perforMed. by freezing MCTs should be used as a diagnostic tool, and to assess the surgical margins. His- tological grades currently used should not be applied when using the freezing technique; rather, histopathology of parafﬁn-embedded tissues is necessary. Sentinel lymph nodes should not be frozen, but should be preserved for histopathological grading according to Weishaar et al. [98]. 2.2. Histopathological Analysis There was 100% agreement among pathologists in classifying low and high malignancy MCT [80]. For standardization purposes, it was suggested that the mitosis ﬁgures should be counted in the hotspot area. This consensus guideline proposes the use of both the classiﬁcation systems suggested by Patnaik et al. [48] and by Kiupel et al. [80], for a better classiﬁcation of the disease (e.g., “MCT grade 3 according to Patnaik and high-grade according to Kiupel”). The recommendation is in line with the recommendation of the American College of Veterinary Pathologists and the Veterinary Cancer Society [54]. g y y The evaluation of the mitotic count also assists in MCT prognostic investigation. The mitotic count must be evaluated in an area of 2.37 mm2, avoiding the use of 10 ﬁelds with high magniﬁcation (400×). Alternatively, the ﬁeld size must be described, as well as the correction and equivalence to the reference work used, since differences between analyzed microscopic areas, which vary with the ﬁeld number of the microscope eyepiece used, can reach up to 400% between one device and another. The importance of correctly applied methodology for deﬁning a variable of prognostic value cannot be neglected [91]: only then can the cutoff values suggested in the literature be validated. The mitotic count has been described as a prognostic factor independent of other histopathological features, having higher indices associated with a worse prognosis [92,93]. Reported cutoff values for the number of mitotic ﬁgures counted in 10 ﬁelds of highest magniﬁcation can range from 5 to 10 [78,92,93]. Until other prospective studies deﬁne statistically calculated cutoff values for MCTs, the authors of this consensus guideline recommend using the methodology of Romansik et al. [92] for classifying the index into ≤5 and >5 mitotic ﬁgures, or of Elston et al. [93], for ranking the index at 0; 1–7, and >7 mitotic ﬁgures. Elevated mitotic indices (>5 mitotic ﬁgures or >7 mitotic ﬁgures) are related to worse prognosis. Most veterinary pathologists agree that the mitotic count is an important element for grading canine cutaneous mast cell tumors and the current approach is to determine the mitotic count in 10 consecutive high-power ﬁelds with the highest mitotic activity. However, there is variability in area selection between pathologists. Bertram et al. 2.2. Histopathological Analysis [94,95] have used computerized calculation of mitotic counts in mast cell tumors and veriﬁed that the mitotic count is area dependent, and the assistance of a computerized system Cells 2022, 11, 618 10 of 37 may improve the interobserver reproducibility and accuracy. Therefore, deep learning techniques aim to help, but not replace, the pathologist evaluation in MCTs. may improve the interobserver reproducibility and accuracy. Therefore, deep learning techniques aim to help, but not replace, the pathologist evaluation in MCTs. q p p p g Considering subcutaneous MCTs, recent studies have sought to trace speciﬁc prog- nostic factors, although these are mostly intermediate to low-grade tumors [84,96,97]. Thompson et al. [84] classiﬁed subcutaneous MCTs into three patterns: 1. Circumscribed; 2. Combined (inﬁltrative/circumscribed); and 2.3. Immunohistochemistry and Histochemical Staining Methods [32] to assess Ki-67 staining in cases of cutaneous MCTs, and the methodology described by Thompson et al. [84] for subcutaneous MCTs. To interpret the labeling in cutaneous MCTs, a Ki-67 value > 23 positive cells in ﬁve ﬁelds of higher magniﬁcation is used (Figure 4A), and is associated with higher risks of recurrence and metastasis development [32]. Figure 4. Ki67 and KIT immunoexpression in canine mast cell tumors. (A) Ki67 expression in a mast cell tumor showing more than 23 positive cells (arrows–brown staining). (B) KIT immunohistochem- istry revealing pattern II (focal cytoplasmic–arrows). Harris hematoxylin counterstaining. Figure 4. Ki67 and KIT immunoexpression in canine mast cell tumors. (A) Ki67 expression in a mast cell tumor showing more than 23 positive cells (arrows–brown staining). (B) KIT immunohistochem- istry revealing pattern II (focal cytoplasmic–arrows). Harris hematoxylin counterstaining. For KIT, studies have determined three patterns of protein expression in canine MCTs: the membranous pattern (KIT pattern 1), focal cytoplasmic pattern (KIT pattern 2) (Figure 4B), and diffuse cytoplasmic pattern (KIT pattern 3) [79,106]. The different patterns correlate with tumor aggressiveness, with focal or diffuse cytoplasmic patterns (KIT pat- terns 2 and 3) being associated with a poor prognosis [106]. However, recent studies have shown that only KIT pattern 3 correlated with a worse prognosis [11]. It is recommended that immunohistochemical reports use the nomenclature determined by Kiupel et al. [106], aiming at standardization of sample analysis. However, different studies have used dif- ferent antibodies, at different concentrations, and using different protocols, which make it difﬁcult to make comparisons. Considering the prognostic and predictive potential of both markers, the authors of this consensus strongly recommend a standardization of the technique used, following the criteria proposed by Kiupel et al. [106]. q g p p y p Considering the possibility of other KIT activation mechanisms, recent studies have evaluated the measurement of phosphorylated KIT (pKIT) as a possible prognostic marker [111,112]. A study by Thamm et al. [112] found that the immunohistochemical detection of pKIT in patients with MCTs may predict prognosis and biological behavior. Further studies are needed to validate this test for routine inclusion. Immunohistochemistry should be used after histopathological analysis, in cases of inconclusive initial diagnosis, or to add prognostic and predictive information in cases with a conﬁrMed. diagnosis, to assist in the deﬁnition of adjuvant therapies. 2.3. Immunohistochemistry and Histochemical Staining Methods Several studies have used immunohistochemistry to analyze markers that aid in the differential diagnosis and understanding of the biological behavior of MCT; however, few antibodies have shown predictive or prognostic value [83]. The most widely used immuno- Cells 2022, 11, 618 11 of 37 11 of 37 histochemical markers in the prognostic evaluation of MCT, and that are recommended by the authors of this consensus, are Ki-67 (proliferative index) and KIT [32,79,105–110]. Ki-67 is a proliferative marker. The methods used for its calculation vary across studies [32,105,107–109], which can generate variation in interpretations and observations. Thus, this consensus recommends using the methodology proposed by Webster et al. [32] to assess Ki-67 staining in cases of cutaneous MCTs, and the methodology described by Thompson et al. [84] for subcutaneous MCTs. To interpret the labeling in cutaneous MCTs, a Ki-67 value > 23 positive cells in ﬁve ﬁelds of higher magniﬁcation is used (Figure 4A), and is associated with higher risks of recurrence and metastasis development [32]. histochemical markers in the prognostic evaluation of MCT, and that are recommended by the authors of this consensus, are Ki-67 (proliferative index) and KIT [32,79,105–110]. Ki 67 i lif ti k Th th d d f it l l ti histochemical markers in the prognostic evaluation of MCT, and that are recommended by the authors of this consensus, are Ki-67 (proliferative index) and KIT [32,79,105–110]. histochemical markers in the prognostic evaluation of MCT, and that are recommended by the authors of this consensus, are Ki-67 (proliferative index) and KIT [32,79,105–110]. (p ) Ki-67 is a proliferative marker. The methods used for its calculation vary across studies [32,105,107–109], which can generate variation in interpretations and observations. Thus, this consensus recommends using the methodology proposed by Webster et al. [32] to assess Ki-67 staining in cases of cutaneous MCTs, and the methodology described by Thompson et al. [84] for subcutaneous MCTs. To interpret the labeling in cutaneous MCTs, a Ki-67 value > 23 positive cells in ﬁve ﬁelds of higher magniﬁcation is used (Figure 4A), and is associated with higher risks of recurrence and metastasis development [32]. p Ki-67 is a proliferative marker. The methods used for its calculation vary across studies [32,105,107–109], which can generate variation in interpretations and observations. Thus, this consensus recommends using the methodology proposed by Webster et al. 2.4. KIT Mutation KIT encodes the receptor tyrosine kinase KIT, a membrane receptor with tyrosine kinase activity for SCF, which stimulates mast cell growth. In 1999, two studies described the presence of mutations in KIT in canine MCTs, after observation of an ITD [28,33]. The KIT protein is composed of an extracellular domain (coded by exons 1–9), a transmem- brane portion (exon 10), juxtamembrane (exons 11 and 12), cytoplasmic (exon 13), and phosphotransferase lobes (exon 17) [39]. Mutations in extracellular domains are called regulatory-type mutations, while mutations in intracellular domains (exons 13–21) are called enzymatic-type mutations [114]. When SCF binds to the KIT receptor, the cytoplasmic portion of the receptor undergoes autophosphorylation. In the presence of the ITD in exon 11, the receptor is phosphorylated, irrespective of whether SCF is bound. pKIT activates signaling pathways that stimulate neoplastic mast cell growth [28]. Therefore, the presence of this mutation is directly responsible for the uncontrolled proliferation of the tumor, which, in this case, presents a worse prognosis [32]. The discovery of an aggressive behavior of MCT by a constitutive activation and signaling of a tyrosine kinase receptor has resulted in research into the use of TKIs in MCTs [38,115]. About 30% of canine MCTs have ITDs in the juxtamembrane (exons 11 and 12) and extracellular (exons 8 and 9) domains of the KIT [37]. ITDs located in exon 11 represent 60–74% of all mutations and are associated with a worse prognosis and greater chances of recurrence and metastasis [11,30,32,40,42]. The prognostic value of detecting mutations in other exons of KIT has not been systematically investigated. Brocks et al. [42,43] compared cases of MCTs with mutations in exon 8 and exon 11 and observed that the presence of ITDs in exon 8 were associated with a more favorable prognosis. p g Other mutations, deletions, and insertions have also been found in this tumor type, but are rarely observed [11,28,30,32–38,116–119]. Only two studies evaluated the presence of mutation in KIT in subcutaneous MCTs, with a negative result in all cases [84,111]. Mutation analysis of KIT (most commonly for exon 11 ITD) is offered by several laboratories and is based on polymerase chain reaction tests. Furthermore, such analyses should be included in a panel of markers for canine MCT, as they have prognostic value [120]. 2.3. Immunohistochemistry and Histochemical Staining Methods Webster et al., 2007 [32], compared three proliferation markers for the assessment of cellular proliferation. The markers were Ki67, the proliferating cell nuclear antigen (PCNA), and the argyrophilic nucleolar organizing region (AgNOR). The study revealed that increased Ki67 and AgNOR counts were both associated with signiﬁcantly decreased Cells 2022, 11, 618 12 of 37 12 of 37 survival. Authors indicated that AgNOR, PCNA, and Ki67 staining provides mutually exclusive and complementary information, and that they could be used together to inform more precisely about the cell cycle than when they are used independently. p y y y p y AgNOR is a silver-based staining that can be easily perforMed. in FFPR whose advantage is the cost, since it is cheaper than immunohistochemistry. However, there are interobserver variabilities, which must be taken into account. In addition, more recently Smith et al., 2017 [113] aiMed. to determine if the extent of surgical excision affected recurrence rate in dogs with grade II MCT with low proliferation activity. The authors suggested combined AgNORxKi67 (Ag67) values to determine low cell proliferation in grade 2 MCTs. 2.4. KIT Mutation These tests must discriminate for which mutation, deletion, or insertion polymorphisms should be searched, as many of them do not lead to constitutive phosphorylation of the KIT [32,37,40,115,120]. 2.5. Staging Staging should always be perforMed. with the objective of deﬁning the extent of the disease, which directly inﬂuences therapeutic decision-making and prognosis [15,52]. The most widely practiced staging involves lymph node evaluation, abdominal ultrasound, and chest radiography. However, Book et al. [121] found that the sensitivity of ultrasound to detect splenic and liver metastases was 43% and 0%, respectively, suggesting that ﬁne- needle aspiration cytology or guided biopsy should be perforMed. to obtain complete staging. However, in clinical routine, the puncture of these organs cannot always be performed. Warland and colleagues [122] also suggested that chest radiography would have less importance in the staging of canine MCT, and is not mandatory for its performance. Cells 2022, 11, 618 13 of 37 13 of 37 However, it is important to remember that, despite being low, the possibility of pulmonary dissemination still exists in cases of MCT. However, it is important to remember that, despite being low, the possibility of pulmonary dissemination still exists in cases of MCT. Computed tomography (CT) is an imaging technique that has been used more fre- quently in recent years as a more sensitive tool in identifying metastatic lesions in different neoplasms. Hughes et al. [123] evaluated CT and spleen and liver cytology results of dogs with MCT and observed that, in most dogs with cytology positive for metastases in the spleen or liver, these organs had a normal appearance on CT, and, therefore, they concluded that the tool was not effective in identifying early forms of metastatic lesions. Dogs with positive cytological evidence of mast cell inﬁltration in the spleen, liver, or both have a worse prognosis [121]. The identiﬁcation of metastases to the lymphatic system plays an important role in the staging of MCTs. Sentinel lymph nodes, considered the ﬁrst lymph nodes in the lymphatic chain to drain the tumor, are important in the staging of several types of human cancer, providing important prognostic information. In veterinary medicine, evaluation of the sentinel lymph node during staging should always be incorporated in malignant neoplasms of lymphatic dissemination, such as carcinomas, melanomas, and MCTs [124]. Krick et al. [125] demonstrated that cytopathological evaluation of lymph nodes from dogs with MCTs provides valuable clinical information and correlates with tumor grade and prognosis, in addition to being a practical and non-invasive technique. 2.5. Staging However, cytopathological analysis of lymph nodes has a sensitivity ranging from 68% to 75%, which may generate conﬂicting results, suggesting that histopathological examination of locoregional lymph nodes should ideally be perforMed. [126,127]. Warland et al. [122] observed that no tumor had distant metastases without affecting the sentinel lymph nodes, suggesting less importance of searching for metastases in distant organs when lymph node metastasis is absent. The identiﬁcation of the sentinel lymph node in clinical routine remains challenging, and it is important to emphasize that locoregional lymph nodes, those in anatomical prox- imity to the tumor, may differ from the sentinel lymph nodes, due to lymphatic drainage changes promoted by the tumor [128,129]. In the study by Worley [128], lymphatic map- ping of dogs with MCT was performed, and it was observed that the sentinel lymph nodes did not correspond to the locoregional lymph node in 42% of cases. Aiming to increase the accuracy of sentinel lymph node detection, Fournier et al. [130] evaluated the effectiveness of contrast ultrasound in identifying these lymph nodes, showing accurate detection in 95.2% of cases. However, further studies are still needed to validate this technique. Regarding tumor staging, different clinical staging systems have been proposed for canine MCT. The World Health Organization (WHO) proposed a clinical staging system (Table 1) for canine MCT, which classiﬁes dogs with the presence of stage II lymph node metastasis and dogs with the presence of multiple tumors as stage III, even with lymph nodes free from metastasis. This classiﬁcation is questioned by several studies, particularly in terms of MCT grade 3 [15,20,52]. Cells 2022, 11, 618 14 of 37 14 of 37 Table 1. Clinical Staging System for Canine Cutaneous MCT proposed by the WHO. Stage Description 0 One tumor incompletely excised from the dermis, identiﬁed histologically, without regional lymph node involvement. a. Without systemic signs. b. With systemic signs. I One tumor conﬁned to the dermis, without regional lymph node involvement. a. Without systemic signs. b. With systemic signs. II One tumor conﬁned to the dermis, with regional lymph node involvement. a. Without systemic signs. b. With systemic signs. III Multiple dermal tumors; large, inﬁltrating tumors with or without regional lymph node involvement. a. Without systemic signs. b. With systemic signs. IV Any tumor with distant metastasis, including blood or bone marrow involvement. MCT, mast cell tumor; WHO, World Health Organization. Table 1. 2.5. Staging Clinical Staging System for Canine Cutaneous MCT proposed by the WHO. MCT, mast cell tumor; WHO, World Health Organization. Another staging system for MCTs was proposed during the Southern European Vet- erinary Conference (SEVC) in Barcelona in 2008 [52], considering as stage IV any patient with metastatic lymph node disease, regardless of the number and size of the formations (Table 2). Table 2. Clinical Staging System for Canine Cutaneous MCT proposed by SEVC. Stage Tumor Lymph Node Metastasis I Single nodule, <3 cm, well circumscribed. − − II +1 nodule, <3 cm, intralesional distance > 10 cm, well circumscribed. − − III 1 or + nodule, >3 cm, intralesional distance < 10 cm, poorly circumscribed or ulcerated. − − IV Any type of lesion. + − V Any type of lesion. + or − + SEVC, Southern European Veterinary Conference; MCT, mast cell tumor Table 2. Clinical Staging System for Canine Cutaneous MCT proposed by SEVC. Given the practical divergences of current staging systems, Horta et al. [11] proposed an improvement in the staging system already established by the WHO, allowing classi- ﬁcation of dogs with MCT according to the risk of recurrence and metastasis. The study demonstrated that patients with multiple lesions without involvement of regional lymph nodes generally have a better prognosis compared to those with a single lesion, but with lymph node involvement. Thus, in the new proposed staging, the presence of metasta- sis in a regional lymph node would classify the patient as stage III. Stage IV would be characterized by the presence of extensive and inﬁltrative formations or multiple forma- tions with lymph node involvement, and stage V, when there is the presence of distant metastases (Table 3). Cells 2022, 11, 618 15 of 37 Table 3. Clinical Staging system for Canine Cutaneous Mast Cell Tumors proposed by Horta et al. [11,27], Adapted from the World Health Organization System. Table 3. Clinical Staging system for Canine Cutaneous Mast Cell Tumors proposed by Horta et al. [11,27], Adapted from the World Health Organization System. Stage Description I Single tumor, without regional lymph node involvement. II Multiple tumors (≥3), without regional lymph node involvement. III Single tumor, with regional lymph node involvement. IV Large and inﬁltrative tumors, without delimitation, or multiple tumors (≥3), with regional lymph node involvement. 3. Prognostic Factors The biological behavior of canine MCT is variable. These MCTs may manifest a low metastatic potential or could show extremely aggressive behavior, leading to metastasis and death. These factors must be evaluated in conjunction with the particular features of each patient and can be used as a tool to anticipate the biological behavior of the tumor, and to guide the treatment [132]. London and Thamm, 2013 [133], presented a useful list of prognostic factors for canine MCTs. p g Horta et al. [11] studied 149 cases of canine MCTs. Their multivariate analysis showed a higher risk of death (62%) related to MCT in patients with tumor recurrences (local or distant) and lymph node metastasis. In the absence of these characteristics, other prognostic factors proved to be reliable predictors of an intermediate risk of death related to MCT (14%). In this context, the histological grading, mitotic count, a high expression of Ki- 67, KIT pattern 3, and ITD in exon 11 of KIT stood out. In the absence of any of these factors, MCT-related risk of death (4%) was reduced. Moore et al. [55] suggested that some dogs with high-grade solitary MCTs (Kiupel criteria) may have a favorable prognosis, particularly those tumors of a smaller size and lower mitotic count. Thus, they concluded that clinical staging and mitotic count can be as useful as histological grading for obtaining prognostic information, although it is essential that they be interpreted together. Factors, such as tumor size and presence of lymph node metastasis, both included in clinical staging, are considered important prognostic factors, directly inﬂuencing the survival obtained with different treatments [133–136]. As previously mentioned, histological grading is currently the most reliable prognostic factor for MCTs. Although some studies found an association of the Kiupel classiﬁcation with a higher prognostic value [63,137], it is still recommended that both classiﬁcation systems be used [15,138]. However, even with the association of both histological classiﬁ- cations, a percentage of MCTs still manifest unpredictable biological behavior, regardless of grading [11,112]. Stefanello et al. [131], emphasized the importance of evaluating the histological grade together with other clinical factors of prognostic relevance, such as tumor size, anatomical location, and the presence of metastases. In addition, other factors that have previously been associated with a worse prognosis also include the presence of multiple lesions, erythema, pruritus, tumor ulceration, and the presence of systemic clinical signs [15]. 2.5. Staging V Any tumor with distant metastasis, including bone marrow invasion and the presence of mast cells in the peripheral blood. Given the challenges in the practical applicability of the staging systems proposed to date, and considering the variable biological behavior of the disease, it is still not possible to standardize a single system for cutaneous MCTs in dogs. Interestingly, other characteristics, such as race and tumor location, are negative prognostic factors that are well established in the literature, and that are not yet considered in current staging systems [11,15,131]. Thus, further studies are needed to validate the staging already proposed or even to propose new adaptations. 3. Prognostic Factors Given the variable behavior of canine MCTs, there is a need to include new tests that provide additional information on biological behavior and clinical prognosis. Im- munohistochemical markers of cell proliferation and differentiation are widely studied and Cells 2022, 11, 618 16 of 37 mainly include Ki-67 immunostaining and KIT receptor localization [11,32,108,109,139,140]. However, some studies have investigated the real prognostic value of these immunomark- ers. Costa-Casagrande et al. [141] evaluated KIT immunostaining patterns and found no prognostic correlation. Similarly, in the study by Horta et al. [11], Ki-67 levels and the KIT expression pattern were not particularly informative or relevant for prognosis. Furthermore, in this study, the presence of mutations in KIT, particularly in exon 11, had a signiﬁcant prognostic impact and was not correlated with aberrant expression of KIT. In an attempt to obtain more robust prognostic information, Ki-67/KIT double-staining was previously evaluated, and was shown to be valuable for prognosis [142]. p y p g Other immunomarkers have also been studied in recent years, including vascular endothelial growth factor (VEGF), tumor suppressor gene TP53, cell nuclear antigen (PCNA), argyrophilic organizer regions (AgNORs), and apoptotic proteins (BAX, BCL-2) [41,138,140,141,143–145]. In the study by Vascellari et al. [140] the expres- sion of Ki-67, BCL-2, and COX-2 were evaluated for their possible prognostic values. They observed that expression of Ki-67 and mitotic count were associated with histological grade and survival, while the expression of BCL-2 and COX-2 showed no signiﬁcant association with prognosis. In contrast, Barra et al. [144] demonstrated that high expression levels of BCL-2 and BAX were associated with a worse prognosis. p g Bergman et al. [143] investigated the Ki-67, PCNA, and AgNOR expression and identiﬁed its positive association with histological grade. However, in the study by Scase and colleagues [107], although AgNOR and Ki-67 levels had prognostic value, PCNA expression showed no correlation with prognosis. Freytag et al. [146] carried out an extensive review and meta-analysis aiming to identify the immunohistochemical markers that have the greatest prognostic value in this context to date. They observed that Ki-67 and KIT expression have prognostic value, and the pro-apoptotic immunomarker BAX also showed promising application as a prognostic factor in MCTs. Regarding prognostic factors of canine subcutaneous MCTs, Thompson et al. [84] found that MCTs with Ki-67 > 21.8 and mitotic count > 4 were more likely to relapse and metastasize. Subsequently, Thompson et al. 3. Prognostic Factors [111] correlated the expression of KIT, VEGFR2, and the presence of mutations in KIT with the prognosis of subcutaneous MCTs. They found that pKIT, aberrant KIT localization, and VEGFR2 expression were associated with shorter survival times, disease-free duration, and increased metastatic rate. Similarly, Gill et al. [97] evaluated negative prognostic factors in subcutaneous MCTs, reporting that proliferation indices, particularly AgNORs and mitotic count, are useful for predicting the biological behavior of this type of presentation. Silva et al. [147] investigated the prognostic value of VEGF-A, VEGFR2, pVEGFR2, VEGF co-receptor Neuropyla-1 (NRP-1), and c- Cbl-1 in a group of canine subcutaneous MCTs associated with patient survival. High expression of VEGFR2, pVEGFR2, and c-Cbl-1 was shown to correlate with the disease-free interval, indicating the potential prognosis of these markers. However, there is a need to establish more routine immunohistochemical markers for subcutaneous MCTs, since the identiﬁcation of KIT expression and mutations in its receptor have not been validated as prognostic factors. Currently, the main validated prognostic factors for subcutaneous MCTs should be considered to be: (1) mitotic count > 4; (2) presence of multinucleation; and (3) inﬁltrative growth pattern [81]. Given the results of previous studies, it can be inferred that there is no single technique or factor that can unambiguously determine the behavior of cutaneous MCTs. Thus, the authors of this consensus statement recommended the combined assessment of different factors, including clinical staging, history of tumor recurrence, histopathological grade (Patnaik and Kiupel gradations and mitotic count) and molecular data, highlighting the assessment of Ki-67 level, expression of KIT, and detection of mutations in KIT, mainly in intermediate- to high-risk cutaneous MCTs [148]. The most studied and validated prognostic factors are summarized in Table 4. 17 of 37 17 of 37 Cells 2022, 11, 618 Table 4. Prognostic Factors Associated with Canine Cutaneous MCT. Table 4. Prognostic Factors Associated with Canine Cutaneous MCT. Table 4. Prognostic Factors Associated with Canine Cutaneous MCT. Prognostic Factor Positive Negative Reference Histopathological Grading Grade 1–low Grade Grade 3–high Grade Patnaik et al. [47,48]; Kiupel et al. [80] History of Tumor Recurrence First clinical presentation Recurrent tumor Horta et al. [11] Tumor Size Higher diameter < 3 cm Higher diameter > 3 cm Hahn et al. [134] Metastasis (regional and/or distance) Absent Present Hume et al. [51]; Book et al. [121]; Warland et al. [7,122] Surgical Margins Free margins Contaminated margins Ozaki et al. 4.1.1. Surgery The main therapeutic modality indicated in cases of canine MCT is surgical exci- sion, which should be perforMed. whenever possible, according to the patient’s clinical conditions and staging [15,149]. Previously, wide excision was advocated, regardless of histopathological grading and associated prognostic factors, with application of a 3 cm lateral margin and a fascial layer for the deep margin. However, recent studies have indi- cated different approaches related to speciﬁc prognostic factors, mainly clinical staging and histopathological grading. p g g g The application of proportional margins was initially suggested as a conservative alternative for dogs affected by cutaneous MCT, in their ﬁrst surgical intervention, and after complete staging. The concept initially consisted of using a lateral margin proportional to the largest tumor diameter, for tumors below 4 cm, and a ﬁxed margin of 4 cm for tumors above this diameter, maintaining a deep margin of at least one fascial plane [150]. Later, the concept was adapted, with a 2 cm lateral and a deep safety margin of a fascial plane being recommended for grade 1 or 2 tumors, and up to 4 cm in diameter, providing effective local control of the disease [151–154], with a recurrence rate of 0–4% [82,150,155]. In cases of high-grade (or grade 3) MCTs, there is a high recurrence rate (36%) regardless of the surgical margins used, as observed in the study by Donelly et al. [156]. Associated lymphadenectomy is recommended during initial surgical intervention. Ferrari et al. [127] demonstrated that approximately half of the patients who did not present alterations on palpation already had metastases when histopathological evaluation was performed. When tumor removal is perforMed. before lymph node removal, it is recom- mended to change the material and surgical drapes, to avoid tumor implantation [133]. Lymphatic mapping by injection of radionucleotides or radiographic contrasts is indicated to aid in the characterization of the sentinel lymph node, whose trans-surgical identiﬁcation can be facilitated by the use of vital dyes, such as patent blue [157]. Regional lymphadenec- tomy, in cases of metastatic lymph nodes, has therapeutic potential, with evidence of reduced local recurrence and the development of distant metastasis [158]. Lymphadenec- tomy, when perforMed. in intracavitary sentinel lymph nodes, can complicate the surgical procedure. Thus, the risk and beneﬁt must be evaluated in each case. 3. Prognostic Factors [133] Mitotic Count <5 or <7 >5 or >7 Romansik et al. [92]; Elston et al. [93] KIT Pattern KIT 1 KIT 2 and 3 Kiupel et al. [19] c-KIT Mutationc-KIT Mutation Absent Present Webster et al. [32,35,36] Ki67 Index <23 >23 Webster et al. [32,139] 4. Therapeutic Approach To deﬁne the best therapeutic approach, all the points raised during the diagnostic in- vestigation should be considered, to determine the ideal approach for each individual case. 4.1. Local Treatments 4.1.1. Surgery 4.1.2. Electrochemotherapy Electrochemotherapy (ECT) has emerged as a viable technique at the experimental level for the treatment of superﬁcial and mucosal tumors in the 1990s [162]. In November 2006, the technique was standardized in the European community for use in humans. The technique combines the intratumoral and/or intravenous application of speciﬁc drugs, fol- lowed by the application of electrical pulses, leading to temporary electropermeabilization of the cell membrane, with the objective of maximizing the entry of the chemotherapeu- tic agent to enhance its cytotoxic effect [163,164]. In general, to perform this technique, the main drugs used are bleomycin and cisplatin, both with intratumoral use, but only bleomycin for intravenous use [165]. The use of conductive gel can facilitate the efﬁ- ciency of the technique by improving the distribution of the electric ﬁeld in the treated region [166,167]. In addition to direct cytotoxic effects, ECT also has indirect antitumor effects through action on the tumor vasculature and local immunity. Vascular effects lead to vasoconstriction and destruction of the local vasculature, and immunological effects lead to signaling for immunogenic cell death [165,168,169]. g g g ECT in MCTs can be used as a single therapy, associated with the trans-operative pe- riod, or as an adjuvant therapy to surgery when margins are compromised [170]. Spugnini et al. [171] used ECT with intralesional bleomycin (1.5 UI per mL) in cases of canine MCT with compromised surgical margins, and observed that, in 85% of cases, the mean time to recurrence was 52.7 months. Kodre et al. [172] reported ECT with intralesional cisplatin (1 mg/cm3 of tumor) as the only therapy to treat MCT and obtained recovery rates similar to those obtained after surgical excision, without associated chemotherapy and lesion regression for a mean of 31.5 months. Spugnini et al. [173] evaluated the effect of ECT with intralesional cisplatin in canines with grade 2 and 3 MCT (relapsing or not) or with compromised margins, and showed a 78% response rate without major adverse effects. p g p j Lowe et al. [174] evaluated the effects of different applications of ECT with IV bleomycin (15 IU per m2) in dogs with MCT, being applied as a single therapy trans-surgically, as an adjuvant to surgery, and in recurrent MCTs. 4.1.1. Surgery When the lesion is located in the scrotum, orchiectomy, and testicular pouch ablation are performed, and when the foreskin is involved, penectomy associated with urethros- tomy is recommended [159]. In the limbs, amputation or tumor exeresis, associated with reconstructive techniques such as subdermal or axial ﬂaps of advancement, rotation, trans- position, and interpolation, or free grafts, can be considered in order to preserve the limb [15,52,159]. Second intention healing can be perforMed. when there is no possibility Cells 2022, 11, 618 18 of 37 of applying reconstructive techniques with adequate repair of the surgical lesion. Second intention healing is preferable in the face of compromised margins [52,159]. Regardless of the technique used, it is necessary to avoid excessive manipulation of the lesion, seeking to avoid stimulation of mast cell degranulation and its consequent adverse effects [159]. In cases of tumor recurrence after initial excision, the main associated factor tends to be histological grading, with low recurrence rates after excision of grade 1 or II MCTs [160,161]. 4.1.3. Radiotherapy Radiotherapy in dogs with MCT can be perforMed. with deﬁnitive protocols (consist- ing of 12–19 fractions perforMed. 3–5 times a week), prior to surgery, or post-operatively in cases of incomplete resection, or with palliative protocols (3–6 fractions perforMed. once a week) in order to reduce tumor volume partially and control pain [177]. In cases where the diffuse and/or extensive nature of the tumor mass precludes surgery, radiotherapy should be considered, although studies on the use of radiation as a sole therapy to treat canine MCT are scarce [178]. The tumor response to radiation is considered to be dose-dependent; thus, increasing the dose can lead to a signiﬁcant increase in tumor response [178]. g g p Adjuvant radiotherapy, in a deﬁnitive protocol, was used to treat grade 2 MCTs in cases where complete surgical resection was not possible, and proved to be effective and safe, with signiﬁcant improvement in local disease control [179–183]. In a study of dogs with grade 2 MCTs, the radiotherapy protocol was 18 daily fractions of 3 gray (Gy) for a total of 54 Gy. This study showed that 94% of patients were in remission within 1 year and 88% within 5 years [179]. The use of this therapy has also been indicated in incompletely excised grade 3 MCTs, with tumor control times and median survival of 17 and 20 months, respectively [134]. The MCTs located in the head and in the distal region of the limbs are important examples of sites in which it is difﬁcult to remove the tumor completely while preserving the local anatomy; therefore, in these cases, radiotherapy is indicated [178]. For best results regarding disease-free intervals, irradiation directly into the primary tumor or surgical scar (with a 3 cm safety margin) and into regional lymph nodes is recom- mended [178,184,185]. The inclusion of regional lymph nodes in the ﬁeld of radiotherapy, whether palpable or not, is extremely important for tumor control [182,184–186]. In a study of dogs with high-grade MCT treated with deﬁnitive radiotherapy, prophylactic treatment of regional lymph nodes signiﬁcantly increased the time to tumor control (>2381 days) as compared to cases where the lymph nodes were not treated (197 days) [186]. A study in dogs with unresectable MCTs of varying degrees used a hypofractionated radiotherapy protocol (4 fractions of 8 Gy) at 7 day intervals and reported an 85% partial or complete response rate [178]. Carlsten et al. [187] perforMed. 4.1.2. Electrochemotherapy The best results were obtained in the adjuvant to surgery group and in the trans-surgical group (93% and 91% complete response [CR], respectively), followed by the single therapy group (80% CR), and ﬁnally, the relapse group, where ECT was applied after local recurrence, at the site where the surgery was perforMed. (64% CR). The group where ECT was used in the trans-surgical period had the longest disease-free interval. Adverse effects, such as degranulation, were reported in two of 28 cases [171]. Kodre et al. [172] stated that the vasoconstriction caused by electrical pulses prevents the release of inﬂammatory mediators contained in the mast cell granules into the bloodstream, with no adverse effects being observed in 12 patients treated in their study. The mean size of the tumors treated in their study was 2.9 cm3, with greater efﬁcacy being observed in tumors < 2 cm. Thus, to prevent the effects of mast cell degranulation, it is important to use a concentric approach, through the application of electrical pulses initially at the periphery of the tumor and moving towards the center. The use of prior antihistamines is recommended in association with any manipulation of MCTs, including for performing ECT, particularly those with larger diameters. p g p y g ECT is an easy, effective, and safe local treatment for MCT cases and can be an alternative treatment to surgery, speciﬁcally for smaller nodules in which a long-lasting CR can be achieved with a single treatment session, or when the nodule cannot be resected due to its location. ECT can also be combined with surgery for larger lesions without signiﬁcant Cells 2022, 11, 618 19 of 37 toxicity, being applied in the surgical bed, after tumor resection, particularly for MCTs where margins may not be reached due to tumor extension or location. Post-surgery, ECT can be applied in MCTs with incomplete resection (compromised or small margins), with the application of therapy being indicated at 2–4 weeks after surgery. Preliminary studies using ECT combined with gene transfer by electroporation of canine IL-12 (electro gene transfer) have shown promising results for the treatment of MCTs [173–176]. 4.1.2. Electrochemotherapy Thus, the authors of this consensus meeting recommend the use of ECT in speciﬁc cases of canine MCT: (1) as a single therapy in small tumors < 3 cm that are difﬁcult to resect surgically either because of the location or the number of tumors (multiple MCTs); (2) intra-operatively, in cases where it is difﬁcult to obtain free margins due to tumor extension or location; (3) as an alternative treatment for cases with compromised or small margins. 4.1.3. Radiotherapy a prospective multicenter study with a combination of hypofractionated radiation treatment, toceranib, and pred- nisone for measurable canine MCT and concluded that this protocol is a viable treatment option for unresectable MCTs, is well-tolerated and effective in most dogs, with response rates and durations higher than those reported for toceranib as a single treatment agent for MCT. Stiborova et al. [188] evaluated the toxicity related to the combination of radiotherapy with vinblastine chemotherapy and concluded that the combination is safe and did not increase chemotherapy-related myelosuppression as a single treatment. Acute side effects resulting from radiotherapy include erythema and epilation in the treated area, which start from the second week of treatment. Alopecia, hyperpigmentation, and thickening of the skin in the affected area can also be seen as late effects [178]. In a study using hypofractionated radiotherapy, many animals developed mild erythema in the treated area, without severe signs related to degranulation and histamine release. Cells 2022, 11, 618 20 of 37 Although still inaccessible in Brazil, radiotherapy proves to be effective as an auxiliary tool in the treatment and control of canine MCTs, particularly in cases where surgical therapy is difﬁcult or incomplete. 4.1.4. Tigilanol Tiglate (Stelfonta®) Tigilanol tiglate (TT) is a natural product extracted from the seed of the Fontainea picrosperma (Blushwood, from the family Euphorbiaceae) native to Australia and puriﬁed by high pressure liquid chromatography. It is a well-characterized small, diterpene ester molecule of the tigliane class [154,155]. The composition of Stelfonta®is 1 mg TT per mL, prepared in 40% propylene glycol, 30 mM acetate buffer, pH 4.2. It must be stored strictly at a temperature of 2–8 ◦C. Its indication for use is in dogs with cytological diagnosis of cutaneous MCT (in any region) or subcutaneous MCT (in the thoracic limbs, below the elbow, or the pelvic limbs, below the tibiotarsal joint), at different stages, but without involvement of regional lymph nodes based on palpation and lymph node cytology if it is palpable. For use, the dog must have been without treatment for at least 3 months. The lesion must have a diameter greater than 1 cm, and a volume ≤10 cm3, without ulceration, so that the drug does not escape after its application. TT causes vascular rupture and acute local inﬂammatory response within 4 h, followed by necrosis within 1–4 days and tumor destruction for a period of 4–7 days, with subsequent resolution of the wound within a period varying from 28 to 35 days [189–192]. TT is applied intratumorally and radially, at a dose of 0.5 mL per cm3 of tumor vol- ume. Tumor volume is determined using the modiﬁed volume calculation for ellipsoid structures = 1/2 × length × width × depth (cm). Prednisone must be used be- fore (0.5 mg/kg, every 12 h, for 2 days) and after (0.5 mg per kg every 24 h, for 3 days) the application, in addition to the use of promethazine (0.5–1.0 mg per kg every 12 h for 7 days); and ranitidine (2.0 mg per Kg, every 12 h for 7 days). When total tumor re- mission does not occur, TT can be reapplied after 28 days, although the volume must be calculated again [190–194]. TT has been the subject of studies mainly in the United States and Europe, with positive results, tolerable toxicity, ease of administration, and being capable of promoting adequate local tumor control [188–193]. The application of TT in felines is contraindicated. Up to the time of writing, TT is not available for routine use in Brazil. 4.2. Systemic Treatments 4.2.1. Anticancer Chemotherapy In most studies, vinblastine is considered to be the ﬁrst-line therapy for treating canine cutaneous MCT, using a dose of 2.0 mg/m2 every 1 or 2 weeks, with or without prednisone [135,201–203]. Generally, the therapeutic dosage is deﬁned as close to the maximum tolerated dose, in order to optimize the cytotoxic effect on tumor cells. In dogs, the dose-limiting toxicity of vinblastine is characterized by neutropenia, with its nadir occurring approximately 7 days after administration [201–205]. Webster et al. [139] described that treatment with vinblastine at a dose of 2.0 mg/m2 once a week for 4 weeks, and prednisone at a dose of 2.0 mg/kg daily, after surgery, was beneﬁcial for dogs with grade 3 MCT, when compared to those treated with surgery alone. Furthermore, dogs with KIT mutations that were treated with this protocol had a longer disease-free interval and survival duration. Thamm et al. [203] evaluated the responses of 61 dogs with high-grade MCT treated with complete excision and with vinblastine as adjuvant treatment at a dose of 2.0 mg per m2, every 7 or 14 days; and prednisone at a dose of 2.0 mg per kg, with a mean overall survival time of 1374 days. In contrast, Rungsipipat et al. [206] observed a partial response rate of 78.2% in dogs with grade 3 MCT who were treated with vinblastine at a dose of 2.0 mg per m2 in weeks 1, 2, 3, 4, 6, 8, 10, and 12, and prednisolone at a dose of 1.0 mg per kg for 4 weeks and then 0.5 mg per kg for 8 weeks. p g p g g p g In the last decade, several studies have sought to optimize the dosage and effective- ness of treatment with vinblastine. It was found that vinblastine can be safely admin- istered at a dose of 3.0 mg per m2, but that hematological and gastrointestinal effects can be observed above this level [55,198,207,208]. Vickery et al. [207] proposed a stag- gered vinblastine–prednisolone protocol with an initial dosage of 2.0 mg per m2, increased weekly to 2.33 mg per m2, 2.67 mg per m2, and 3.0 mg per m2, and maintaining the last dosage at 2-week intervals. Similarly, Serra Varela and others [209] also increased the weekly dosage from 2.3 mg per m2 to 2.6 mg per m2, but the dosage of 3.0 mg per m2 was given weekly. 4.2.1. Anticancer Chemotherapy 4.2.1. Anticancer Chemotherapy Systemic therapies used adjuvant to surgical treatment are indicated in speciﬁc cases, which present an intermediate to high risk of death related to cutaneous MCT, partic- ularly those in advanced stage, grade 3–high grade tumors with a high mitotic count. Although histopathological grading is considered to be the most reliable criterion of prognostic value in MCTs, other prognostic factors must be considered when making chemotherapy decisions, such as the proliferative activity, tumor location, and presence of multiple tumors [15]. The use of chemotherapy can also be indicated in the preoperative period, terMed. neoadjuvant chemotherapy, used in cases of large tumors or located in anatomical sites that are difﬁcult to approach surgically, aiming at controlling disease progression in the perioperative period and reducing its tumor dimensions, allowing for more adequate local control [67,195]. However, the reduction in surgical margins based on tumor shrinkage after debulking is a controversial topic in the current literature. Stanclift and Gilson [196], used prednisolone for tumor debulking and concluded that the adoption of 3 cm lateral surgical margins based on tumor size after neoadjuvant treatment were sufﬁcient to obtain free margins. However, it is known that microscopic reduction may not follow macroscopic tumor reduction, and may not occur proportionally. Thus, further studies are needed to determine the adequate extension of the surgical margins necessary after debulking. Cells 2022, 11, 618 21 of 37 Importantly, the response to neoadjuvant chemotherapy treatment tends to be temporary, with an average duration of 40–70 days, with surgical intervention being recommended during this period [15,52]. Chemotherapy, with or without other treatment modalities, is also indicated for advanced and/or inoperable disease, whether due to tumor dimensions, the number of lesions, or the presence of metastases, thus aiming at palliative control, disease stabilization, and maintenance of quality of life [197,198]. Multidrug therapy protocols promoted a greater response than single-agent chemotherapy [199]. However, simple protocols with a single chemotherapeutic agent, usually associated with prednisone or prednisolone, have become more popular. Sequential association of these protocols in an adjuvant modality demonstrated increased disease-free interval and survival in high-risk dogs [200]. Numerous protocols have been described in the literature; however, data on disease-free interval and other important criteria, to compare their effectiveness, are scarce [197]. It is known that MCTs have different response rates to different chemotherapy protocols and the occurrence of adverse effects may be common among patients [139]. 4.2.1. Anticancer Chemotherapy In their study, the protocol was well tolerated, with 4% of patients having grade 3 or 4 toxicity. Bailey et al. [210] described the highest tolerated dose as 3.5 mg/m2 every 2 weeks; however, grade 4 toxicity commonly occurred among these patients. Fur- thermore, Klutchkovsky et al. [211] evaluated the efﬁcacy and safety of using vinblastine at an experimental dose of 3.0 mg per m2 every 7 days, as compared to the conventional dose of 2.0 mg per m2 every 7 days. The experimental dose protocol was well tolerated, with 5.6% of the patients manifesting grade 4 neutropenia. Comparing the two groups, the animals treated with a dose of 3.0 mg per m2 had a lower recurrence rate, but there was no signiﬁcant difference in the mean overall survival time between the groups. Lomustine is also widely used in cases of MCT, but it is considered as a second therapeutic in terms of adjuvant chemotherapy. The recommended dose, when used as a single agent, is 60–90 mg per m2 every 21 days. Hosoya et al. [212] used lomustine at a dose of 60 mg per m2 every 3 weeks, and prednisone at a dose of 40 mg per m2 once a day (SID) for 7 days, followed by 20 mg/m2 every other day for partially resected grade 2 MCTs. Cells 2022, 11, 618 22 of 37 22 of 37 The animals received treatment for 4–6 months, with no local recurrence or regional or distant metastasis, and with 100% of the animals being disease-free at 1 year and 77% being disease-free at 2 years. Hay and Larson [213] evaluated the efﬁcacy and toxicity of the protocol using six sessions of lomustine at a dose of 70 mg per m2, every 4 weeks; and prednisone at a dose of 0.5–1mg per kg SID, for 6 months, for completely excised, high-grade MCTs. The results showed that the protocol was well tolerated, providing a mean overall survival time of 904 days, with 60% of dogs alive at the end of the ﬁrst year and 40% of dogs alive at the end of the second year. g y Protocols alternating vinblastine, lomustine, and prednisone were also tested. Cooper et al. [214] evaluated the effect of chemotherapy with lomustine at a dose of 60 mg per m2 and 2.0 mg per m2 vinblastine in dogs with grade 2 and 3 MCTs. 4.2.1. Anticancer Chemotherapy The authors showed that this protocol was well tolerated and adequate for disease control, as the response lasted longer than that of other agents. Rassnick et al. [198] evaluated the protocol comprised of lomustine at a dose of 70 mg per m2, vinblastine at a dose of 3.5 mg per m2, and prednisone at a dose of 1–2 mg per kg in dogs with unresectable MCT, and reported observing response rates of 65% (ﬁve with CR and six with partial remission) in non-operated animals. Lejeune et al. [185] perforMed. a retrospective study to assess the clinical response of animals with grade 2 and stage 2 MCTs to the combined protocol of vinblastine at a dose of 2.0 mg per m2, lomustine at a dose of 60–80 mg/m2, and prednisone at a dose of 40 mg per m2, alternated every 14 days. Patients subjected to this protocol had a satisfactory disease-free time and mean survival time: 2120 days (range: 169–2325 days) and 1359 days (range: 188–2340 days), respectively. y g y p y Chlorambucil has also been used in multidrug therapy protocols, with controversial results. Taylor et al. [197] treated 21 dogs with unresectable grade 2 and 3 MCTs with a combination of prednisolone 40 mg per m2 SID for 14 days and 20 mg per m2 every other day, followed by chlorambucil at a dose of 5.0 mg per m2 SID. In cases where CR was achieved, treatment was stopped after 6 months; otherwise, treatment was continued. Three dogs achieved CR; nine remained in stable disease, ﬁve in partial remission, and four in disease progression (overall response rate 38%). According to the authors, this protocol yielded a worse response than other protocols. However, Horta et al. [200] observed that patients with high-risk MCT (grade 3 or grade 2 with metastasis) treated with the lomustine protocol at a dose of 60–90 mg per m2 every 21 days, for a total of 3–4 sessions; followed by chlorambucil at a dose of 4–6 mg per m2, every 48 h, for 8 weeks, reached a higher disease-free interval (686 days) than those treated only with lomustine at the maximum dose (107 days). y In addition to being part of conventional chemotherapy protocols for canine MCTs, corticosteroids are also frequently used as neoadjuvant therapy, aiming at reducing peritu- moral edema and debulking the tumor. Teng et al. 4.2.1. Anticancer Chemotherapy [215] evaluated prednisone as the only treatment for MCTs, and observed a signiﬁcant reduction in tumor size, albeit with gradual resistance, as the response rate was short (81.5% of patients had a maximal response by 3 weeks). Stanclift and Gilson [196] reported an average tumor volume reduction of 80.6%, with no signiﬁcant difference in the percentage reduction between the groups treated at a dose of 1.0 mg per kg and 2.2 mg per kg. Linde et al. [216] studied the effects of the use of prednisone at a dose of 1.0 mg per kg SID in canines with MCTs, in a period of 7–14 days before the biopsy. The results of the study revealed that the use of neoadjuvant prednisone in low to intermediate grade MCTs did not exert signiﬁcant effects on tumor grading, mitotic count, or cell atypia. However, more studies are needed to validate these ﬁndings, and to evaluate the effects of cytoreductive therapy in reducing surgical margins. Horta et al. [217] correlated prognostic factors with response to neoadjuvant glucocor- ticoid treatment in MCTs, and noted that a partial response was achieved in 63.3% of cases, while 36.7% did not respond. In their study, the response to treatment with glucocorticoids was positively correlated with the presence of already established favorable prognostic factors (patients at an early stage of the disease, with low histological grade, KIT pattern 1, and Ki-67 score < 23). Cells 2022, 11, 618 23 of 37 To date, few studies have been published regarding mechanisms of multiple drug re- sistance (MDR) related to canine MCT. Despite the use of different methods to detect genes and key proteins involved in MDR of canine MCT (MDR1, P-glycoprotein, glutathione- S-transferase pi, protein associated with multi-resistance 1, p53, etc.), no study has con- ﬁrMed. the exact mechanism involved in chemotherapy resistance. Apparently, there is no histological association with the expression of MDR mechanisms that could explain why undifferentiated tumors respond poorly to chemotherapy [137,218–220]. It remains controversial whether the use of glucocorticoids as the only treatment for canine MCT induces MDR. The primary outcomes of metronomic chemotherapy protocols have been studied to date [221,222]. Tripp et al. [223] evaluated a metronomic protocol using lomustine at a dose of 2.84 mg per m2 in different canine tumors, including MCT, and observed good tolerability in terms of adverse effects. Leach et al. 4.2.1. Anticancer Chemotherapy [224] evaluated the metronomic protocol using chlorambucil at a dose of 4 mg per m2 per day for 35 weeks, in different tumor types, including four cases of canine MCT, and observed CR in one case, stable disease in two cases, and progressive disease in one case. Current knowledge about metronomic therapies indicates that their use, particularly in combination with other cytotoxic therapies, can increase response rates and optimize the treatment of cancer patients. Nevertheless, more studies are needed to validate their real effectiveness [225]. The main adjuvant and cytoreductive chemotherapy protocols (neoadjuvant and/or inoperable disease) are reported in Tables 5 and 6, respectively. Table 5. Chemotherapy Protocols Evaluated in Canine Mast Cell Tumors and Mean Overall Survival. Table 5. Chemotherapy Protocols Evaluated in Canine Mast Cell Tumors and Mean Overall Survival. Chemotherapeutic Protocol n Histopathological Grading Overall Survival Median Prednisolone and Vinblastine [168,169] 61 (a, b) a. Grade 2/3 b. Grade 3 a. 1374 days b. 800 days Prednisone and Vinblastine [179,180] 41 Grade III and Grade 2/High grade 904 days Prednisolone, Vinblastine and Lomustine [149,150] 21 Grade 2 1359 days Lomustine and Vinblastine [180,181] 20 Grade 2/3 336 days Table 6. Published Chemotherapy Protocols for Treating Advanced Mast Cell Tumors in Dogs and the Associated Remission Rates (RR) (Adapted from [162,197]). Chemotherapeutic Protocol n RR (C + P) Prednisone/Prednisolone [216] 60 63% Prednisone [195,196] 49 70% Prednisone [215,216] 16 63% Prednisone and Vinblastine [225,226] 41 47% Prednisolone, Vinblastine, and Cyclophosphamide [226,227] 35 63% Prednisone, Vinblastine, and Lomustine [197,198] 35 65% Prednisone, Vinblastine, and Lomustine [213,214] 56 57% Lomustine [227,228] 19 40% Chlorambucil and Prednisolone [196,197] 21 38% Table 6. Published Chemotherapy Protocols for Treating Advanced Mast Cell Tumors in Dogs and the Associated Remission Rates (RR) (Adapted from [162,197]). 4.2.2. Tyrosine Kinase Inhibitors y The previously mentioned family of tyrosine kinase receptors are involved in the mech- anisms of proliferation, differentiation, migration, angiogenesis, and cell activation [15]. Thus, studies suggest that these receptors play a fundamental role in the neoplastic trans- formation process [229]. The main strategy developed in veterinary medicine to inhibit these receptors was the use of the so-called “small-molecule tyrosine kinase inhibitors” Cells 2022, 11, 618 24 of 37 (TKIs). These act by blocking the binding of ATP to the receptor by a competitive inhibi- tion mechanism (reversible or not) preventing phosphorylation and cell proliferation and angiogenesis signaling [230]. g g g g The TKIs currently available in veterinary medicine are toceranib (Palladia®) and masitinib (Kinavet®/Masivet®), licensed for the treatment of unresectable or relapsed MCTs of grades II and III. There are also studies of imatinib (Gleevec®), licensed for use in humans, with a similar mechanism of action. These medications are also indicated in other circumstances, such as resistance to multiple drugs, presence of metastases, ﬁrst-line adjuvant therapy for high-risk tumors, unresectable tumors, and as salvage therapy for recurrent tumors [38,229,231–233]. The ﬁrst studies indicated that the presence of the mutations in KIT and the aberrant localization of the KIT protein (patterns 2 and 3) in MCTs were predictive factors of response to treatment with TKIs. London et al. [234] evaluated the efﬁcacy of toceranib in several tumor types, and obtained response rates of approximately 90% in MCTs with mutations in the KIT and 25% in MCTs without such mutations. Later, London et al. [38] evaluated the effect of toceranib in dogs with relapsed or metastatic MCTs, of high or intermediate grade, and concluded that the presence of mutation in KIT and the absence of lymph node metastases were signiﬁcantly associated with a greater response, objectively. Although initial results indicate a positive correlation between the presence of mu- tation in KIT and response to treatment with TKIs, recent studies have indicated that the response to treatment is not associated with mutational status. In order to assess the inﬂuence of the presence of mutation in KIT and KIT location on the therapeutic decision, Weishaar et al. 4.2.2. Tyrosine Kinase Inhibitors [235] compared the response of dogs with MCTs (resectable and unresectable, at various stages) to treatment with the protocol using vinblastine (2.5 mg per m2, four ses- sions every 7 days, and four sessions every 14 days) and toceranib (2.75 mg per kg, every 48 h). Mutation status in KIT and KIT location were not predictive of response to treatment with toceranib, and, therefore, were not considered sufﬁcient for the therapeutic decision between toceranib and vinblastine use in dogs with macroscopic MCTs. Therefore, it can be concluded that the presence of mutation in KIT is not predictive of treatment response, and that dogs with MCT that do not have KIT mutations can also beneﬁt from TKIs. This is due to dysregulation in other cell pathways, particularly the platelet- derived growth factor (PDGFR) receptor, or even the anti-angiogenic effect achieved by the inhibition of the vascular endothelial growth factor receptor (VEGFR), and the possible re-establishment of antitumor immunity from the depletion of regulatory T lymphocytes and increased serum concentration of interferon-γ [38,231–233,236]. Other causes may be involved in the tumorigenesis of MCTs, such as the overexpression of receptors and increased production of SCF. It is also important to mention that the blockade of KIT and PDGFR occurs through the mechanisms of action of both toceranib and masitinib; however, blockade of VEGFR occurs only through the use of toceranib [36,237]. y g Other TKIs have also been studied. Horta et al. [238] evaluated the response of high-risk MCTs treated with TKIs (toceranib and masitinib) and correlated the response to treatment with different prognostic factors. In the population analyzed, the presence of mutation in KIT, KIT location, and Ki-67 value were not prognostic or predictors of response to TKIs. In their study, the only prognostic factor considered reliable was the initial response to treatment. Their data corroborated the study by Smrkovski et al. [239], in which the initial response to treatment was the most signiﬁcant prognostic factor for dog survival. Similarly, Hahn et al. [233] evaluated the therapeutic potential of masitinib in dogs with MCT, noting that there was an increase in the time to disease progression, regardless of the presence or absence of mutations in KIT. The ideal doses of TKIs have also been investigated. The commercial indication for toceranib dosage is 3.25 mg per kg on Mondays, Wednesdays, and Fridays. 4.2.2. Tyrosine Kinase Inhibitors There is clinical evidence of good efﬁcacy and greater safety at lower doses of toceranib. London et al. [234] perforMed. the ﬁrst study on the efﬁcacy and safety of toceranib in dogs with different tumor types using staggered doses, ranging from 1.25 to 3.75 mg per kg, every 48 h. In this Cells 2022, 11, 618 25 of 37 study, dogs treated with a dose of 2.5 mg per kg every 48 h had a similar response and fewer adverse effects than dogs treated with a dose of 3.25 mg per kg. London et al. [38] used toceranib at a dose of 3.25 mg per kg every 48 h, in dogs with recurrent and metastatic MCTs, obtaining an objective response of 42.8%, and with 34.5% of patients developing grade 3 to 4 adverse effects. A dosage of 2.5 to 2.75 mg per kg, every 48 h, can be advantageous due to its greater safety, better adherence by owners, and a lower probability of breaks during treatment [240]. g Smrkovski et al. [239] evaluated the efﬁcacy of masitinib in the treatment of grade 2 and III MCTs, at a dose of 12.5 mg per kg, as rescue therapy or ﬁrst-line treatment, and obtained a mean survival time of 630 days. In contrast, in the study by Grant et al. [241], dogs with macroscopic MCTs treated with masitinib at a dose of 12.5 mg per kg had a mean survival time of only 159 days, considering that, in most cases, masitinib was instituted as rescue therapy. Recently, the efﬁcacy and safety of the association of TKIs with other conventional chemotherapeutics have been evaluated, in order to maximize their antitumor effects. To determine the maximum tolerated dose, the study by Robat et al. [242] reported that the use of vinblastine (1.6 mg per m2 every 14 days) and toceranib (3.75 mg per m2 every 48 h) was well tolerated, with signiﬁcant activity against MCT (71.4% response objective). Olsen et al. [229] evaluated the efﬁcacy of the protocol of vinblastine (1.6 mg per m2, every 14 days, in 8 sessions), prednisolone (1.0 mg/kg SID), and toceranib (2.5 mg per m2, every 48 h, for 16 weeks) as neoadjuvant therapy for large MCTs and as adjuvant to surgery for high-grade MCTs, resulting in an 88% response in the neoadjuvant therapy group and a mean survival time of 893 days in the adjuvant therapy group. 4.2.2. Tyrosine Kinase Inhibitors The therapeutic combination of TKIs with lomustine was also evaluated. Pan et al. [243] carried out a study in dogs with different tumors, combining the use of toceranib (2.75 mg per kg, every 48 h) and lomustine, with a deﬁned maximum tolerated dose of 50 mg per m2, every 3 weeks. Similarly, Burton et al. [195] reported that the maximum tolerated dose of lo- mustine, when combined with toceranib (2.75 mg per kg, days 1, 3, and 5 of a 21-day cycle) was also 50 mg per m2, with an objective response rate of 46% in unresectable MCTs. In both studies, the protocol was well tolerated, the most common adverse effect being neutropenia, with an increase in alanine transaminase enzyme levels in more than 50% of patients. Additionally, Bavcar et al. [244] observed that the protocol combining toceranib (2.7 mg per kg, every 48 h), lomustine (60 mg per m2, every 21 days), and prednisolone (1.0 mg per kg, every 48 h, alternating with the toceranib) had a 50% satisfactory objective response rate, but resulted in signiﬁcant toxicity, leading to the need for some protocol change in all study animals, and with 30% deaths/euthanasia due to therapy-related toxic- ity. In that study, neutropenia was also the most frequent adverse effect. The association of TKIs with conventional chemotherapy has already been proven to be effective and safe, if tolerable doses are respected, but in some cases, it can cause serious adverse effects. p Similar to chemotherapeutic agents, TKIs also have adverse effects [38]. These effects result from chronic inhibition of the receptor tyrosine kinase in normal cells, which depend on these kinases for survival and proliferation under normal conditions. The main adverse effects are gastrointestinal problems and neutropenia, but dermatological signs, hepatotoxi- city, nephrotoxicity, hypoalbuminemia, and hypertension have also been reported [230]. In the face of adverse effects, the dose of toceranib can be reduced by 0.5 mg per kg (up to a minimum dose of 2.2 mg per kg) and treatment can be interrupted for up to 2 weeks, according to the manufacturer’ recommendations. Importantly, the response to TKIs may be slower and, therefore, maintenance of the medication for at least 3 weeks is suggested. However, in cases of progressive disease, the duration of treatment should be reassessed. 4.3. Support and Palliative Treatment As previously mentioned, the clinical signs most commonly associated with MCTs are directly related to mast cell degranulation, mainly leading to gastrointestinal ulcerations, delayed wound healing, hypotensive shock, and coagulopathies. Therapeutic management of these complications through supportive therapies is employed to prevent and minimize these deleterious systemic effects, improving the quality of life of patients [15,67]. With a view to prevention and treatment of systemic signs caused by hyperhistaminemia, the use of histamine receptor inhibitors (H1 and H2) is indicated. To prevent and treat gastrointestinal ulcerations, the use of H2 inhibitors is recommended (famotidine, at a dose of 0.5–1 mg per kg, BID; cimetidine, at a dose of 4–5.5 mg per kg TID; or ranitidine, at a dose of 2 mg per kg, BID). Occasionally, dog’s refractory to antihistamine therapy may beneﬁt from the use of proton pump inhibitors (omeprazole, at a dose of 0.5–1 mg per kg, BID–SID), as they also inhibit gastric acid production, but via a proton bomb effect. In cases where there is evidence of active gastrointestinal ulceration, sucralfate can be added to the protocol, at a dose of 0.5–1 g BID [14,132]. H1 inhibitors (diphenhydramine, at a dose of 2–4 mg/kg BID) are indicated to minimize the adverse effects of histamine release on the peripheral vasculature and wound healing, as well as for the treatment of erythema, edema, and pain locations [67]. In cases with a higher degree of inﬂammation, local pain management can also be achieved with the use of analgesics and systemic anti-inﬂammatory drugs. It is important to emphasize that the release of histamine by tumor manipulation during surgery can lead to unwanted cardiovascular effects, such as a decrease in vas- cular resistance, systemic blood pressure, and the development of arrhythmias, as well as hypersensitivity reactions and, more rarely, anaphylactic shock. Therefore, the use of H1 inhibitors (diphenhydramine 1–2 mg per kg, SC) should be instituted for dogs with MCTs at 30–60 min before the procedure [66,132,245,246]. Furthermore, some anesthetic drugs, such as some opioids (morphine and meperidine), have the potential to maximize histamine release, and should be avoided in patients with MCTs [247]. In addition to histamine, other vasoactive substances may also be involved. Heparin release by neoplastic mast cells mainly contributes to the development of local coagu- lopathies. Heparin released during surgical manipulation of the tumor can also lead to considerable bleeding. 4.2.2. Tyrosine Kinase Inhibitors The authors of this consensus guideline recommend treatment with TKIs, particularly for patients with unresectable or metastatic cutaneous MCTs or as adjuvant therapy (alone or associated with chemotherapy). However, given the absence of predictive factors for the response to TKIs and chemotherapy, it is indicated that the choice of treatment should be based on each patient’s individual data, considering the clinical criteria (size, Cells 2022, 11, 618 26 of 37 ulceration, growth rate), histopathological data (grading by Kiupel and Patnaik, mitotic count, and lymph node status), and as necessary, immunohistochemical data (KIT pattern and Ki-67 count). 4.3. Support and Palliative Treatment Protamine sulfate, a heparin antagonist, is indicated for use in cases of prolonged bleeding [248]. Therefore, this consensus indicates the institution of supportive therapeutic manage- ment in patients with MCTs who: 1. Manifest systemic and/or local clinical signs; 2. Are larger; g 3. Will undergo considerable surgical manipulation or tumor section (cytoreductive surgery); g g p ( y g y) 4. With a greater probability of local degranulation (chemotherapy or radiotherapy in macroscopic disease); and/or 5. Patients with metastatic disease [15,67,132,245]. 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. 5. Future Perspectives New studies are underway to test new therapies that can improve the treatment of canine MCT. Researchers have evaluated the efﬁcacy of therapies by the intramuscular and intratumoral routes, using an electrogene with plasmid encoding interleukin-12 (IL-12), resulting in CR in 2/3 canines with MCT, and volume reduction in about 83% of the tumor [249]. Advances in epigenetics have shown the existence of histone hypoacetylation in can- cer. Thus, experimental treatments with a histone deacetylation inhibitor, AR-42, induced growth reduction and apoptosis in vitro in a strain of canine MCT, which may be a promis- ing therapy for this tumor [250]. A Brazilian study evaluated Trichostatin A (TSA), a Cells 2022, 11, 618 27 of 37 histone deacetylase inhibitor that has an antiproliferative effect and induces apoptosis in cancer cells. The action of the drug in vitro on MCT grade III cells had deleterious effects on the growth and proliferation of tumor cells, suggesting a good chemotherapeutic potential [251]. Furthermore, the JAK2/STAT5 signaling pathway has been the target of studies in different neoplastic types, with evidence of its activity in cases of canine MCT, representing a possible target for the development of new therapies [252,253]. Author Contributions: Conceptualization: A.B.d.N., R.d.S.H., M.L.Z.D.; methodology, A.B.d.N., R.d.S.H.; formal analysis, A.B.d.N., R.d.S.H., C.E.F.-A.; investigation, A.B.d.N., R.d.S.H., M.L.Z.D., C.E.F.-A., F.N.d.P., L.C.M.L., B.F.F., L.R.B., F.A.R.S., K.D.d.O., S.V.L., R.D.F.S., C.H.M.B., M.M.M.R., P.C.J., J.L.C.C., R.U., K.B., R.A.S., N.O.d.C., A.T.N., S.C.F., S.C.d.S.C., D.G.G., G.S.G.C., L.R.B., R.L.-A., P.R.d.O.P., G.E.L., R.R.H., F.G., C.H.d.C.V., D.S.d.A., Â.C.M.L., J.M.M., M.V.; resources, A.B.d.N., R.d.S.H., M.L.Z.D., C.E.F.-A., F.N.d.P., L.C.M.L., B.F.F., L.R.B., F.A.R.S., K.D.d.O., S.V.L., R.D.F.S., C.H.M.B., M.M.M.R., P.C.J., J.L.C.C., R.U., K.B., R.A.S., N.O.d.C., A.T.N., S.C.F., S.C.d.S.C., D.G.G., G.S.G.C., L.R.B., R.L.-A., P.R.d.O.P., G.E.L., R.R.H., F.G., C.H.d.C.V., D.S.d.A., Â.C.M.L., J.M.M., M.V.; data curation, A.B.d.N., R.d.S.H., M.L.Z.D., C.E.F.-A., F.N.d.P., L.C.M.L., B.F.F., L.R.B., F.A.R.S., K.D.d.O., S.V.L., R.D.F.S., C.H.M.B., M.M.M.R., P.C.J., J.L.C.C., R.U., K.B., R.A.S., N.O.d.C., A.T.N., S.C.F., S.C.d.S.C., D.G.G., G.S.G.C., L.R.B., R.L.-A., P.R.d.O.P., G.E.L., R.R.H., F.G., C.H.d.C.V., D.S.d.A., Â.C.M.L., J.M.M., M.V.; writing—original draft preparation, A.B.d.N., R.d.S.H., F.N.d.P., L.C.M.L., B.F.F.; writing—review and editing, A.B.d.N., C.E.F.-A., M.V., M.L.Z.D.; visualization, A.B.d.N., R.d.S.H., M.L.Z.D., C.E.F.-A., F.N.d.P., L.C.M.L., B.F.F., L.R.B., F.A.R.S., K.D.d.O., S.V.L., R.D.F.S., C.H.M.B., M.M.M.R., P.C.J., J.L.C.C., R.U., K.B., R.A.S., N.O.d.C., A.T.N., S.C.F., S.C.d.S.C., D.G.G., G.S.G.C., L.R.B., R.L.-A., P.R.d.O.P., G.E.L., R.R.H., F.G., C.H.d.C.V., D.S.d.A., Â.C.M.L., J.M.M., M.V.; supervision, M.L.Z.D.; project administration, A.B.d.N., L.R.B., M.L.Z.D.; funding acquisition, A.B.d.N., M.L.Z.D. All authors have read and agreed to the published version of the manuscript. 5. Future Perspectives Funding: The publication fee for this article was provided by the Brazilian Association of Veterinary Oncology, ABROVET. Institutional Review Board Statement: The study did not require ethical approval. Data Availability Statement: Data are available upon request. Data Availability Statement: Data are available upon request. Acknowledgments: ABROVET thanks the attendees of the II MCT consensus meeting for their active participation in the discussions. References 1. Macy, D.W. Canine Mast Cell Tumors. Vet. Clin. North Am. Small Anim. Pr. 1985, 15, 783–803. [CrossRe 2. Pakhrin, B.; Kang, M.-S.; Bae, I.-H.; Park, M.-S.; Jee, H.; You, M.-H.; Kim, J.-H.; Yoon, B.-I.; Choi, Y.-K.; Kim, D.-Y. Retrospective study of canine cutaneous tumors in Korea. J. Vet. Sci. 2007, 8, 229–236. [CrossRef] [PubMed] y J [ ] [ ] 3. Gamlem, H.; Nordstoga, K.; Glattre, E. Canine neoplasia—Introductory paper. 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https://openalex.org/W2728154421	https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-017-4349-x	English	null	From fear to resilience: adolescents’ experiences of violence in inner-city Johannesburg, South Africa	BMC public health	2,017	cc-by	11,942	* Correspondence: fscorgie@wrhi.ac.za 1Wits Reproductive Health and HIV Institute (WRHI), Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa 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. From fear to resilience: adolescents’ experiences of violence in inner-city Johannesburg, South Africa Fiona Scorgie1*, Deborah Baron1, Jonathan Stadler1, Emilie Venables2, Heena Brahmbhatt3, Kristin Mmari3 and Sinead Delany-Moretlwe1 The Author(s) BMC Public Health 2017, 17(Suppl 3):441 DOI 10.1186/s12889-017-4349-x The Author(s) BMC Public Health 2017, 17(Suppl 3):441 DOI 10.1186/s12889-017-4349-x Background Much of the research on young people and violence in South Africa has come from low-income urban communi- ties in the Cape [4, 25, 26], an area that has very specific socio-cultural dynamics relating to gang violence and poverty. In this article, we report on qualitative findings from the Johannesburg site of the WAVE study, a cross-sectional study of adolescents in low-income urban settings, conducted between 2011 and 2013. Specifically, we examine how violence – both in public spaces and in the home – is experienced differently by adolescent boys and girls living in Hillbrow, a high-density inner-city area. Placing adolescents’ own narratives at the centre of our analysis, we explore how they conceptualise ‘dangerous’ and ‘safe’ spaces in their neighbourhood, and how gender and space shape their everyday experiences of living here. By considering how adolescents make sense of and cope with living in a violent environment, we begin to unpack the forms that resilience might take in this setting. To our knowledge, this concept has not been used before to understand the lives of adolescents in South African inner-cities. Finally, we take a brief look at the social ser- vices and support available to vulnerable adolescents in Hillbrow, focusing on the gaps identified both by adoles- cents and by service providers. There are multiple causal pathways to violence in South Africa, with historical roots in “structural inequality, socio-cultural tolerance of violence, militarised masculin- ity, disrupted community and family life, and the erosion of social capital” [9]. Gender power inequity [10] and a history of racial discrimination and marginalisation also play an integral role. Furthermore, the poor are generally more vulnerable to violent crime than those in middle and upper income neighbourhoods owing to environmental factors such as poor public policing, poor street lighting, and overcrowding [1, 11]. g [ ] Adolescents exposed to interpersonal violence1 – whether directly or indirectly, as witnesses – have sub- stantial long-term health consequences, many of which remain unrecorded and unquantified [12]. Exposure also puts adolescents at higher risk of becoming either victims or perpetrators of further violence in adult life [13–16]. Experiencing violent crime is a major cause of post-traumatic stress syndrome (PTSS) and depression, and even living in high crime areas may induce fear and anxiety, negatively impacting on mental health in the long term [1]. Background with depression three times more likely in girls with ex- perience of IPV than those without [8]. g South Africa is one of the most violent countries in the world: in 2005, as many as 39% of all deaths recorded at national mortuaries were due to violence [1] and an esti- mated 1.75 million people in the country seek healthcare annually for non-fatal injuries resulting from violence [2]. Death rates per 100,000 due to homicide and violence are five times as high as the global average (72.5 in South Africa, versus 14.0 globally in 2000) [3]. For young people growing up in poor communities, violence is often a part of daily life [4]. In a national household survey of over 3500 children aged 10–17 years, more than half (56.3%) reported lifetime physical abuse and 9% lifetime sexual abuse [5]. Data from the recent five-country WAVE study (Well-being of Adolescents in Vulnerable Environments) found that adolescents in Hillbrow, Johannesburg, were deeply concerned about their personal safety [6] – and for good reason. Two-thirds (67%) of adolescent males and 48% of adolescent females in this setting reported being a victim of violence in the past 12 months [7]. Prevalence of intimate-partner violence (IPV) in the past year was a staggering 36.6% among adolescent girls, the highest of all five cities in the study [8]. In the literature on violence, child abuse and social protection, more attention is being paid to the development of ‘resilience’ among victims [18–21]. In this context, resili- ence may be defined as the ability to develop “patterns of positive adaptation in the context of significant risk or ad- versity” [22]. While still nascent, the literature recognises that resilience is not only an individual personality trait but also a context-dependent interplay between vulnerability and protective factors in the social environment, such as the availability of social capital and cohesion [23, 24]. In theory, adolescents with greater resilience are at lower risk of developing PTSS, since they effectively have a ‘buffer’ to lessen the impact of violence [25], although it remains unclear how resilience is affected by instances of repeated exposure to violence. Also poorly understood is the ques- tion of how gender and other demographic factors shape levels of resilience among adolescents living in violent environments. Background A community study in Khayelitsha, Cape Town, found that 95% of children between the ages of 6 and 16 years had witnessed at least one violent event, and 22% met the criteria for PTSS, a substantially higher rate than that recorded among similar age cohorts in North America and Europe [17]. In inner-city Johannesburg, girls’ exposure to intimate-partner violence and non-partner sexual violence was associated with sub- stance abuse, condom non-use, multiple sexual partners, pregnancy, transactional sex and poor mental health – Abstract Background: For adolescents growing up in poor urban South African settings, violence is often a part of daily life and has lasting effects on physical and mental health outcomes in adulthood. We conducted a qualitative study to document and understand the forms of interpersonal violence experienced by adolescents living in Hillbrow, Johannesburg. In this article, we explore how violence is experienced differently by adolescent boys and girls, how they conceptualise ‘dangerous’ and ‘safe’ spaces in their neighbourhood and what gaps exist in available services for youth in Hillbrow. Methods: The article draws on data collected in the formative phase of the ‘Wellbeing of Adolescents in Vulnerable Environments’ (WAVE) Study of challenges faced by adolescents (15–19 years) growing up in impoverished parts of five cities. This article reports on analysis using only data from the Johannesburg site. Using both purposive and snowball sampling to select participants, we conducted in-depth interviews (n = 20) and community mapping exercises with female (n = 19) and male (n = 20) adolescents living in Hillbrow, as well as key informant interviews with representatives of residential shelters, CBOs, and NGOs working with youth (n = 17). Transcripts were coded manually and analysed using an inductive thematic analysis approach. Results: Both girls and boys reported high exposure to witnessing violence and crime. For girls, the threat of sexual harassment and violence was pervasive, while boys feared local gangs, the threat of physical violence, and being drawn into substance-abuse. Home was largely a safe haven for boys, whereas for girls it was often a space of sexual violence, abuse and neglect. Some adolescents developed coping mechanisms, such as actively seeking out community theatres, churches and other places of sanctuary from violence. Community-based services and shelters that support adolescents reported a lack of resources, overall instability and difficulties networking effectively. Conclusions: Adolescents in Hillbrow commonly witnessed and had direct experience of many forms of violence in their environment, and these experiences differed markedly by gender. Interventions that build young peoples’ social capital and resilience are essential for reducing violence-related trauma and long-term health and social consequences for adolescents in this community. Keywords: Adolescents, Violence, Inner-city, Gender, Low-income, SOUTH Africa, Johannesburg The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Page 52 of 122 Page 52 of 122 Data analysis Audio files of the IDIs and community mapping discus- sions were transcribed and translated into English, where necessary, with quality checks on the translations con- ducted by the site Principal Investigator. Analysis was carried out manually by two authors (FS and DB), who read the transcripts to identify initial themes and develop a coding structure using an inductive thematic analysis approach [31]. Transcripts of IDIs with adolescents and community mapping activities were combined for the pur- poses of analysis and then coded using a set of key themes, with additional themes added iteratively as the analysis proceeded. Girls’ transcripts were analysed separately from boys’ transcripts, and the coding structure was set up to In-depth interviews d h In-depth interviews (IDI) were conducted with 20 ad- olescents at a research clinic in Hillbrow, using a semi-structured discussion guide with questions about personal perceptions and experiences of danger and safety in Hillbrow. In addition, key informants were interviewed about their experiences working with youth and about the experiences and life challenges faced by adolescents in the area. Interviews were facilitated by two trained female researchers from WRHI and lasted roughly an hour each. IDIs were conducted in English, and two local languages (IsiZulu and Sesotho). Notes were taken and interviews recorded using digital audio recorders. Recruitment and data collection During provisional mapping of shelters and services for young people in Hillbrow, the research team met with representatives of a diverse range of organisations work- ing with youth in Johannesburg spanning the health, education, social welfare services and housing sectors. Through a combination of purposive and snowball sampling, these representatives were invited to partici- pate as key informants, and were asked to introduce the researchers to adolescents who could potentially be invited to take part in the study. Additional adolescent participants were recruited directly from schools and youth shelters in the area by two trained female re- searchers, following the distribution of recruitment fliers in local streets, parks, churches, and the research clinic. Methods Data presented in this article were collected during for- mative research in the WAVE study, the full methods of which are described in detail elsewhere [6]. Study protocols, researcher training and consent procedures were standar- dised across sites. Methods included key informant inter- views, in-depth interviews, community mapping and linked focus group discussions. In the Johannesburg site, commu- nity mapping involved only adolescents (15–19 years) living The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Page 53 of 122 Page 53 of 122 in Hillbrow at the time of the study (n = 39), as did the in- depth interviews (n = 20). Key informants (n = 17) included representatives of residential shelters, community-based or- ganisations (CBOs), and non-governmental organisations (NGOs) working with youth.2 at a local residential shelter. The groups were gender distinct and divided by age (15–16 years old and 17– 19 years old). Mapping was facilitated by two female re- searchers, trained by the lead qualitative investigator on the main WAVE study (KM), through the use of video clips and role-play exercises using the research instru- ments [6]. Discussion in the groups took place in English and two local languages – isiZulu and seSotho – and each session lasted between one and two hours. Partici- pants were provided with flip chart paper, pens and stickers to produce the maps. Following the community mapping exercise, a focus group discussion was held with each group to collect additional information. Study setting Hillb i Hillbrow is a densely populated inner-city neighbour- hood in central Johannesburg (an estimated 100,000 in- habitants in one square kilometre), with many high-rise apartment blocks, entertainment venues, small retail shops and informal traders. It is often the first place in which newcomers to the city settle. Hillbrow’s large mi- grant population incorporates economic migrants from other provinces of the country, as well as refugees and immigrants from across the continent and beyond. These newcomers are at the receiving end of pervasive regional xenophobia, and they are socially and econom- ically marginalised, living in makeshift, overcrowded and often unsafe dwellings [27]. Participants were not expected to produce cartograph- ically accurate maps of Hillbrow, but rather to draw a schematic representation of the neighbourhood based on how they experienced it as residents (See also [29, 30]). Recreational areas, apartment blocks, schools, libraries, and other venues were drawn, and participants circled those where they felt safe or unsafe by day and night. The activity created much animated discussion, which was audio-recorded. Images of two maps are included here as examples of the end products generated through this process (see Figs. 1 and 2). In quantitative findings of the WAVE study, many Johannesburg adolescents interviewed were living in unstable housing, often in single-parent households or lodging with relatives [28]. They reported low levels of support from caring family members and from neighbour- hood resources, and had a much poorer perception of their physical environment than their counterparts in other cities [7]. Hillbrow, as they described it, was charac- terised by dirty and decayed buildings, overcrowded living conditions, garbage and sewage spilling onto the streets, and chronically neglected public infrastructure. Community Mapping Only adolescent boys (n = 20) and girls (n = 19) took part in these mapping activities. Eight mapping groups of between three and six participants were formed, and these met separately at the research clinic in Hillbrow or Page 54 of 122 The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Fig. 1 Girls’ map of Hillbrow. Legend: Red stickers show residential places; blue stickers = schools; yellow stickers = churches and religious buildings; orange stickers = places where young people hang out; brown stickers = libraries, cyber cafes. Circled in yellow = feel safe during day; circled in blue = feel safe at night; circled in red = don’t feel safe during day; circled in black = don’t feel safe at night Fig. 1 Girls’ map of Hillbrow. Legend: Red stickers show residential places; blue stickers = schools; yellow stickers = churches and religious buildings; orange stickers = places where young people hang out; brown stickers = libraries, cyber cafes. Circled in yellow = feel safe during day; circled in blue = feel safe at night; circled in red = don’t feel safe during day; circled in black = don’t feel safe at night enable comparison of adolescents of different ages. Coding of transcripts concluded when all data had been assigned to a code and saturation achieved. the terrain of Hillbrow. Key coding themes from analysis of key informant interview transcripts included: nature and scope of work; interaction with adolescents; greatest challenges in service provision; and the impact of the Hillbrow environment on young residents. The final set of key themes included: public violence and crime; gang violence; sexual abuse; nature of home life; greatest concerns or worries; positive/negative as- pects of living in Hillbrow; reflections on how girls’ and boys’ experiences of violence differed; availability of local support services; and safe spaces. Analysis of the maps developed during community mapping exercises in- volved assessment of their layout, a count of ‘safe’ and ‘dangerous’ spaces, and their physical distribution across Our analysis was also partly informed by narrative analysis, taking the position that adolescents’ accounts about everyday life are not solely descriptive but are meaningful acts, potentially both socially powerful and personally empowering [32, 33]. Community Mapping As such, their narratives are commentaries about the lived world as well as idea- lised worlds [34], and provide insight into young peoples’ Page 55 of 122 The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Fig. 2 Boys’ map of Hillbrow. Legend: Red stickers show residential places; blue stickers = schools; yellow stickers = churches and religious buildings; orange stickers = places where young people hang out; brown stickers = libraries, cyber cafes. Circled in yellow = feel safe during day; circled in blue = feel safe at night; circled in red = don’t feel safe during day; circled in black = don’t feel safe at night Fig. 2 Boys’ map of Hillbrow. Legend: Red stickers show residential places; blue stickers = schools; yellow stickers = churches and religious buildings; orange stickers = places where young people hang out; brown stickers = libraries, cyber cafes. Circled in yellow = feel safe during day; circled in blue = feel safe at night; circled in red = don’t feel safe during day; circled in black = don’t feel safe at night characteristics, we tackle each of these themes below. Firstly, we report on the multiple forms of violence ex- perienced by young people in Hillbrow’s public spaces, followed by a look at what domestic life holds for them: is home necessarily a safe haven in this setting? Then, we consider how Hillbrow’s adolescents identify ‘safe’ coping strategies, resistance to victimisation, and aspira- tions for the future. Sample overview In total, 59 adolescents took part in the study (commu- nity mapping = 39; IDIs = 20). Equal numbers of girls and boys took part in the IDIs, while the community mapping involved 19 girls and 20 boys. On average, the 17 key informants interviewed (10 female, 7 male) had worked with youth for 10 years, with experience ranging from 1 to 36 years. Among the adolescent IDI participants, four boys and three girls reported having at least one deceased parent. Some participants lived with relatives as their parents ei- ther could not afford to take care of them or lived too far away, and two of the girls lived in a shelter because they had been expelled from home by relatives. Virtually all participants made some reference to financial hard- ship in their households, but for at least half of the girls interviewed, these hardships were acute: where money for food, school fees and rent was often inadequate. Two girls had recently experienced interrupted schooling owing to a lack of finances. Earning a small income through in- dependent, part-time work appeared to be more common among boys (fixing cars, selling flowers or recording music for local DJs) than girls (braiding hair). Two girls men- tioned receipt of state child support grants, but claimed these funds were being collected by relatives and used for other purposes. p y Not all gangs were regarded as violent, however. Local groups such as the ‘Pantsulas’, ‘Bourgeois’ and ‘Hip-hop- pers’ were apparently better known for their music and clothing preferences, and for characteristic styles of dan- cing. But intense competition among local gangs, even those defined more by dancing than by a penchant for violence, meant that physical conflict routinely erupted between them, particularly when sexual liaisons devel- oped across gang boundaries. Boys explained that young people who were simply bystanders could inadvertently be drawn into these competitive and intrinsically violent interactions. One popular gang in the neighbourhood, they claimed, was the ‘Izikhothane’, modelled on a no- torious youth sub-culture that originated in Soweto in the early 2000s [35]. Izikhothane members would wear luxury branded clothes and nihilistically flaunt wealth they did not have. They were said to tear up banknotes and clothes “and throw [them] at you”. Sample overview Boys said they felt “ashamed” when confronted with Izikhothane, and hinted at how the gang ethos fused local constructions of masculinity with the glorification of ostentatious dis- plays of consumerism: Results Four distinct thematic areas emerged from an analysis of our findings. Following a brief overview of sample The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Page 56 of 122 Page 56 of 122 from rugby practice. I was coming there by Shoprite and it was dark and I think it was probably around eight thirty in the evening…I was traumatised…I was alone and I was shocked.” (Tshepo,3 15 year-old boy) and ‘unsafe’ areas in the neighbourhood, and navigate these spaces to minimise risk. This section reflects on how these young people make sense of their environ- ment and deal with negative stereotypes about Hillbrow in surprisingly positive ways. Finally, we present data on the availability and quality of support services in Hillbrow, from the perspectives of service providers and of the young people they are intended to serve. Participation in petty crime and substance abuse was allegedly required for membership to some male gangs that populated the Hillbrow landscape, intimidating people, stealing cell-phones and instigating violent clashes. A gang known as the ‘Vandals’ apparently used plastic rulers to search for phones hidden on the bodies of their victims. Their modus operandi was simple and effective: “They take people’s phones, they hit people, they ask you for money, you say no, the next thing they hit you” (Karabo, 16 year-old girl). Another gang known as the ‘CMFs’ (“Crazy Mother Fuckers”) were known for stealing and stabbing. They were “…always drinking alcohol and if you quarrel with one of them they all come to you because their slogan is ‘touch one, touch all’” (Busi, 16 year-old girl). Participants described how the determination of certain gangs to protect their turf meant that spatially, Hillbrow was divided into a number of tightly controlled ‘no-go’ areas. Those who strayed into their territory – whether members of other gangs or not – were likely to be punished with threatened or actual physical violence. “…they took my cell phone from me and they had a knife and that happened when I was coming back “Outside these walls it’s dangerous”: violence in Hillbrow’s public spaces In the narratives of adolescent participants, the social environment of Hillbrow was dominated by violent crime, substance abuse, and sex workers, a place where drugs were routinely sold in the open. Police intervention in Hillbrow was described by most as virtually non-existent, with the only effective protection against violent crime be- ing that offered by armed guards hired by private security companies and reserved for the neighbourhood’s enter- tainment venues. Drug- and alcohol-fuelled fighting in public spaces was reportedly a common occurrence, along with mugging and petty theft. One boy recounted his experience of the latter just a few days prior to the interview. “…some of us want to live a peaceful life and it’s not a nice thing if you go out there and someone says ‘look at this one, the poor one, he’s got nothing and we’ve got this and that’. And that won’t make you feel good about yourself…you will end up beating them up because that person will be embarrassing you. And it “…they took my cell phone from me and they had a knife and that happened when I was coming back Page 57 of 122 The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Page 57 of 122 would be worse if there were girls around because you don’t want to be embarrassed in front of women.” (Boys CM 2402) that xenophobic attitudes were preventing these migrants from accessing local health services. Finally, in addition to direct, personal experiences of violence, both boys and girls reported commonly witnes- sing acts of violence in public. While none of the boys reported membership to a gang or direct experience of violence inflicted by gang members, some had witnessed others being attacked and mugged. They expressed anxiety about the need to avoid encountering these gangs in their day-to-day interac- tions. Overall, boys’ detailed descriptions of adult male gang behaviour revealed an acute awareness of how their presence in Hillbrow was impacting on the ability of all residents to feel safe. “…we see a lot of things. We see people fighting, drinking, we see pornography live. “They know that if they speak something, uncle will kill me”: Violence in the home Others spoke of their reluctance to visit bars in the area, because they could expect to encounter men who were “too touchy-touchy [laughter]” (Girls CM 0404). Rose (17 year-old girl) described being targeted sexually by the leader of a local gang: Compared to descriptions of violence in the public spaces of Hillbrow, violence in the home was charac- terised by an even more profound gender asymmetry, in that it was raised almost exclusively by girls. Their stor- ies took three broad forms: direct experiences of sexual violence; witnessing domestic violence; and experiencing emotional abuse and neglect. “Every time I come to Hillbrow, I am scared that I might meet him. […] When I see him I have to kiss him, like really…I’m scared he will hit me, like really bad. […] Yes! ‘I will slap you, if you don’t kiss me now like really I will point you, I will take out my knife, I will stab you…’.” Accounts of sexual violence in the home mostly in- volved perpetrators who were known to the girls. Fifteen year old Thembi, who was perinatally infected with HIV and currently not attending school, stayed with her aunt and uncle following her mother’s death from AIDS. While her aunt was away from home for some time, Thembi was subjected to repeated rape by her uncle – Many adolescents drew connections between sexual violence, transactional sex, and the drug trade – which they believed had been boosted in the area by the pres- ence of foreign men. Indeed, allegations that foreigners were the primary source of crime in Hillbrow were com- mon. Portia (19 year-old girl) spoke about harassment by “foreign guys selling drugs” in public: “…one evening [he] came home and started touching my private parts and told me that he is going to teach me how to please a man and he raped me. And this happened several times until I told my aunt about it and she didn’t believe me, but took me to the clinic … she asked that I be given family planning injection.” (Thembi, 15 year-old girl) “…like if you are walking by them they will be pulling you, [saying] ‘come here my friend I will give you this, anything you want in this world.’ What is that? ‘Cos the guy knows he sells drugs, all he wants to do is spoil my life. “Outside these walls it’s dangerous”: violence in Hillbrow’s public spaces We just see it there, people raping each other …” (Cécile, 16 year-old girl) While boys residing at the Twilight Shelter returned by 18 h00 every evening – for their own safety – they still frequently witnessed violence from the confines of the shelter; they “would see people fighting outside, if looking out the window” (Boys CM 0302). Unsurpris- ingly, the witnessing of such violence was said to have clear mental health consequences: By contrast, girls’ narratives focused much more on their personal experiences of sexual harassment in the public spaces of Hillbrow – mentioned by almost all girls in the IDIs. This was commonly referred to as be- ing “pulled on the street”, and reflected how the simple act of moving around in public was a deeply gendered one. Girls in a community mapping group explained, “Kids experience a lot of fighting in the community, when someone is fighting someone, and taking the bottle to strike someone with this bottle, that is not something good for a child to see. It goes deep into them and it depresses them.” (KI09; male orphanage director) “When you walk around they [men] call you…Yes and say things such as ‘my size, hey my size come here’, I will give you money.” (Girls CM 0304) “They know that if they speak something, uncle will kill me”: Violence in the home They know that if they speak something, uncle will kill me…The children are living in fear.” (KI09, male orphanage director) This absence of a positive adult role model was also keenly felt by 17 year-old Zama, who lived with her sister and her sister’s partner, whose emotional abuse led her to frequent thoughts about running away. Her mother died in 2000, and she has never met her father. A second form of violence in the home involved the witnessing of domestic violence. Rose described watch- ing her sister being beaten up by her partner, to the point where she was unable to walk. Although the police were called, “they didn’t come”, forcing Rose to go to the police station the following morning to open a case of assault. Similarly, Busi described her failed attempts to intervene when her father was abusive: “At times I get angry when I am at home because we live with my sister’s boyfriend and I don’t like him much, we don’t get along...When he gets home drunk he will start telling my sister about how useless I am and that he doesn’t like me.” (Zama, 17 year-old girl) “My dad abuses my mom when he is drunk… He swears at my mom and it happens every time he is drunk...It makes me feel terrible, bad and sad. I try to stop him and he always tells me not to defend my mother because he will hit me.” (Busi, 16 year-old girl) None of the boys reported having being rejected or chased away by their families, nor did they report experi- encing physical abuse in the home. In the final sections below, we explore how adolescents in our study have responded to these experiences of violence – whether remaining silent, tapping into support services in their environment, or forging their own strategies for coping and staying safe. Such responses are crafted against the backdrop of how residents of Hillbrow imagine and inter- act with its physical spaces. Nineteen year old Sello recalled witnessing his father’s violent attacks on his mother over the course of several years, eventually culminating in his parents’ separation. Sello was the only boy who reported domestic violence in his home. “They know that if they speak something, uncle will kill me”: Violence in the home You know, he wants to sleep with me…” Efforts to tell other adults in her surroundings, includ- ing a neighbour, led to further victimisation: “ I told her what my uncle is doing to me and she asked my aunt about it and she [the aunt] said that I This perception contrasted starkly with key informants’ main concern in relation to foreigners in Hillbrow, namely, Page 58 of 122 The Author(s) BMC Public Health 2017, 17(Suppl 3):441 am lying, that I want to take her man from her, so she beat me after the lady left… she told me that I am lying and called me a ‘whore’.” (Thembi) her of using witchcraft against his own children. She then went to stay with an aunt, “I had to clean the house, wash her clothes, her son’s clothes, her daughter’s clothes and her husband’s clothes and I had to cook and everything, and sometimes she would not give me food.” (Cécile, 16 year-old girl) Thembi’s was the only direct experience of rape re- ported by participants, but it nonetheless highlights a more general absence of positive adult role models for young people in this area, including recourse for young people experiencing sexual violence in their homes. It is perhaps not surprising that key informants remarked on the under-reporting of abuse in the home as a significant challenge in their work. The director of a local orphan- age alluded to even greater violence that awaited victims who spoke out about their abuse, although did not pro- vide specific examples of such extreme retribution: Cécile then left her aunt’s house and moved into the shelter. In the interview, she reflected that it “hurts a lot” to not live with her mother and siblings anymore, and that growing up without parental guidance was a real challenge. “…living life away from them, it’s painful because life in this place is hard. You get into the adolescent stage and you don’t know who to trust, who to go to give you advice then the things people do here, you don’t know if this is good or this is bad.” (Cécile) “You see the problem here is that so many girls have these problems [sexual abuse] and they don’t talk. They keep it a secret…these children are small, but they are scared of people. “They know that if they speak something, uncle will kill me”: Violence in the home Thirdly, and perhaps less easy to categorise, was a form of emotional abuse and neglect experienced at home by girls such as Lerato, Cécile and Zama, who had been separated from their parents, either by death, poverty or conflict in the family. As a consequence, they stayed with relatives in somewhat precarious home circumstances, were often made to carry out the major- ity of the housework, including caring for younger sib- lings, and at times made to feel unwanted. Living in a shelter at the time of the interview, Cécile had been thrown out of her home by her step-father who suspected Geographies of danger: finding safe spaces and reframing violent realities At first glance, the community maps produced by ado- lescent participants in our study simply lay out the spatial arrangement of Hillbrow: the streets, apartment buildings, shops, the police station, churches, parks, and schools, and so on. Closer examination reveals that they also depict a ‘geography of personal danger and safety’. As the social realities of living in Hillbrow were plotted onto representations of its physical spaces, isolated pockets of safety emerged amidst this complex terrain. As with the The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Page 59 of 122 exposure to violence in public and at home, there were clear gendered differences in how this precarious geography was lived and imagined. Boys tended to avoid places where they were likely to encounter peer pressure to engage in physical conflict or substance use. One boy even framed this strategy as a need to be safe not only from “the outside world” but also “from my own danger” (Boys CM 2402). Churches were identified as potential ‘sanctuaries’ regardless of gender, while schools were only mentioned by boys as offering protection from outside dangers. Girls cited in- stances of sexual harassment by schoolmates, suggesting that in this respect at least, schools were no different to other public spaces, where such harassment was the norm. g Overall, boys’ and girls’ maps differed mainly in relation to the number and dispersal of ‘safe’ spaces identified across Hillbrow. Figures 1 and 2 illustrate this pattern well: the girls’ map (Fig. 1) identifies only a few places concentrated in a small area of the neighbourhood as ‘safe’, suggesting that girls’ movement beyond this cluster is probably quite limited. Areas identified as ‘unsafe’ by the girls are specific sections of streets that are main thoroughfares or access/ exit points to Hillbrow, which in turn corresponds to their complaints of being “pulled on the street” by men, and of needing to find alternative routes when “being sent” to run errands, or when walking to and from school (see below). The boys’ map in Fig. 2, by contrast, identifies more loca- tions as ‘safe’, and these are scattered across a wider area. Retail and entertainment venues, which pose the risk of muggings and alcohol-fuelled violence, were cited most as ‘unsafe’. Geographies of danger: finding safe spaces and reframing violent realities Age did not appear to be a strong factor shaping these maps – except in one respect, namely, that older girls (17–19) seemed more aware than their younger counter- parts of which entertainment venues served alcohol – de- scribed as dangerous but also enticing – and hinted at their own patronage of these venues. While one might expect young people to regard their homes as ‘safe spaces’, importantly, this association was heavily contested within the girls’ mapping groups. Those who asserted that they felt safe “at home” or “in flats” were almost always challenged by fellow group members who had experienced otherwise. One girl specified, “I feel safe only on my floor, because we lock” (Girls CM 1805), while others contended, “no, I don’t feel safe at our flats” (Girls CM 0404) and even claimed, “your flat is full of thugs… they mug you around there” (Girls CM 0404). By contrast, boys from a shelter were unanimous in identifying this home as a safe haven from street violence, “from people who might mug you and also from gunshots”. Another said, “[In Twilight Shelter] there is always protection… We are safe from drugs, crossfires, crime, human trafficking and a lot of bad things out there.” (Boys CM 2402) Since virtually all public spaces were regarded as dan- gerous after dark, few participants had direct experience of the city at night, particularly those who lived in shelters, which had strict curfews. For girls, freedom of movement was also limited after school and on weekends to their own homes, the homes of friends, churches, or well- populated shopping areas. The observation of sixteen-year old Busi was a common one: “I am only with my friends on weekends and we never go anywhere. We just play around our flat”. Geographies of danger: finding safe spaces and reframing violent realities In contrast to those who blamed foreign migrants for the unsafe streets of Hillbrow, other participants embraced the diversity of this neighbourhood, crediting its cosmopolitan make-up with broadening their exposure to a variety of people, languages and diverse points of view: In the long run, these perspectives point to promising signs of resilience among some adolescents – both male and female – for it is the ability to reframe their sur- roundings in more positive ways that may ultimately serve to lessen the risk, fear and trauma associated with living in the inner-city. An imperfect stop-gap: support services for vulnerable youth Beyond the construction of certain physical spaces as ‘safe’, however, adolescent participants also commented on the scarcity of external support services and interventions available to help them should they ever become victims of violence. A dominant theme here was that while psycho- social support – particularly counselling services – was known to exist in theory, many adolescents were either not aware of how and where these services could be accessed, or (based on prior experience) they doubted their ability to meet their needs. Some were unaware that services targeting youth existed in Hillbrow at all. “…there are lots of opportunities and it’s all about what you make out those opportunities… So Hillbrow is a fantastic place to us…But it’s all about what you make out of it.” (Boys CM 2402b) Mbali (18 year-old girl), who had moved from Kwa-Zulu Natal to live with an aunt in Hillbrow, stated that “staying in places like these, where there are lots of people opens your mind; it’s not the same as living in the rural areas”. In a girls’ mapping group, one participant even exclaimed, “it’s a cool place to be….I am proudly Hillbrowian! (laugh- ter)” (Girls CM 0304). From the perspective of service providers, however, the nature of this gap between demand for and supply of psy- chosocial services was more complex than ‘mere’ lack of awareness. One key informant who had grown up in the neighbourhood was running a peer education organisation in Hillbrow which was struggling to secure the necessary funding and support. In his estimation, adult residents in Hillbrow did not “have time to come and donate and see what their children are getting up to when they are out”. Geographies of danger: finding safe spaces and reframing violent realities It’s just I take it as a joke.” it as like insulting or something like that. It’s just I take it as a joke.” it as like insulting or something like that. It’s just I take it as a joke.” levels of anxiety and stress and indicated that coping with constant fear was a challenge. Sixteen year old Busi reflected that: “There is nothing easy about staying in Hillbrow, everything is difficult”, while Thembi’s struggles to cope with trauma following sexual violence were heightened by the intense isolation and lack of support she experienced living at a shelter. She occasionally vis- ited an aunt and some cousins on weekends, but this was about to change: Evidence of self-motivation and agency in the face of adversity appeared also in the narrative of Cécile (16 years), a young Congolese woman living in a Hill- brow shelter for girls. A sense of hope was palpable in her vision for the future, which involved a commitment to improve both the shelter and the neighbourhood that had taken her in. “…they said they don’t want me there anymore and they told the social worker that. […] No one visits me. I don’t have friends.” (Thembi) “I want to be an attorney because of the things I see here. I see that there is lot of injustice and unfair trials in this place, so I want to make a difference and my first pay check should be given to this [shelter] to help it become better.” (Cécile) Despite this somewhat grim picture, however, other narratives embodied a strong sense of survival against the odds, and even an expression of community pride in Hillbrow. Tshepo (15 year-old boy) reckoned, “it’s not safe living in Hillbrow but we survive”, while Sandile (18 year-old boy) claimed, “I enjoy it a lot. It’s a good place even though there is crime”. “…there is just something that’s lacking between the service provider and the people needing this service. Geographies of danger: finding safe spaces and reframing violent realities Even one of the boys commented on the apparent ‘informal curfew’ that applied to girls in Hillbrow and limited their mobility: Encounters with potentially violent petty criminals on the streets of Hillbrow were seen as almost inevitable, but steering clear of well-known dangerous routes dur- ing the day was a common tactic, particularly for girls: 15 year-old Lerato’s mother could not allay her fears of “kids who stay at Umshangani Park”, whom she passed when running errands for adults in her household, “be- cause she [her mother] knows that if they come for me then they’ll come.” Lerato’s only strategy – and one that demonstrated agency – was to “take a different direction” on her route in the hope of avoiding them. “Girls have to be home around six because of their safety, while we as boys can be home around ten in the evening.” (Joe, 16 year-old boy) For others, coping with life in a violent environment was easier when one had become desensitised to its effects. Vusi (15 year-old boy) had witnessed so much violence in his neighbourhood it had become ‘normal’: The Hillbrow Theatre was a space identified by all girls’ mapping groups as ‘safe’. This complex, which hosts after- school drama, dance and music programmes for young people in the inner-city, is a popular spot, particularly in the afternoons and on weekends. For boys, libraries and internet cafes were popular retreats, along with parks, which enabled them to “relax” and “refresh your mind”. “I guess for me it’s normal because I have lived here most of my life. Like some of my friends when they come here they tell me that Hillbrow is dangerous and stuff like that, but for me I guess I know that it can be dangerous. I have seen people die here and whatever but you know, it’s something I am used to.” “…because during the day there are people there [in Berea Park] doing different activities such as exercising and all that…the atmosphere is great…it makes you be at ease. And there are security guards there.” (Boys CM 2402b) Vusi’s apparent nonchalance was not shared by all. Some participants – girls in particular – alluded to high The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Page 60 of 122 it as like insulting or something like that. Geographies of danger: finding safe spaces and reframing violent realities For him, there was no shortage of potential resources – organisations, services and safe spaces – on which young people in Hillbrow could draw. But these resources were under-funded and under-used, owing to chronic apathy and a series of “disconnects” between the services and their target populations. Regardless of adolescents’ own thoughts on Hillbrow, however, they were often confronted with the negative perceptions held by others. As one girl put it, “there is a perception that when you say you stay in Hillbrow, you prostitute yourself or you drink alcohol or you take drugs” (Mbali). Most participants recounted being teased by peers who lived in townships, and having to either ig- nore the teasing, defend their reputations, or even pre- tend that they lived elsewhere – acts that in themselves became forms of agency and resilience. Vusi was one of the few who seemed able to brush off these remarks: “…at school people call me a drug dealer and say things like I am a criminal and stuff like that, you know, just the typical stereotype. […]Well I don’t take “…there is just something that’s lacking between the service provider and the people needing this service. Page 61 of 122 Page 61 of 122 The Author(s) BMC Public Health 2017, 17(Suppl 3):441 You know people still need to be convinced as to why do we need this service.” (KI10; male community organisation leader) mentors. She offered specific recommendations for these mentors to lead “groups that would motivate youth as to how they should handle life”. Her suggestion highlights one way in which existing services and organisations could become more responsive to the needs of adolescents: by addressing the longer term problem of absent positive adult role models, rather than only providing a temporary ‘stop-gap’ service for youth who are victims of violence. Other key informants spoke of numerous challenges facing services that attempt to target local youth with recreational activities. These included chronic under- funding and inadequate coordination with the police and other social service agencies. Key informants work- ing in psychosocial services noted the continuing stigma around sexual abuse, particularly for male victims, which accounted for the profound under-reporting to police services, and the low uptake of sexual abuse counselling. A female manager at a national counselling organisation commented on her observation of serious mental health problems among youth. Discussion Adolescents’ narratives about everyday life in Hillbrow painted a picture of pervasive violence, with armed mug- gings, gang-related violence, crime linked to substance use, sexual assault, and domestic violence all unfolding against the background of flawed public policing and so- cial service systems, which have largely failed the people of Hillbrow. Local service providers describe valiant ef- forts to run programmes for youth while facing limited resources, virtually non-existent engagement from adults in the community, and at times, direct obstruction from public sector representatives. The broader context is one in which a weakened civil society is confronting a growing lack of infrastructure and accountability. Many NGOs and CBOs in South Africa experienced serious funding de- clines after the democratic transition in 1994. Rising antagonism from government, particularly intense in the last decade, has further undermined the ability of these organisations to effectively advocate on behalf of the increasingly marginalised communities they repre- sent [36]. Such underlying structural factors compound the poverty-linked vulnerability of young people in Hillbrow, but could also be generalizable to other urban slums – particularly those in Latin America, which mir- rors South Africa’s levels of poverty and inequality, and has similar examples of poorly controlled urbanisation and spatial segregation [37]. Indeed, evidence emerging from our study suggests that everyday violence in Hillbrow is “invisible” and “normalised” in much the same way that Scheper-Hughes has shown in her study of violence in impoverished, inner-city Brazil [38]. Echoing the key informant who was concerned about self-harming behaviour, another noted that young boys “…don’t know what to do with their feelings…it leads to quite a lot of underlying anger simmering from hopeless- ness” (KI016; female counsellor). While we did not expli- citly pose the question of young people themselves perpetrating violence, examples of this did emerge in the data, particularly in relation to children living on the streets: “…when they first arrive, you will see that a child [is] not violent, but once they start staying on the streets they learn all those tricks to survive on the streets, maybe they are fighting to wash somebody’s car…they are also fighting to carry somebody’ bag. It's a serious issue. Geographies of danger: finding safe spaces and reframing violent realities Among the greatest problems was a pattern of suicidal ideation and self-harming be- haviour, which she attributed to being “exposed to things that they don’t have the emotional capacity to deal with”. Often undiagnosed, even when these cases do come to light, “there are no resources” to support refer- rals into proper care. This was also true of referrals for young people with substance abuse problems. Without prompting, Sandile (18 year-old boy) expressed aware- ness that his own drinking had become harmful – “I drink too much, but can’t seem to stop” – and which he was attempting to address alone. Discussion The question then becomes: in settings of heightened vulnerability for young people, would compliance with these gendered roles potentially safeguard their access to networks and sources of social capital, thereby shoring up resilience – or render them even more vulnerable? harmful patterns and build new positive social norms. Arguably, this requires structural interventions that directly engage with the gendered patterns of violence in this setting and the gendered differences in local al- cohol and drug use, as suggested by this study and confirmed by other research [40]. Outside of the home, individual strategies developed by both girls and boys in Hillbrow to minimise risks of violence are similar to those documented more than 15 years ago among women working in this area (mainly rubbish collectors and sex workers) [41]. To cope with the daily risk (and fear) of rape, they created “avoidance zones” around particular streets and public spaces, largely based on prior experience. Adolescents in our study simi- larly constructed ‘safe’ spaces amidst a plethora of areas in the neighbourhood known to be dangerous, and structured their daily movements accordingly, to minimise their risk of exposure to violence. These strategies have limited use in the long term, however, as they restrict the physical mobility of young people – particularly of girls, who are subjected to sexual harassment in public spaces in this setting in ways that boys simply do not experience. This limited mobility has implications for girls’ ability to access information, care and other public resources, constraining their access to social networks and other sources of support to the few areas of Hillbrow in which they feel ‘safe’. Studies of low-income urban communities in the Cape, where male gang activity and community vigilant- ism are rife, have found that boys experience higher levels of violence-related trauma than girls in the same areas [4]. Our findings on male gangs in Hillbrow sug- gest a need to revisit the assumption that gang-related violence is restricted to the Cape. And while we did not measure trauma levels among participants per se, the qualitative findings on the high burden of sexual vio- lence experienced by girls suggest that Hillbrow may be a place where this gender asymmetry is inverted. Discussion In this respect, our findings echo the quantitative analysis in the larger WAVE study, which found that adolescents in the Johannesburg site displayed extremely high rates of depression (41.1% of males and 44.6% of females above the cut-off point for mild depressive symptomology) and PTSD symptoms (54.5% of males and 67.0% of females) [28]. The experiences of girls in our study highlight the extent to which their sexual availability for men is as- sumed is this setting, along with the expectation that girls will occupy the position of domestic servitude within the household reserved for them. How do we craft appropriate and effective responses to this situation? Aside from the critical need for improve- ments in the physical environment, properly resourced, gender sensitive and youth-appropriate services are also required to assist young people who have been exposed to violence. Arguably, this should take the form of interven- tions to build social capital and deepen adolescents’ exist- ing capacities for resilience, rather than being content with efforts to ameliorate its effects at an individual level alone, such as trauma counselling [42, 43]. The success of such resilience-based interventions will depend largely on the availability of sustained adult and community support. This could take the form of structured adult mentorship programmes, which may help to protect against trauma and risk-taking behaviours later in life: research has shown that a relationship with a caring and committed adult “can buffer a young person who grows up in a violent and abu- sive environment from its consequences” [44]. In the absence of positive adult role models, where families are disrupted and violence against women and children becomes normalised, the young people of Hillbrow are left in a precarious position. Yet girls’ lack of personal safety in the home and the experience of witnessing domestic violence point to generations of trauma experience. Repeated cycles of poverty and violence over time create environments that entrench negative social norms and expectations across genera- tions – in spite of the aspirational legislation created after 1994 to counter this. The challenge remains one of generating structural changes that can break these In reflecting upon adolescents’ narratives about Hillbrow, we were struck by their descriptions of a community that yields diverse opportunities for young people, and even elicits expressions of neighbourhood pride. These senti- ments existed side-by-side with stories of streets marked by violence and fear. Discussion You will hardly find a child who has stayed on the streets who has never fought with any other child.” (KI12; male shelter worker) Overall, the home circumstances of the girls in this study appeared less stable and more burdensome than those of their male counterparts, with the effects of poverty, alcohol abuse, gender-based violence, domestic responsibilities and the absence of adult role models strikingly evident in their descriptions of everyday life. Indeed, the findings on orphanhood and the lack of a secure home for a sizeable portion of our sample war- rant further investigation. We may ask whether some of the young people living in unstable housing and shelters in the inner-city are there because extended families are struggling to absorb those orphaned by the HIV epidemic. From the limited data presented here, we might also posit that in this setting, boys are more Other key informants considered existing counselling and support services in Hillbrow to be “geared for adults” and therefore not youth-friendly, leading to further limitations around access and impact. On a similar theme, Rose (aged 17) believed that what young people in Hillbrow needed most to help them cope with the challenges of their environment and build resilience, were opportunities for collective problem-solving under the guidance of adult The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Page 62 of 122 Page 62 of 122 successful than girls at securing an independent income – however modest – whereas girls appear to be more dependent on others for financial support. This may be due in part to the boys’ greater freedom of mobility in Hillbrow’s public spaces. It also follows a pattern of traditional gender roles, in which men are expected to be ‘providers’ for women, and the ability to earn an in- come becomes a key marker of masculinity [39]. The stereotype emerges even in the ethos of some male gangs that appear to have infiltrated the social fabric of Hillbrow, which reward men who can show evidence of wealth, and shame those who cannot. Local gender norms appear to reward male aggression and female disempowerment, in turn fuelling male gangs and deepen- ing girls’ vulnerability to sexual violence. Competing interests Co pet g te ests The authors declare that they have no competing interests. Endnotes 1 1In this article, we use the WHO definition of inter- personal violence: “the intentional use of physical force or power, threatened or actual, against another person or against a group or community that results in or has a high likelihood of resulting in injury, death, psychological harm, maldevelopment, or deprivation” (Dahlberg LL, Krug EG. Violence: a global public health problem. In: Krug EG, Dahlberg LL, Mercy JA, Zwi AB, Lozano R, editors. World Availability of data and materials Th d l d d h Our study reaffirms the agency of adolescents in Hillbrow, even in the contexts of vulnerability currently at play in these inner-city areas, where exposure to violence is part of daily life. Young people are reaching out to find positive recreational spaces, and searching for sanctuaries from violence in their neighbourhoods to provide them with a sense of belonging and collective affirmation. Nascent signs of underlying resilience among adolescents could be leveraged and expanded through appropriate and ad- equately resourced community-level interventions, located within safe spaces close to where they live. For these inter- ventions to succeed, strong commitment from local and national stakeholders are needed, along with positive adult role models and regenerated public institutions, fully ac- countable to the communities they serve. The datasets analysed during the current study are available from the corresponding author on reasonable request, and with permission of the WAVE Study Coordinators and Publications Committee. Ethics approval and consent to participate The study received ethics approval from the Human Research Ethics Committee at the University of the Witwatersrand, Johannesburg. Written informed consent was obtained from adolescents aged 18 and 19 years. Adolescents aged 15–17 years gave written assent and their parents or legal guardians gave written consent. Those who reported personal experiences of violence were referred for counselling and care at local centres. All participants were reimbursed 50 ZAR (3.25 USD) as stipend. Funding This research was supported by Young Health Programme, a partnership between AstraZeneca, Johns Hopkins Bloomberg School of Public Health, and Plan International, a leading global children’s charity. SDM, JS, FS and DB were supported in part by a grant from the UK Department for International Development to the STRIVE Research Programme Consortium (Ref: Po 5244). However, the views expressed do not necessarily reflect the Department’s official policies. WRHI Research Capacity Building funds were used to fund the publications charges for the supplement. Authors’ contributions SDM, EV, HB and KM designed the study and secured ethical approval, while SDM and EV led data collection. FS and DB analysed and interpreted the data, with assistance from SDM and EV. FS and DB prepared the first draft of the manuscript, and all co-authors provided input and assisted in finalising the paper. All authors read and approved the final manuscript. Acknowledgements f The authors are grateful to all participants in this study, who generously shared their experiences with us. We also wish to thank Mmarashia Beleng for conducting interviews, facilitating focus groups, and transcribing and translating data. Discussion The re-framing of Hillbrow as a “cool place to be”, as a culturally-diverse neighbour- hood that was a “good place even though there is crime”, hints at adolescents’ capacity for resilience and The Author(s) BMC Public Health 2017, 17(Suppl 3):441 Page 63 of 122 Page 63 of 122 report on violence and health. Geneva (Switzerland): World Health Organization; 2002. p. 1–21). report on violence and health. Geneva (Switzerland): World Health Organization; 2002. p. 1–21). report on violence and health. Geneva (Switzerland): World Health Organization; 2002. p. 1–21). report on violence and health. Geneva (Switzerland): World Health Organization; 2002. p. 1–21). strength in an environment that would appear to offer little more than hopelessness and despair. These indications of “everyday resilience” [45] perhaps offer some challenge to conventional understandings of post-traumatic stress syndrome as an inevitable response to violence, a response that “underestimates the human capacity not only to survive, but to thrive, during and following states of emergency, extreme adversity, and everyday as well as extraordinary violence” [45]. 2A Photovoice exercise with 10 adolescents was also carried out as part of this phase of the study. We do not present any results from this exercise here. 3All participant names in this article are pseudonyms. “Boys CM” and “Girls CM” refers to Boys’ and Girls’ Community Mapping groups respectively. “KI” refers to Key Informant. 3All participant names in this article are pseudonyms. “Boys CM” and “Girls CM” refers to Boys’ and Girls’ Community Mapping groups respectively. “KI” refers to Key Informant. Study limitations CBO: Community-based organisation; CM: Community mapping; IDI: In-depth interview; IPV: Intimate partner violence; KI: Key informant; NGO: Non-governmental organisation; PTSD/S: Post-traumatic stress disorder/syndrome; WAVE: Well-being of adolescents in vulnerable environments study A number of limitations must be borne in mind when considering our findings. Firstly, the sample of adolescents interviewed for the study was small and not randomly selected, thus limiting the extent to which broader gener- alisations can be made. 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W3155420137.txt	https://ojs.cvut.cz/ojs/index.php/cej/article/download/7165/5930	en		TRAFFIC VOLUMES TEST OF AIRPORT RUNWAY	Stavební obzor	2,021	cc-by	5,748	Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------- TRAFFIC VOLUMES TEST OF AIRPORT RUNWAY Xingang Shi, Liangcai Cai and Guanhu Wang University of Air Force Engineering, Department of Airport Construction Engineering , Xi’an, China; kgdwxsxg@163 .com, 994174763@163.com, 794599756@qq.com ABSTRACT Currently, airport pavement design only considers the different horizontal standard deviation at the end and the middle of the runway, which had ignored the longitudinal distribution of airport runway traffic volume (ARTV). Especially for short take-off and landing aircraft, the take-off and landing distribution are not full of the whole runway. In allusion to the characteristics of ARTV, this paper developed a test system for the wheel track distribution and conducted a test of a short take-off and landing aircraft. Based on the statistics and analysis of test results, the horizontal and longitudinal distribution statistical laws of ARTV were obtained. At last, the longitudinal passage factor was proposed, and the planar distribution model was established to evaluate the ARTV at each point of the runway. By comparison with current design specifications, it is indicated that the pavement thickness will be designed more conservative without considering the planar distribution of traffic volumes. KEYWORDS Airport, Pavement design, Traffic volume, Planar distribution, Influence factors INTRODUCTION The design of a new runway requires information on many parameters: including airport runway traffic volume (ARTV), coverage and pass-to-coverage ratio, mixed traffic analysis if there is more than one significant aircraft and design life. Among all the parameters, ARTV was defined as number of aircraft wheel passes the pavement [1]. In the beginning, the ARTV model was simplified as the uniform distribution [2]. As early as the late 1950s, the USA Air Force had carried out tests on ARTV. Based on the test data, it was suggested that the airplane wheel tracks were uniformly distributed in the 1/3 width range of the middle runway [3]. In 1960, Vedros, professor of USA Army Engineering Laboratory, put forward the Pass-to-coverage ratio to build the uniform distribution model, which was widely recognized and concerned [4]. The field survey results of aircraft distribution in Buffalo International Airport and Atlanta International Airport [5][6] shown that the APTV model was better fitted by a normal distribution. Review of the pavement design methods in various countries, different considerations were given to the lateral distribution of ARTV. Airport Pavement Structural Design System in Australia suggested that the transverse distribution standard deviations of wheel track were 1.8m to 3.4m, 0.8m to 1.8m and 2.4m to 3.2m in runway, taxiway and connection lane, respectively [7]. In 2003 Boeing, in conjunction with FAA, conducted the B747 aircraft wheel track lateral offset tests using laser testing technology at New York's Kennedy International Airport and Anchorage International Airport [8][9]. According to the results of previous tests, the standard deviation of transverse distribution was determined as 30.435 inch (773 mm) in FAA consultation bulletin (150/5320-6E) [10]. The specification revised in 2016 (AC 150/5320-6F) also adopted the same distribution model [11] [12]. It is assumed that the lateral distribution of wheel tracks is standard Beta distribution in DOI 10.14311/CEJ.2021.01.0005 63 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------the Guideline on PCN Assignment of Netherlands [13]. The wheel tracks standard deviations of take-off and landing are recommended as 2.4 m and 2.6 m for 45 m runway width, 2.4 m and 3 m for 60 m runway width. In the pavement design guidelines for Japanese airports, the distribution of front landing gear in the cross direction of runway is classified according to the design load. The range of runway standard deviations for take-off and landing are 0.42 m to 0.91 m and 1.31 m to 1.74 m [14] [15]. In China, Multiple aircraft wheel tracks had been recorded by Lu and Wang, suggesting the normal distribution of APTV [16] - [18]. The newly revised asphalt pavement design in China (MH/T 5010-2017) [19] also adopted the same distribution model as FAA. Normal distribution of APTV was more suitable in coverage calculation than uniform distribution based on the research of Li and Lin [20][21]. Lei [22][23] conducted a comprehensive comparison of the lateral distribution test systems including infrared, video, piezoelectric and laser. Finally, laser testing technology was determined to carry out the lateral offset tests in Hongqiao Airport. The measured results showed that the wheel track distribution obeys a negative skew distribution with a mean value of 0.17m and a standard deviation of 0.99. Furthermore, Shi et al. [24][25] used the used the test statistical results for the pavement response study. It can be seen from the above reviews that in the field of pavement thickness design [26], runway width design [27] and pavement remaining life prediction [28], the lateral distribution of APRV had been widely used. The distribution of ARTV evolved from uniform to normal, which declared the method progressed from “point” to “line”. However, it is quite clear that ARTV could not bestrew the whole runway especially for military aircraft using both sides and whose taking off distance was less half of the runway. There is not only lateral distribution in the ARTV, but also longitudinal distribution due to different take-off and departure spots, landing locations and aircraft sideslips, especially for short take-off and landing aircraft. There is limited information in literatures describing the longitudinal distribution of APRV. This paper developed a test system for the wheel track distribution and conducted a test of a short take-off and landing aircraft. Then based on the statistics and analysis of test results, the horizontal and longitudinal distribution statistical laws of ARTV were obtained. The planar distribution model was established to evaluate the ARTV at each point of the runway, which provided a new method for the description of APTV. CHARACTERISTICS OF ARTV Compared with the traffic volume of highway, the ARTV has the following characteristics: First, ignoring the influence of short-distance acceleration, it is approximated that speed and load of the vehicle are constant when driving in the lane. That is, the motion states of vehicles in different cross-sections are unchanged. However, the lift of an aircraft is subject to changes in speed, resulting in loads variation acting on different cross-sections. Second, the road traffic volume is calculated by the direction coefficient, the lane coefficient and the vehicle type distribution coefficient. The distribution coefficient is constant, that is, the vehicle wheel track is assumed to be evenly distributed in the lane. The ARTV is calculated by multiplying the traffic coverage rate and the number of traffic passes. The traffic coverage rate reflects that the statistical law of the wheel track in the cross section, which obeys the normal distribution. Besides, same state of motion and same distribution of traffic volume is assumed when the vehicle is driving in the lane, while the lateral standard deviation of ARTV has changed because of the aircraft taking off offset the runway centerline, and landing alignment the runway centerline. Last but not least, the road traffic volume is constant in the longitudinal direction, while the ARTV in longitudinal direction is different due to diversification in aircraft take-off starting position, take-off running distance, landing grounding point position, and landing slip. However, the traffic volume distribution is similar to the highway traffic volume distribution in the taxiway, connecting lane, towing lane and tarmac, etc. DOI 10.14311/CEJ.2021.01.0005 64 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------From above analysis, there are different traffic volumes in every point of the runway. In fact, the characteristics of ARTV consist of lateral and longitudinal distributions. The current specification only considered the lateral variance. Longitudinal distribution had been simplified as the different offset standard deviations at the edge of the runway and the middle of the runway. It is necessary to consider the longitudinal traffic distribution in pavement design. ARTV TEST Test methods and principles Distance measuring techniques including infrared, video, piezoelectric and laser had been used to capture the wheel-track of the aircraft [22][23]. Infrared ranging method is that the transmitter sent infrared beam and when the wheels pass through cutting the beam, receivers cannot receive the infrared signal to trigger the event and record the wheel track position. It is clear that the infrared ranging method has the advantages of high propagation speed and low cost. However, the receiving and transmitting devices of the system are arranged at a long distance, and the system has poor anti-interference ability. Besides it is difficult to measure the offset of the complex axial type, and the test accuracy is greatly affected by the environment. The video test technology arranges high-speed cameras at the end of the runway to capture the position of the landing point. And the distribution of the wheel trace was obtained by analyzing the video images. Thus, the system is easy to install, simple to maintain, and intuitive to process the test results, while there are also high requirements for the camera, high cost, and heavy data processing task. What more, it is hard to extract of the landing point accurately. Pressure sensors or distributed optical fiber sensors were deployed under the pavement to measure the pavement response by Piezoelectric testing technology. The use of piezoelectric testing technology to test aircraft wheel tracks has low cost and high precision. But there are also defects such as large pre-distribution of the sensor and difficulty in repairing once damaged. Laser testing technology use laser transmitter to transmit laser beam. When the wheel passes by, the laser reflects, and the receiving device receives the echo signal. The distance between the wheel and the rangefinder is calculated by the product of wave velocity and time difference. This method has stable performance, strong antiinterference, simple adjustment, high precision and moderate cost. The paper intends to use laser testing technology to measure the APTV. Laser test system As shown in Figure 1, the laser test system consists of five parts: laser ranging sensor, data storage unit, visible laser calibration device, instrument stents and power supply unit. The system adopts FSA-ITS02 laser sensor, produced by Shenzhen Lanshi Laser Radar Co., Ltd. The test accuracy of the system can reach 5cm and the measuring frequency is 2 kHz to 4 kHz. It can trigger the measurement of high-speed moving objects within 120m range. In order to meet the requirements of high-frequency acquisition and recording, industrial-grade serial data recorder produced by Shenzhen Jingmei Technology Co., Ltd was employed. The data transmission rate can reach 921600bps. After power-on, the trigger data will be stored in an SD card. The calibration system is a DANGER laser pointer which emits visible laser light before measurement to ensure perpendicularity between the test system and the runway. The power supply unit is a 12V/60A lithium battery, which can work continuously for 48 hours. The instrument bracket is made of aluminium alloy, which can provide sufficient support strength and reduce the weight of the equipment to facilitate equipment transportation and installation. The height of the sensor can be 75-100cm. The pitch angle of the device can be adjusted from -45o-45o. DOI 10.14311/CEJ.2021.01.0005 65 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------- Fig. 1 - Field installation of test equipment Test scheme Test Profile and device arrangement The test site was selected at an airport in Tianjin, whose runway length is 2800m×60m. The wheel track test of aircraft A was carried out according to the flight plan. A total of 436 flights were recorded. During the test period, more southerly winds were observed at the test site, so the north end of the runway was the main take-off and landing direction. The equipment was arranged at the north end of the runway according to the following principles: The laser ranging device was arranged in the flat area on one side of the runway. The distance from the instrument to the center line of runway meet the flight safety requirements, which is generally not less than 60m. In order to avoid the bird-riding vehicle passage and the other inconvenient installation area, the distance from the equipment to the runway center line ranged from 63.2m to 93.3m. Layout distances of all test equipment are shown in Table 1. The longitudinal layout distance was arranged according to the principle that half of the runway should be covered, the landing site at the end of the runway and the take-off site in the middle of the runway should be densely arranged, and the taxiing region should be appropriately sparse. the test area of 1285m was divided into three parts: (1) Landing region: the initial position was 191m away from the end of the runway, and 6 instruments were installed in total, covering the landing area. (2) Middle taxiing region: a total of 5 sets of equipment were installed in the high-speed taxiing area; (3) Take-off region: a total of 6 instruments were installed to cover the take-off position of the test aircraft. Tab. 1 - Layout distances of all test equipment Device number 1 2 3 4 5 6 7 8 9 Distance from the centerline of the runway (m) 72.2 73.5 78.2 78.6 82.7 82.9 83.1 87.8 63.2 Distance from the end of the runway (m) 191 239 277 368 430 483 562 652 740 Device number 10 11 12 13 14 15 16 17 Distance from the centerline of the runway (m) 87.8 88.8 88.5 89.01 90.9 91.3 92.2 93.3 Distance from the end of the runway (m) 814 881 972 1036 1101 1165 1222 1285 DOI 10.14311/CEJ.2021.01.0005 66 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------Installation Steps (1) Hammer the bracket and wedge it into the soil foundation in the flat area. Install the fixed laser ranging sensor, ensure the sensor slope is 0 (Figure 2 (a)). (2) The tester holds the self-made equipment debugging board (Figure 2(b)), stands near the centerline of the runway, and debugs the vertical line of the runway. The other tester turns on the calibration laser pointer and adjusts the height of the device so that the visible laser projection point is 25 ± 5 mm above the center of the runway (Figure 2(c)). (3) Adjust the angle of the sensor from left to right to make laser projection move around the debugging board. When the sensor reading is at the minimum, the device is perpendicular to the middle line of the runway (Figure 2(d)). (4) Install the memory card after power off and then restart to conduct the test (Figure 2(d)). （a）（b）（d）（c）（e） Fig. 2 - Installation step LATERAL STATISTICAL REGULATION OF APTV The Cartesian coordinate system was setup at the mid-point of the runway edge. x-direction is along the lateral edge of the runway, and y- direction is along the center line of the pavement. Figure 3 illustrates the coordinate system. Fig. 3 - Coordinate system of the planar distribution model The runway was divided into groups along x direction with the interval of 2 m length. And the coverage of each group was counted divided into the states including landing, single-aircraft take-off and double-aircrafts take-off (two fighter jets taking off at the same time, side by side). DOI 10.14311/CEJ.2021.01.0005 67 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------Figures below show the horizontal distribution of APTV during landing recorded by equipment No.7 and No.14. Fig. 5 - Lateral distribution histograms and fitting curves of landing wheel track by equipment No.7 and equipment No.14, respectively As can be seen from the Figure 5, 333 aircraft passes through equipment No. 7. The test results have an average value of -2.290m and a standard deviation of 3.253m. Equipment no. 14 tested 357 sets of data, with an average value of -4.506m and a standard deviation of 3.034m. The frequency distribution histograms of the two groups were found to have good normality fitting. What is more the k-s test showed that the wheel offset of all the two sections follow the normal distribution. Track volume for landing The landing track volumes of the 17 groups were analyzed by the same statistical and hypothesis-test method. The equation and curve shown in Figure 5 were obtained by fitting the variation of the mean and standard deviation in y direction. Fig. 5 - Mean value and standard deviation in y direction for landing track volume The landing wheel coverage recorded by equipment No. 1 was only 49 times. There are insufficient data to acquire the statistics regulation of the cross section. From Figure 5, the average values of equipment from No. 2 to No. 7, -2.0385 was used to illustrate the beginning average. Average value from No. 8 to No. 17 decreased as the aircraft slowed down and deviated from the DOI 10.14311/CEJ.2021.01.0005 68 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------runway centerline. It can also be seen from Figure 5 that the standard deviation decreased constantly after landing, as the aircraft aligned with the runway centerline. Besides, when the aircraft pass through equipment No. 12, the standard deviation maintained a constant. The variation mean value  yl and standard deviation  yl were represented by the following piecewise functions. 2.0385 ， 0  y  634.5  1.5972  0.00573 y ， 634.5  y  2500 (1) 3.5673  5.6750  10 4  y ， 0  y  938.3 ， 938.3  y  2500 3.0348  (2)  yl    yl   The above track value of landing refers to the outside edge of the main landing gear wheel. The average track value at the center of a wheel needs to add half of the width of the wheel, while the standard deviation remains unchanged. f Xl ( x, y )  1  yl 2  e ( x   yl w /2)2 2 yl 2 (3) Where, W is the width of the wheel. Track volume of single-aircraft take-off The same Analysis was used to process the horizontal deviation statistical data of single take-off wheel track. The fitting equation and curve shown in Figure 6 were obtained by statistical analysis of mean value and standard deviation of single take-off wheel track. Fig.6 - Mean value and standard deviation in y direction for single-aircraft take-off track volume The single take-off wheel coverage recorded by equipment No. 13 to No.14 was not more than 50 times. There are not enough data to acquire the statistics regulation of these cross sections. As shown in Figure 6, the average values of equipment No. 2 to No. 7 float around -1.9 m. Mean value of the average, -1.953 was used to illustrate the average. Besides, with the increase of the flight distance, the standard deviation of the take-off wheel-track increases gradually. Following formula (6) exhibited good linear fit between standard deviation and ycoordinate. μyq  1.953 DOI 10.14311/CEJ.2021.01.0005 (4) 69 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 ---------------------------------------------------------------------------------------------------  yq  2.00825+8.8728  104  y (5) The wheel-track distribution of the middle point of the main landing gear on one side near the flat area can be expressed as f Xq ( x, y )  1  yq 2  e ( x   yq  0.106 )2 2 yq 2 (6) Track volume for dual-aircrafts take-off Same methods were used to analysis the take-off track volumes of two aircrafts. Figure 7 displays the fitting equation and fitting curve. (a) Fitting equation and curve of mean values for left and right take-off tracks (b) (b) Fitting equation and curve of standard deviation for left and right take-off tracks Fig. 7 - Fitting equation and curve of standard deviation for take-off track volume of two aircrafts The take-off wheel coverage of two aircrafts recorded by equipment No. 14 to No.17 was not more than 50 times. There are not enough data to acquire the statistics regulation of these cross sections. As shown in Figure 7, Mean value of the average, -11.603m and 9.528m were used to illustrate the traffic volume of close to the flat area and away from the flat area. What is more, similar to the single aircraft, the standard deviation increases gradually with the increase of the flight distance. It is interesting that the deviation which close to flat area had good linear fitting with high R-square of 0.986, while the deviation which away from the flat area had poor linear DOI 10.14311/CEJ.2021.01.0005 70 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------fitting with R-square of 0.8478. The interference between aircraft skidding may led to the fluctuations.  yq1  11.603 (7)  yq 2  9.528 (8)  yq1  2.2720+7.3566 104  y (9)  yq 2  2.3256+4.9466  104  y (10) The wheel-track distribution of the middle point of the main landing gear for two aircrafts can be expressed as: f Xq1( x, y )  f Xq 2 ( x, y )  1  yq1 2  e  1  yq 2 2 e ( x   yq 1  0.106 )2 2 yq 12 (11) ( x   yq 2  0.106 )2 2 yq 22 (12) LONGITUDINAL STATISTICAL REGULATION OF ARTV The design of a runway length is determined by the accelerate-stop distance associated with an aborted take-off under the most adverse environmental conditions. For the majority of the airports, the runway length is surplus, which will result in that ARTV cannot cover the entire pavement. Longitudinal passage factor is the ratio of flight sorties passing through a certain crosssection to the total flight sorties and can be calculated by the following formula: fY ( y )  Where N y Ny (13) N is the flight sorties passing through a certain cross-section, N is the total flight sorties. Longitudinal traffic volume of landing The tire marks in the middle of the runway reflect that the landing point of the aircraft is basically within the test range of the first 7 equipment. Remove the days of occasional damage, the longitudinal passage factor of landing in each cross-section is calculated as shown in Table 5. It can be seen from the Figure 8 that all aircrafts have landed and skidded after the 7th cross-section. That is to say, the longitudinal passage factor identically equal to 1 before the aircraft leaves the runway at the linking taxiway. Polynomial fitting of longitudinal passage factor along y direction can be obtained as shown in Figure 8. DOI 10.14311/CEJ.2021.01.0005 71 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------- Fig. 8 - Fitting equation and curve of landing longitudinal passage factor The Piecewise function (14) is adopted to represent the longitudinal passage factor of the whole runway 0 , y  180.95   2 3 fYl ( y )  1.5586  0.0109 y  1.3308E  5 y  3.7081E  9 y , 180 .95  y  459 .65  1 , y  459.65  (14) Longitudinal traffic volume of single-aircraft take-off When a single aircraft takes off, the beginning positions are basically at the runway take-off line, and the longitudinal distribution of the take-off point can be ignored. Through observation and statistics, it is found that there are little aircraft take off before the 10th cross-section. In other words, the departure point of the aircraft is basically located at the equipment No. 10 to No. 17. Figure 9 shows good linear fitting of the longitudinal passage factors along the runway, which can be express by the following piecewise function (15). Fig. 9 - Fitting equation and curve of longitudinal passage factor for single aircraft take-off DOI 10.14311/CEJ.2021.01.0005 72 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------1   fYq单 ( y )   2.5995  0.00205 y  0  , 150  y  780.24 , 780.24  y  1268.05 , (15) y  1268.05 Longitudinal traffic volume of dual-aircrafts take-off It has been known that the take-off point is also basically at the runway take-off line, similar to single-aircraft take-off. The longitudinal distribution of the beginning point can be ignored. Through observation and statistics, it was found that the dual-aircrafts left the ground successively after the cross-section of equipment No. 10. Figure 10 shows the polynomial fitting of dual-aircraft longitudinal passage factor along y direction. Fig. 10 - Fitting equation and curve of longitudinal passage factor for dual-aircraft take-off Adopt the following piecewise function (15) to represent the longitudinal passage factor of the whole runway: 1 , 150  y  840.56   2 fYq双 ( y )  1.126  0.00106 y  1.4394E  6 y , 840.56  y  1326 .25  0 , y  1326.25  (16) PLANE DISTRIBUTION MODEL OF ARTV Previous sections have studied the lateral and longitudinal traffic volume of landing, singleaircraft take-off and dual-aircraft take-off. Normal fitting curves of ARTV were obtained, which reflect the probability of wheel-track migration on each cross-section. Longitudinal passage factor is defined as the probabilistic representation of the total flight sorties passing through a crosssection. The joint distribution of lateral and longitudinal directions represented the traffic volume probability at a certain point of the pavement surface. The total number of flights operated by the airport in its design life is assumed to be N times, including Ns times of single-aircraft take-off and Nd times of dual-aircraft take-off, where N  Ns  2Nd . Take-off and landing ratio of flight sorties at one side to the other is a : b ( a  b  1 ). The ARTV of landing, single-aircraft take-off and dual-aircraft take-off can be represented by Eq. (17), Eq. (18), Eq. (19) and Eq. (20) respectively. DOI 10.14311/CEJ.2021.01.0005 73 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------N x, yl  aN  f Xl ( x, y )  fYl ( y )  bNf Xl ( x, L  y ) fYl ( L  y ) (17) N x, yqs  aN s  f Xqs ( x, y )  fYqs ( y )  bNf Xqs ( x, L  y ) fYqs ( L  y ) (18) N x, yqd1  aN d  f Xq1( x, y )  fYqd ( y )  bN d f Xq1( x, L  y ) fYqd ( L  y ) (19) N x, yqd2  aN d  f Xq 2 ( x, y )  fYqd ( y )  bN d f Xq 2 ( x, L  y ) fYqd ( L  y ) (20) The traffic volumes of main landing gear left wheel were calculated by the upper planar distribution model. It is necessary to add right wheel traffic volume to the formulas to get the whole coverage. Neglect the effect of aircraft drift and wheel roll, the normal distribution mean at the center of right wheel needs to add the width of the two main landing gears, while the standard deviation remains unchanged. It is known that the take-off and landing sorties of this airport support aircraft in the design life are 100,000 times. The probability of taking off and landing at one side to the other is 7:3 and the take-off ratio of single-aircraft to dual-aircraft is 1:3. The airport had a runway length of 2800m, and width of 60m. Assumed that all aircrafts leave the runway from the contact channel (the longitudinal coordinates at both ends are 300m and 2500m respectively). Based on the wheel track test statistical regulation and the planar distribution model, the coverage of the main landing gear wheels under three states of landing, single-aircraft take-off and dual-aircraft take-off can be obtained, as shown in Figure11, Figure12 and Figure 13 respectively. Fig. 11 - Traffic distribution surface of landing Fig. 12 - Traffic distribution surface of single-aircraft take-off DOI 10.14311/CEJ.2021.01.0005 74 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------- Fig. 13 Traffic distribution surface of dual-aircraft take-off As seen in Figure 11 to Figure13, it is shown that the wheel tracks distributed throughout the runway. Coverage times at every point of the pavement can be clearly calculated using the above model, with maximum value of 23986 times for landing, 5423 times for single-aircraft takeoff, 7575 times and 7535 times for dual-aircraft take-off near the flat area and for away from the flat area. If the lateral distribution of the wheel track is calculated according to the specification, with an average value of 0.02m and a standard deviation of 2.83m, neglecting the longitudinal distribution, the ARTV is shown in following Figure 14. Fig. 14 - Traffic volume curve of lateral distribution As can be seen from the figure above, the traffic volume curve only considering the transverse distribution is one case of the planar distribution model, which has not considered the aircraft's take-off and landing point, taxiing offset and longitudinal passage probability of each cross-section. It can be calculated that the maximum coverage times of the traffic volume distribution curve are 25,370 times. Compared with the above planar model, it is found that without considering the longitudinal distribution of APTV will make an excessive estimate of the traffic volume and more conservative for the pavement thickness design. What is more, the planar model can also be used to determine the width of airport runway based on runway risk probability. These are also our future research directions. CONCLUSIONS This paper pointed out that there are not only lateral distributions, but also longitudinal distributions of ARTV based on the analysis of its characteristics. An airport runway traffic DOI 10.14311/CEJ.2021.01.0005 75 Article no. 5 THE CIVIL ENGINEERING JOURNAL 1-2021 --------------------------------------------------------------------------------------------------measurement system was developed, which adopted laser testing technology to test the wheel track distribution in landing, single-aircraft take-off and dual-aircraft take-off. The regulation of lateral distribution of wheel tracks was analyzed statistically. The longitudinal passage factor was proposed to illustrate the longitudinal distribution of ARTV. Then, the planar distribution model of ARTV was established. The example showed that the planar distribution model could evaluate the coverage times of every point on the pavement. In addition, by comparing with traffic volume calculation method in current design specification, it was found that only considering the lateral distribution of traffic volume will make the pavement design more conservative. The model proposed in this paper developed the traffic volume calculation method from transverse distribution curve to planar distribution surface, without considering the load of different aircraft weight. Combined with the planar distribution of load, it can provide the traffic volume basis for pavement segmentation design and establish the pavement design method based on the cumulative damage surface. ACKNOWLEDGMENTS The authors give special thanks to Tianjing Yangcun Airport for their supply of the test site. REFERENCES [1] Richard G. Ahlvin Origin of Developments for Structural Design of Pavements Report No. GL-91-26 U.S.Army Corps of Engineers, Waterways Experiment Station, Vickburg, MS.1991. [2] Zhang Yaohua. 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https://openalex.org/W3038464427	https://europepmc.org/articles/pmc7370094?pdf=render	English	null	Self-Control Moderates the Association Between Perceived Severity of Coronavirus Disease 2019 (COVID-19) and Mental Health Problems Among the Chinese Public	International journal of environmental research and public health/International journal of environmental research and public health	2,020	cc-by	7,184	Received: 22 May 2020; Accepted: 2 July 2020; Published: 4 July 2020 Abstract: Coronavirus disease 2019 (COVID-19) has caused thousands of deaths in China. Prior research suggests that individuals’ perceived severity of COVID-19 is related to a range of negative emotional and behavioral reactions among the Chinese public. However, scant research has examined the underlying mechanisms. Drawing upon the risk-resilience model, this study proposes that self-control, as a resilient factor, would potentially moderate the association between perceived severity of COVID-19 and mental health problems. Data from a national survey was used to examine this idea. Participants were 4607 citizens from 31 regions in China (Mage = 23.71 years, 72.5% female) who completed a national survey at the beginning of February 2020. Results of hierarchical regression showed that after controlling for a number of demographic variables, perceived severity of COVID-19 and self-control were positively and negatively related to mental health problems, respectively. More importantly, self-control moderated the “perceived severity of COVID-19–mental health problems” association, with this link attenuating as the levels of self-control increased. These ﬁndings suggest that compared to those with high self-control, individuals with low self-control are more vulnerable and are more in need of psychological aids to maintain mental health in the encounter of the COVID-19 outbreak. Practically, enhancing individuals’ self-control ability might be a promising way to improve individuals’ mental health during the early period of the COVID-19 outbreak. ywords: risk factor; resilience; cognitive appraisal; self-control; COVID-19; public health concerns Self-Control Moderates the Association Between Perceived Severity of Coronavirus Disease 2019 (COVID-19) and Mental Health Problems Among the Chinese Public Jian-Bin Li 1 , An Yang 2, Kai Dou 3,* and Rebecca Y. M. Cheung 1 Jian-Bin Li 1 , An Yang 2, Kai Dou 3,* and Rebecca Y. M. Cheung 1 Jian-Bin Li 1 , An Yang 2, Kai Dou 3,* and Rebecca Y. M. Cheung 1 1 Department of Early Childhood Education, Center for Child and Family Science, The Education University of Hong Kong, Hong Kong, China; lijianbin@eduhk.hk (J.-B.L.); rymcheung@eduhk.hk (R.Y.M.C.) 2 Department of Applied Psychology, Guangdong University of Foreign Studies, Guangzhou 510006, China; yangan@gdufs.edu.cn 3 1 Department of Early Childhood Education, Center for Child and Family Science, The Education University of Hong Kong, Hong Kong, China; lijianbin@eduhk.hk (J.-B.L.); rymcheung@eduhk.hk (R.Y.M.C.) 2 Department of Applied Psychology, Guangdong University of Foreign Studies, Guangzhou 510006, China; yangan@gdufs.edu.cn 1 Department of Early Childhood Education, Center for Child and Family Science, The Education University of Hong Kong, Hong Kong, China; lijianbin@eduhk.hk (J.-B.L.); rymcheung@eduhk.hk (R.Y.M.C.) 2 Department of Applied Psychology, Guangdong University of Foreign Studies, Guangzhou 510006, China; yangan@gdufs.edu.cn 3 Department of Psychology and Research Center of Adolescent Psychology and Behavior, School of Education, Guangzhou University, Guangzhou 510006, China 3 Department of Psychology and Research Center of Adolescent Psychology and Behavior, School of Education, Guangzhou University, Guangzhou 510006, China International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health 1. Introduction A recent study reveals that individuals’ perceived severity of the COVID-19 outbreak is related to more undesirable emotional and behavioral outcomes in the Chinese public [1], suggesting that perceived severity of COVID-19 may impose a negative main eﬀect on mental health outcomes. This is in line with past studies which found that individuals’ negative appraisals about the incident (e.g., perceived risk, perceived threat, etc.) were related to more mental health problems during the outbreak of severe acute respiratory syndrome (SARS) and Ebola [4–7]. These studies mainly adopt the main eﬀect tenet to examine the risk and protective factors of outcomes during the encounter of emergent public health concerns. Beyond this, the risk-resilience tenet suggests the necessity and importance to take into account how assets may buﬀer the negative main eﬀect of risk precursors on health outcomes. This motivated us to examine the potential moderation eﬀect of self-control. As an asset, self-control ability may serve to regulate the undesirable mental health consequences brought by COVID-19. Previous research suggested that individuals with high self-control have better inhibition and initiatory ability [8], use more positive coping strategies and fewer negative coping strategies [9], and persist more in important life domains [10,11]. In addition, self-control supports the retrieval of restrain standards and deliberative evaluations [12]. Therefore, good self-control is robustly related to a wide range of life outcomes, including better mental health [13]. Besides the main eﬀect of self-control, several aspects of good self-control are particularly relevant to the moderation in the association between risk factors and health outcomes. For instance, planning and forethought could help individuals anticipate and prepare for diﬃcult situations; emotional self-regulation could provide better emotional control in problem situations; the ability to restrain and initiate action is important to deal with the diﬃculties in problem situations [14]. Prior studies have found that good self-control buﬀers the inﬂuence of negative environment (e.g., peer deviance, negative life events) on undesirable outcomes such as substance use [14,15]. 1. Introduction The outbreak of coronavirus disease 2019 (COVID-19) has not only led to thousands of deaths in China, but it also caused tremendous psychological stress to the Chinese public. In order to medically and mentally combat COVID-19, professional input from various disciplines is needed, including the perspectives from psychological science. A recent study conducted among 4607 Chinese reveals that individuals’ cognitive appraisals, perceived severity of COVID-19 in particular, are related to a number of undesirable emotional (e.g., increase in negative emotion) and behavioral (e.g., increase in sleep problems) reactions [1]. However, little is known about what factors can buﬀer the negative inﬂuence of perceived severity on individuals’ mental health. Studying this issue is important and necessary, as the ﬁndings will shed light on at speciﬁc factors for intervention programs to target. In this study, www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020, 17, 4820; doi:10.3390/ijerph17134820 www.mdpi.com/journal/ijerph 2 of 10 Int. J. Environ. Res. Public Health 2020, 17, 4820 drawing upon the risk-resilience model [2], we propose that self-control—one’s ability to override or change his/her inner responses and to interrupt undesired behavioral tendencies [3]—could be a candidate that buﬀers the negative inﬂuence of perceived severity of COVID-19 and mental health problems among the Chinese public. The aim of this study is to examine this idea. The risk-resilience model proposes that risk and adversity increase the propensity of undesirable outcomes; individuals who have suﬃcient assets to oﬀset the negative inﬂuence of the risk could overturn undesirable outcomes, thus showing resilience [2]. In this model, risk can be deﬁned in diverse ways, including both intrinsic and extrinsic factors such as adverse living condition, the negative life events that have occurred in recent months, massive community trauma, and cumulative risk calculations that combine diﬀerent kinds of risk factors. The main eﬀect tenet suggests that risk factors are associated with undesirable outcomes such as maladjustment. Nevertheless, the compensatory eﬀect (or moderation eﬀect) tenet assumes that suﬃcient positive internal and/or external assets could mitigate the burden of an individuals’ life due to risk inﬂuences, and thus these individuals would be less aﬀected by the risk and have better outcomes than those who have the same levels of risk but do not possess enough assets. For instance, children reared in risky environments commit more aggressive acts, but assets like positive parenting (e.g., authoritative parenting) buﬀer the negative inﬂuence of risky environment on children’s antisocial behavior [2]. 2.1. Participants and Procedure We used snowball sampling to recruit participants online to take part in a national survey that investigated Chinese citizens’ emotional and behavioral reactions as well as risk and protective factors during the outbreak of COVID-19 between 2 and 9 February 2020. A total of 4826 Chinese individuals visited our online survey website during the survey period. A total of 219 participants were excluded because they met one of the following criteria: (1) they did not show interest to participate in the study after reading the information sheet; (2) they were not old enough to provide consent form (i.e.,≤16 years); or (3) they showed an obvious responding pattern across multiple consecutive items (e.g., select “3” for all items). Thus, 4607 participants comprised the ﬁnal sample, 1265 men (27.5%) and 3342 women (72.5%) aged from 17 to 90 years (Mage = 23.71 years, SD = 7.29). These participants came from 31 regions in China, with the sample size ranging from 16 (0.3% of the total sample, Ningxia Hui Autonomous Region) to 1386 (30.1% of the total sample, Guangdong Province). Most participants (73.2%) held a bachelor’s degree or above. Regarding physical and mental health condition, 77.3% of participants reported that their current physical health was good or very good. In addition, 94.6% and 99.2% of participants reported that they did not have any history of chronic physical diseases or history of psychiatric/psychological disorders, respectively. Moreover, 97.7% of participants reported they had not been diagnosed with COVID-19 or involved in the pandemic; 2.3% of participants reported they were suspected/diagnosed with COVID-19 or had relatives/friends who were suspected/diagnosed cases. The ethical committee of the Guangzhou University reviewed and approved this study before data collection (Ethical approval number: GZHU2020001). This study was part of the large survey study which aimed to examine Chinese public’s emotional and behavioral outcomes and its risk and protective factors towards the COVID-19 outbreak [1]. This paper and the large survey study share only one main variable (i.e., perceived severity) and the demographic variables. No other variables overlapped. Thus, we deem that the two papers are substantially distinctive. Over 200 volunteers who majored in psychology in various universities in China helped distribute the survey link on a number of internet platforms, including WeChat, Weibo, QQ, Facebook, forums, etc., after receiving a 3-hour training to get familiar with the research procedure and the contents of the questionnaires. 1. Introduction Applying the moderation role of self-control to the case of COVID-19, individuals with good self-control, compared to their low self-control counterparts, are more likely to plan ahead (e.g., preparing for facing the pandemic actively), regulate the negative emotions (e.g., anxiety, fear) induced by the high morbidity and uncertainty at the time when we conducted this study, and strictly adhere to government’s guidelines of prevention and protection (e.g., wearing facial masks, staying at home as much as possible, and maintaining social distancing). These actions may help mitigate the negative inﬂuences of perceived risk on individuals’ mental health. According to the literature reviewed above and the logic of the risk-resilience model, we hypothesized that perceived severity of COVID-19 and self-control would be related to mental health problems in positive and negative directions, respectively. Moreover, we assumed that the negative association between perceived severity of COVID-19 and mental health problems would be less 3 of 10 Int. J. Environ. Res. Public Health 2020, 17, 4820 pronounced among those with high self-control compared to their low-self-control counterparts. To examine these hypotheses, we would use data from a recent national survey [1] which examines the Chinese public’s emotional and behavioral outcomes and its antecedents in the encounter of the COVID-19 outbreak. Given that these data were collected from diﬀerent regions in mainland China, the individual data were nested in regions and therefore we would employ a hierarchical regression analysis to control for the potential inﬂuence of the cluster, with a number of demographic variables included in the model as covariates to rule out their inﬂuence on the outcome. 2.1. Participants and Procedure For instance, a volunteer might post the survey link on their WeChat Moment (a function in WeChat which is similar to Facebook and Twitter) so that friends and relatives of that volunteer could participate and further shared the link to their social networks. Participants provided their electronic consent form prior to participation. Voluntary participation was emphasized, and no incentive reward was given to participants. We also stressed anonymity and did not collect any identiﬁable personal particulars to protect participants’ privacy. 2.2. Measures Mental health problems. The Chinese version of the 12-item General Health Questionnaire (GHQ-12) [16] was used to measure participants’ overall mental health problems over the past 10 days. The scale contains 12 items either in negative or positive wordings. Participants were asked to indicate their situation on these items over the past 10 days compared to their general situation. All items were 4 of 10 Int. J. Environ. Res. Public Health 2020, 17, 4820 rated on a four-point scale. According to the scoring system used in Zhang and Wang [16], the ﬁrst two options of each question indicate that participants feel better than, or as much as, usual. Thus, these two options were coded 0 (i.e., no signiﬁcant changes in mental health condition). The third and the fourth options indicate participants feel/experience a little and much poorer than usual, respectively, and thus they were coded 1 (i.e., mental health condition slightly worse than usual), and 2 (i.e., mental health condition much worse than usual), respectively. Averaging the items yields a mean score and a higher total score reﬂects more mental health problems. Sample items are “feeling unhappy and depressed” (negative wording) and “been able to face up to your problems” (positive wording). This measure has been used in prior research showing good internal consistency reliability [16]. Perceived severity. Participants’ perceived severity about COVID-19 was measured with ﬁve items developed by our team. Participants indicated their evaluation of how severe they think COVID-19 is in various aspects, including the infection rate, morbidity, mortality, its negative inﬂuence on social order, and its negative inﬂuence on the economics, on a ﬁve-point scale (from 1 = not severe at all to 5 = very much severe). A higher mean score indicates participants perceived COVID-19 to be more severe. Sample items are “how severe you think of the infectious rate of COVID-19 is?” and “how severe you think of the morbidity of COVID-19 is?” This measure has been used in prior research showing good internal consistency reliability [1]. Self-control. The Chinese version of the Brief Self-Control Scale (BSCS) was used to measure participants’ self-control ability [17]. The scale consists of 13 items rated on a ﬁve-point scale (from 1 = not like me at all to 5 = like me very much). A higher mean score indicates better self-control ability. 2.2. Measures Sample items are “I am good at resisting temptation” and “I have a hard time breaking bad habits”. This measure has been used in prior research showing good internal consistency reliability [17]. 3. Results 3.1. Mean Levels of and the Bivariate Correlations Between Mental Health Problems, Perceived Severity, and Self-Control 3.1. Mean Levels of and the Bivariate Correlations Between Mental Health Problems, Perceived Severity, and Self-Control As shown in Table 1, participants reported low levels of mental health problems (0.19 out of 2) over the past 10 days, relatively high levels of perceived severity of COVID-19 (4.09 out of 5), and medium levels of self-control (3.03 out of 5). Moreover, the results of correlation analyses found that perceived severity of COVID-19 (r = 0.19, p < 0.001) and self-control (r = −0.21, p < 0.001) was positively and negatively related to mental health problems, respectively. The eﬀect sizes for these correlation coeﬃcients were small-to-medium, according to Cohen’s (1992) standard [19]. Table 1. Descriptive statistics and bivariate correlations of mental health problems, perceived severity, self-control, and demographic variables. Variables 1 2 3 4 5 6 7 8 9 10 1. Sex 2. Age −0.08 * 3. Phy. history 0.06 * −0.10 * 4. Psy. history −0.01 0.01 0.08 * 5. Health con. −0.04 −0.03 0.18 * 0.08 * 6. Education −0.00 −0.13 −0.03 * 0.03 * 0.02 7. Rel. w. COVID-19 0.01 0.09 ** −0.02 −0.03 * −0.03 * −0.01 8. Mental health problems 0.07 *** 0.04 * −0.08 * −0.09 * −0.19 *** 0.02 0.03 * 9. Perceived severity 0.11 * 0.09 * 0.00 −0.00 −0.04 ** −0.03 * −0.00 0.19 * 10. Self-control −0.01 0.21 * 0.05 0.06 * 0.21 * −0.06 * 0.03 −0.21 * −0.10 * Cronbach’s α - - - - - - - 0.86 0.84 0.84 Min. 1.00 17 1.00 1.00 1.00 1.00 1.00 0.00 1.00 1.31 Max. 2.00 90 2.00 2.00 5.00 6.00 2.00 2.00 5.00 5.00 M - 23.71 - - - - - 0.19 4.09 3.03 SD - 7.29 - - - - - 0.27 0.59 0.50 Note. Sex: 1 = male, 2 = female; Phy. history = history of chronic physical diseases: 1 = yes, 2 = no; Psy. histroy = history of psychiatric/psychological disorder: 1 = yes, 2 = no; Health con. = current physical health condition, from 1 = very poor to 5 = very good; Education: 1 = junior middle school and below, 2 = high school degree, 3 = college degree, 4 = bachelor’s degree, 5 = master’s degree, and 6 = doctoral degree. Rel. w. 2.3. Data Analysis We analyzed the data in SPSS 18.0 (IBM, Armonk, NY, USA) and Mplus 7.0 (Muthén & Muthén, Los Angeles, CA, USA) in several steps, with 0.05 as the signiﬁcance level throughout the analyses. First, we carried out descriptive statistics to capture the levels of each variable. Second, we performed correlation analysis to examine the association between perceived severity, self-control, and mental health problems. Last, given that the individual data were nested in diﬀerent provinces/regions, we carried out hierarchical regression models to examine the moderation eﬀect of self-control in the association between perceived severity and mental health problems, controlling for a number of demographic variables (i.e., biological sex, age, the history of chronic physical problems, the history of psychiatric/psychological disorders, current physical health condition, educational levels, and relationship with COVID-19). To this end, we ﬁrst calculated the intraclass correlation and the variance at the within- and between-level. Then, we centered the covariates, independent variable and the moderator using the “groupmean” function [18]. Subsequently, we calculated the product term between the centered independent variable and the centered moderator as the interaction term. Based on this, we ﬁt a full model, with the independent variable, the moderator, the interaction term, and the covariates as level-1 variables. The intercept of the dependent variable was also estimated at level-2 to control for the variance of the dependent variable accounted for by the cluster. Finally, we conducted simple slope tests with 1 standard deviation (SD) below and above the centered mean of the moderator and examined the diﬀerences in the magnitude of the slopes (i.e., Slope 1 SD vs. Slope mean, Slope 1 SD vs. Slope −1 SD, and Slope mean vs. Slope −1 SD) to determine if the simple slopes diﬀer against each other. Since Mplus 7.0 does not accommodate bootstrapping with multi-level models, we used R to calculate the 95% bootstrapping (N = 20,000) conﬁdence interval of the simple slopes and their diﬀerences. If the conﬁdence interval does not include zero, signiﬁcant diﬀerences between slopes by the levels of the moderator would be tenable. 5 of 10 Int. J. Environ. Res. Public Health 2020, 17, 4820 3. Results 3. Results COVID-19 = relationship with COVID-19: 1 = not relevant/infected, 2 = suspected/diagnosed cases or had relatives/friends who were suspected/diagnosed cases. * p < 0.05; p < 0.01; * p < 0.001. Table 1. Descriptive statistics and bivariate correlations of mental health problems, perceived severity, self-control, and demographic variables. Table 1. Descriptive statistics and bivariate correlations of mental health problems, perceived severity, self-control, and demographic variables. 1. Descriptive statistics and bivariate correlations of mental health problems, perceived severity, ntrol and demographic variables Note. Sex: 1 = male, 2 = female; Phy. history = history of chronic physical diseases: 1 = yes, 2 = no; Psy. histroy = history of psychiatric/psychological disorder: 1 = yes, 2 = no; Health con. = current physical health condition, from 1 = very poor to 5 = very good; Education: 1 = junior middle school and below, 2 = high school degree, 3 = college degree, 4 = bachelor’s degree, 5 = master’s degree, and 6 = doctoral degree. Rel. w. COVID-19 = relationship with COVID-19: 1 = not relevant/infected, 2 = suspected/diagnosed cases or had relatives/friends who were suspected/diagnosed cases. * p < 0.05; p < 0.01; * p < 0.001. Note. Sex: 1 = male, 2 = female; Phy. history = history of chronic physical diseases: 1 = yes, 2 = no; Psy. histroy = history of psychiatric/psychological disorder: 1 = yes, 2 = no; Health con. = current physical health condition, from 1 = very poor to 5 = very good; Education: 1 = junior middle school and below, 2 = high school degree, 3 = college degree, 4 = bachelor’s degree, 5 = master’s degree, and 6 = doctoral degree. Rel. w. COVID-19 = relationship with COVID-19: 1 = not relevant/infected, 2 = suspected/diagnosed cases or had relatives/friends who were suspected/diagnosed cases. * p < 0.05; p < 0.01; * p < 0.001. 3.2. The Moderation of Self-Control in the Association Between Perceived Severity and Mental Health Problems 3.2. The Moderation of Self-Control in the Association Between Perceived Severity and Mental Health Problems The Moderation of Self-Control in the Association Between Perceived Severity and Mental Health Problems We conducted hierarchical regression models to examine the association between perceived severity of COVID-19 and mental health problems as well as the moderation of self-control in Mplus. Results of the null model with mental health as an outcome revealed that the intraclass correlation (ICC) of mental health problems was 0.04. The within-level and the between-level model explained 7.1% (p < 0.001) and 0.3% (p = 0.012) variance of mental health problems, respectively. p p p p y The results of the full model which included the independent variable, moderator, dependent variable, and demographic variables are summarized in Table 2. This model was a saturated model. The results showed that after controlling for a number of demographic variables, the main eﬀect of perceived severity on mental health problems was signiﬁcant yet small (B = 0.07, SE = 0.01, p < 0.001). The main eﬀect of self-control was also signiﬁcant yet small (B = −0.08, SE = 0.01, p < 0.001). More importantly, the interaction eﬀect between perceived severity and self-control was signiﬁcant as well, B = 0.05, SE = 0.01, p < 0.001. p Given the signiﬁcance of the interaction term, we further conducted simple slope tests and the results are displayed in Table 3 and Figure 1. The results showed that the simple slopes were signiﬁcant when the levels of self-control were low (B = 0.10, SE = 0.01, p < 0.001), medium (B = 0.07, SE = 0.01, p < 0.001), and high (B = 0.05, SE = 0.01, p < 0.001). Furthermore, we compared the diﬀerences in the magnitude of these slopes, ﬁnding that the slope of perceived severity when self-control was low was signiﬁcantly larger than the slopes when self-control was at medium and high levels, and that the slope when self-control was at medium levels was also signiﬁcantly larger than the one when self-control was at high levels. Taken together, the results suggest that perceived severity was linked with only 6 of 10 Int. J. Environ. Res. Public Health 2020, 17, 4820 modest mental health issues among the Chinese public. Moreover, the ﬁndings also suggest that as individuals’ self-control ability increased, the association between perceived severity and mental health problems decreased. 3.2. The Moderation of Self-Control in the Association Between Perceived Severity and Mental Health Problems In other words, the negative association between perceived severity of COVID-19 and citizens’ mental health problems was more pronounced among those with low self-control than their counterparts with high self-control. Table 2. Hierarchical regression model of the association between perceived severity and mental health problems and the moderation eﬀect of self-control. Predictors B SE p Sex 0.04 0.01 <0.001 Age 0.00 0.00 0.048 Phy. history −0.04 0.02 0.025 Psy. history −0.17 0.07 0.013 Health con. −0.04 0.01 <0.001 Education 0.01 0.01 0.062 Rel. w. COVID-19 0.08 0.03 0.013 Perceived severity 0.07 0.01 <0.001 Self-control −0.08 0.01 <0.001 Perceived severity × self-control −0.05 0.01 <0.001 Note. Sex: 1 = male, 2 = female; Phy. history = history of chronic physical diseases: 1 = yes, 2 = no; Psy. histroy = history of psychiatric/psychological disorder: 1 = yes, 2 = no; Health con. = current physical health condition, from 1 = very poor to 5 = very good; Education: 1 = junior middle school and below, 2 = high school degree, 3 = college degree, 4 = bachelor’s degree, 5 = master’s degree, and 6 = doctoral degree. Rel. w. COVID-19 = relationship with COVID-19: 1 = not relevant/infected, 2 = suspected/diagnosed cases or had relatives/friends who were suspected/diagnosed cases. J. Environ. Res. Public Health 2020, 17, x 7 o ble 2. Hierarchical regression model of the association between perceived severity and mental health oblems and the moderation eﬀect of self-control. Note. Sex: 1 = male, 2 = female; Phy. history = history of chronic physical diseases: 1 = yes, 2 = no; Psy. histroy = history of psychiatric/psychological disorder: 1 = yes, 2 = no; Health con. = current physical health condition, from 1 = very poor to 5 = very good; Education: 1 = junior middle school and below, 2 = high school degree, 3 = college degree, 4 = bachelor’s degree, 5 = master’s degree, and 6 = doctoral degree. Rel. w. COVID-19 = relationship with COVID-19: 1 = not relevant/infected, 2 = suspected/diagnosed cases or had relatives/friends who were suspected/diagnosed cases. Environ. Res. Public Health 2020, 17, x 7 o Note. Sex: 1 = male, 2 = female; Phy. history = history of chronic physical diseases: 1 = yes, 2 = no; Psy. histroy = history of psychiatric/psychological disorder: 1 = yes, 2 = no; Health con. 3.2. The Moderation of Self-Control in the Association Between Perceived Severity and Mental Health Problems = current physical health condition, from 1 = very poor to 5 = very good; Education: 1 = junior middle school and below, 2 = high school degree, 3 = college degree, 4 = bachelor’s degree, 5 = master’s degree, and 6 = doctoral degree. Rel. w. COVID-19 = relationship with COVID-19: 1 = not relevant/infected, 2 = suspected/diagnosed cases or had relatives/friends who were suspected/diagnosed cases. . Environ. Res. Public Health 2020, 17, x 7 o Figure 1. The association between perceived severity of coronavirus disease 2019 (COVID-19) and meatal health problems by self-control. Note. IV = perceived severity of COVID-19; Mod = moderator (i.e., self-control). 0.1 0.15 0.2 0.25 0.3 0.35 -1SD IV Mean 1SD IV Mental health problems Perceived severity of COVID-19 -1SD Mod Mean 1SD Mod Figure 1. The association between perceived severity of coronavirus disease 2019 (COVID-19) and meatal health problems by self-control. Note. IV = perceived severity of COVID-19; Mod = moderator (i.e., self-control). Mean Perceived severity of COVID-19 Perceived severity of COVID-19 Figure 1. The association between perceived severity of coronavirus disease 2019 (COVID-19) and meatal health problems by self-control. Note. IV = perceived severity of COVID-19; Mod = moderator (i.e., self-control). Figure 1. The association between perceived severity of coronavirus disease 2019 (COVID-19) and meatal health problems by self-control. Note. IV = perceived severity of COVID-19; Mod = moderator (i.e., self-control). Figure 1. The association between perceived severity of coronavirus disease 2019 (COVID-19) and meatal health problems by self-control. Note. IV = perceived severity of COVID-19; Mod = moderator (i.e., self-control). Figure 1. The association between perceived severity of coronavirus disease 2019 (COVID-19) and meatal health problems by self-control. Note. IV = perceived severity of COVID-19; Mod = moderator (i.e., self-control). Int. J. Environ. Res. Public Health 2020, 17, 4820 7 of 10 Table 3. Summary of simple slope tests by the levels of self-control and the diﬀerence in simple slopes. Simple Slope Tests and Comparison B SE p 95% Bootstrapping CI (N = 20,000) b Simple slopes Low self-control (−1SD) 0.10 0.01 <0.001 [0.080, 0.116] Medium self-control (mean) a 0.07 0.01 <0.001 [0.058, 0.086] High self-control (1SD) 0.05 0.01 <0.001 [0.028, 0.064] Comparison between simple slopes Diﬀerence between low and medium self-control 0.03 0.01 <0.001 [0.015, 0.037] Diﬀerence between low and high self-control 0.05 0.01 <0.001 [0.029, 0.075] Diﬀerence between medium and high self-control 0.04 0.01 <0.001 [0.015, 0.037] Note. 4. Discussion COVID-19 was declared a Public Health Emergency of International Concern by the World Health Organization at the end of January 2020 [20]. The current study, conducted soon after the declaration, was one of the pioneer surveys around the globe that examined the inﬂuence of COVID-19 on individuals’ mental health and well-being. COVID-19 was still largely conﬁned within mainland China when this study was conducted, but it has been spreading worldwide rapidly. At the end of May 2020, there have been already over ﬁve million conﬁrmed cases and it has caused more than 350,000 deaths around the world. Now, an increasing volume of studies have examined the inﬂuence of COVID-19 on individuals’ mental health and behavior around the world [21–23]. However, very few studies have examined the role of perceived risk in mental health and what buﬀers the inﬂuence of perceived risk [1]. In this sense, a unique contribution of this study was the identiﬁcation of individuals’ perception of risk of COVID-19 as a risk factor of mental health problems and self-control as a buﬀer of this association. Prior research found that perceived severity of COVID-19, as a negative appraisal of the event, was associated with more undesirable emotional and behavioral outcomes [1]. This result is also consistent with the ﬁndings that negative evaluation of an event, such as high levels of perceived severity, is associated with negative mental health outcomes when individuals encounter a novice virus [5,7,24]. Deepening our understanding of this association, the current study sought to examine self-control as a candidate to moderate the link between perceived severity and mental health problems among the Chinese public. Supporting our hypotheses, the results showed that even after controlling for a range of demographic variables, the association between perceived risk and mental health problems was mitigated by self-control. This ﬁnding also supports the main eﬀect and the compensatory eﬀect tenets of the risk-resilience model [2], such that the inﬂuence of risk factors (e.g., perceived risk) on individuals’ life outcomes could be buﬀered by protective factors (e.g., self-control) and thus individuals showed resilience. We need to point out that the levels of mental health problems found in this study were low. Similarly, research conducted in Italy during the early lockdown period also suggested that the levels of mental health problems (as measured with the Strength and Diﬃculties Questionnaire) were relatively low [25]. 3.2. The Moderation of Self-Control in the Association Between Perceived Severity and Mental Health Problems a: medium self-control refers to the centered mean. b: 95% CIs were calculated with R. Table 3. Summary of simple slope tests by the levels of self-control and the diﬀerence in simple slopes. 4. Discussion In addition, research conducted in India during the early lockdown also showed that negative sentiments were few and positive sentiments prevailed [26]. Nevertheless, as the lockdown continues and the national economies suﬀer, individuals’ mental health problems could increase due to various reasons, such as unemployment, reduced salary, and restricted social interaction. Thus, individuals’ mental health problems may change as the situation of COVID-19 develops. In this sense, it is important to continue monitoring individuals’ mental health and oﬀer continuous psychological aid to mitigate the potential negative inﬂuence during the post-COVID-19 period. This study bears both theoretical and clinical implications. Theoretically, the signiﬁcant interaction suggests that besides the main eﬀect model, the interaction eﬀect model is also important in studying mental health in the context of emergent public health concerns. This is because the eﬀect of a risk factor on health outcomes could be masked or averaged without examining its interaction with other 8 of 10 Int. J. Environ. Res. Public Health 2020, 17, 4820 risks and assets, and thereby the likelihood of detecting such an eﬀect is reduced. Clinically, high levels of self-control buﬀer the association between perceived severity and mental health problems, suggesting that individuals with low levels of self-control are more vulnerable. This implies that those with low levels of self-control would be more in need of psychological aids, which echoes the call by the Chinese government “to provide mental aid to those who need it”. In addition, since self-control is a crucial asset, enhancing individuals’ self-control ability might help buﬀer the inﬂuence of negative appraisal and improve the public’s mental health. We need to point out that although self-control has its heritable underpinnings, self-control can still be shaped by the environment [27] and develops within individuals throughout the life span [28]. Prior research found some programs to be useful in improving individuals’ self-control eﬀectively, such as mediation [29] and comprehensive self-control training [30]. In addition, this study also identiﬁed the populations who are vulnerable to mental health problems during the outbreak of COVID-19. These people are females, the elderly, individuals with history of physical, psychiatric, and psychological disorder, and those involved in COVID-19 (e.g., those diagnosed with COVID-19 and relatives/friends of conﬁrmed cases of COVID-19). Hence, these vulnerable populations, especially those with low levels of self-control, are in higher need of receiving psychological aids. 4. Discussion When practitioners develop and implement the intervention and prevention programs, they should be highly ﬂexible in terms of modes, materials, and schedules. This is because experts have thought that COVID-19 will last for a substantial period of time [31], which implies that there will be a regular anti-COVID-19 period and activities such as intervention and prevention programs are expected to cater for this special circumstance. We must acknowledge that the current study has several limitations. First, the self-report data increases the common method bias and the cross-sectional nature prevents us from inferring causality. Therefore, future research should employ multiple informant methods and longitudinal design to further strengthen the ﬁndings. We need to stress again the need to continue monitoring individuals’ mental health because of social changes induced by various reasons (e.g., the emergence of COVID-19) can be closely linked with human development [32]. Hence, it would be promising if future research investigates the dynamic relationship between the development of COVID-19 and individuals’ mental health as well as other developmental outcomes. Second, we did not employ a probability-sampling technique to recruit participants and sample sizes from diﬀerent provinces/regions vary greatly, which may cause selection eﬀect and aﬀect the estimation of the between-level variance. In this sense, readers should interpret the ﬁndings with caution and a more balanced sample size from diﬀerent provinces/regions in future research is desirable. Third, some populations were over-representative, such as females and university students, which may cause some bias in the results. 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We believe that these ﬁndings bear important implications in understanding and improving the public’s mental health during the encounter of emergent public health concerns. 9 of 10 Int. J. Environ. Res. Public Health 2020, 17, 4820 Author Contributions: Conceptualization, J.-B.L. and K.D.; Funding acquisition, J.-B.L. and K.D.; Investigation, J.-B.L. and K.D.; Methodology, J.-B.L., A.Y. and K.D.; Project administration, K.D.; Resources, J.-B.L. and K.D.; Software, J.-B.L.; Validation, A.Y. and K.D.; Writing—original draft, J.-B.L. and K.D.; Writing—review and editing, A.Y. and R.Y.M.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the FEHD’s Internationalization & Exchange Research Scheme of the Education University of Hong Kong, the MOE (Ministry of Education in China) Project of Humanities and Social Sciences (17YJCZH040), the 13th Five-Year Plan of Philosophy and Social Science of Guangzhou (2020GZYB92), the Key Research Items from Guangzhou University (YK2020025), and the grant of Guangdong University of Foreign Studies (299-X5216193 and 299-GK19G062). Conﬂicts of Interest: The authors declare no conﬂict of interest. References 2016, 97, 130–133. [CrossRef] 10. Duckworth, A.L.; Seligman, M.E.P. Self-discipline outdoes IQ in predicting academic performance of adolescents. Psychol. Sci. 2005, 16, 939–944. [CrossRef] 11. Moﬃtt, T.E.; Arseneault, L.; Belsky, D.; Dickson, N.; Hancox, R.J.; Harrington, H.; Houts, R.; Poulton, R.; Roberts, B.W.; Ross, S.; et al. A gradient of childhood self-control predicts health, wealth, and public safety. Proc. Natl. Acad. Sci. 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https://openalex.org/W4210616419	https://strathprints.strath.ac.uk/79681/1/Stock_etal_NM_2022_Author_correction_SARS_CoV_2_infection_and_COVID_19_vaccination_rates_in_pregnant_women.pdf	English	null	Author Correction: SARS-CoV-2 infection and COVID-19 vaccination rates in pregnant women in Scotland	Nature medicine	2,022	cc-by	294	Nature Medicine \| www.nature.com/naturemedicine https://doi.org/10.1038/s41591-022-01730-5 Author Correction: SARS-CoV-2 infection and COVID-19 vaccination rates in pregnant women in Scotland Sarah J. Stock , Jade Carruthers, Clara Calvert, Cheryl Denny , Jack Donaghy, Anna Goulding , Lisa E. M. Hopcroft, Leanne Hopkins, Terry McLaughlin, Jiafeng Pan, Ting Shi , Bob Taylor , Utkarsh Agrawal, Bonnie Auyeung, Srinivasa Vittal Katikireddi , Colin McCowan, Josie Murray, Colin R. Simpson , Chris Robertson, Eleftheria Vasileiou, Aziz Sheikh and Rachael Wood Srinivasa Vittal Katikireddi , Colin McCowan, Josie Murray, Colin R. Simpson , Chris Robertson, Eleftheria Vasileiou, Aziz Sheikh and Rachael Wood on to: Nature Medicine https://doi.org/10.1038/s41591-021-01666-2, published online 13 January 2022. In the version of this article initially published, a Statistical Analysis subsection was not present in the Methods and has now been included. Further, the Data availability statement has been amended to clarify that patient-level data may be available to approved researchers after securing relevant permissions from Public Health Scotland via the Public Benefit and Privacy Panel. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regula- tion or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/ by/4.0/.
https://openalex.org/W2010958885	https://www.frontiersin.org/articles/10.3389/fnhum.2015.00097/pdf	English	null	Electrocorticographic representations of segmental features in continuous speech	Frontiers in human neuroscience	2,015	cc-by	12,850	HUMAN NEUROSCIENCE published: 24 February 2015 doi: 10.3389/fnhum.2015.00097 Fabien Lotte 1†, Jonathan S. Brumberg 2†, Peter Brunner 3,4, Aysegul Gunduz 5, Anthony L. Ritaccio 4, Cuntai Guan 6 and Gerwin Schalk 3,4 1 Inria Bordeaux Sud-Ouest/LaBRI, Talence, France 2 Department of Speech-Language-Hearing, University of Kansas, Lawrence, KS, USA 3 National Center for Adaptive Neurotechnologies, Wadsworth Center, New York State Department of Health, Albany, NY, USA 4 Department of Neurology, Albany Medical College, Albany, NY, USA 1 Inria Bordeaux Sud-Ouest/LaBRI, Talence, France 2 Department of Speech-Language-Hearing, University of Kansas, Lawrence, KS, USA 3 National Center for Adaptive Neurotechnologies, Wadsworth Center, New York State Department of Health, Albany, NY, USA 4 Department of Neurology, Albany Medical College, Albany, NY, USA 1 Inria Bordeaux Sud-Ouest/LaBRI, Talence, France 2 Department of Speech-Language-Hearing, University of Kansas, Lawrence, KS, USA 3 National Center for Adaptive Neurotechnologies, Wadsworth Center, New York State Department of Health, Albany, NY, USA 4 Department of Neurology, Albany Medical College, Albany, NY, USA 5 J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA 6 ASTAR Agency for Science, Technology and Research, Institute for Infocomm Research, Singapore, Singapore Acoustic speech output results from coordinated articulation of dozens of muscles, bones and cartilages of the vocal mechanism. While we commonly take the ﬂuency and speed of our speech productions for granted, the neural mechanisms facilitating the requisite muscular control are not completely understood. Previous neuroimaging and electrophysiology studies of speech sensorimotor control has typically concentrated on speech sounds (i.e., phonemes, syllables and words) in isolation; sentence-length investigations have largely been used to inform coincident linguistic processing. In this study, we examined the neural representations of segmental features (place and manner of articulation, and voicing status) in the context of ﬂuent, continuous speech production. We used recordings from the cortical surface [electrocorticography (ECoG)] to simultaneously evaluate the spatial topography and temporal dynamics of the neural correlates of speech articulation that may mediate the generation of hypothesized gestural or articulatory scores. We found that the representation of place of articulation involved broad networks of brain regions during all phases of speech production: preparation, execution and monitoring. In contrast, manner of articulation and voicing status were dominated by auditory cortical responses after speech had been initiated. These results provide a new insight into the articulatory and auditory processes underlying speech production in terms of their motor requirements and acoustic correlates. ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Frontiers in Human Neuroscience Edited by: Edited by: Srikantan S. Nagarajan, University of California, San Francisco, USA Reviewed by: Edmund C. Lalor, Trinity College Dublin, Ireland Elana Zion Golumbic, Bar Ilan University, Israel Correspondence: Jonathan S. Brumberg, Department of Speech-Language-Hearing, University of Kansas, 1000 Sunnyside Ave., 3001 Dole Human Development Center, Lawrence, KS 66045, USA e-mail: brumberg@ku.edu †Th h h ib d Srikantan S. Nagarajan, University of California, San Francisco, USA Reviewed by: Edmund C. Lalor, Trinity College Dublin, Ireland Elana Zion Golumbic, Bar Ilan University, Israel Correspondence: Jonathan S. Brumberg, Department of Speech-Language-Hearing, University of Kansas, 1000 Sunnyside Ave., 3001 Dole Human Development Center, Lawrence, KS 66045, USA e-mail: brumberg@ku.edu †These authors have contributed Srikantan S. Nagarajan, University of California, San Francisco, USA Reviewed by: Edmund C. Lalor, Trinity College Dublin, Ireland Elana Zion Golumbic, Bar Ilan University, Israel Correspondence: Jonathan S. Brumberg, Department of Speech-Language-Hearing, University of Kansas, 1000 Sunnyside Ave., 3001 Dole Human Development Center, Lawrence, KS 66045, USA e-mail: brumberg@ku.edu Correspondence: Jonathan S. Brumberg, Department of Speech-Language-Hearing, University of Kansas, 1000 Sunnyside Ave., 3001 Dole Human Development Center, Lawrence, KS 66045, USA e-mail: brumberg@ku.edu †These authors have contributed equally to this work. †These authors have contributed equally to this work. Keywords: electrocorticography (ECoG), speech processing, place of articulation, manner of articulation, voicing 1. INTRODUCTION phonological constructs and lexical relationships (Indefrey and Levelt, 2004). Speech and language are realized as acoustic outputs of an aeromechanical system that is coordinated by a vast brain and muscular network. The interaction between neural structures, facial and vocal tract musculature, and respiration provides humans with a dynamic speech production system capable of forming simple sounds (e.g., mono-syllabic words) and com- plex sounds (e.g., ﬂuent conversation). These sounds are often represented by phonemes and syllables, which are fundamen- tal linguistic bases for constructing both simple and complex speech production (e.g., the “b” in “bad” is an example of a phoneme while the “ba” is an example of a consonant-vowel (CV) syllable), which in turn correspond to stereotyped vocal- tract movements resulting in acoustic speech output. Examples of such vocal-motor articulations range from the compression of the lungs for producing the air pressure needed for vocalization, to movements of laryngeal muscles during phonation, to con- ﬁgurations of the upper vocal tract for ﬁnal shaping of speech output. These muscular actions are the behavioral consequences of the speech neuromotor system, which is in turn driven by This type of communication relies on neural processes that construct messages and sensorimotor commands to convey and receive communicative information. These processes have previ- ously been characterized in a theoretical neurolinguistic model, the Levelt-Roelofs-Meyer (LRM) model (Levelt et al., 1999). Using this model as a framework, it is possible to investi- gate the behavioral, neurological, linguistic and motor processes involved in vocal communication. The model consists of the following processing components: conceptual preparation, lexi- cal selection, morpho-phonological code retrieval, phonological encoding, phonetic encoding and articulation (Levelt et al., 1999; Indefrey and Levelt, 2004). The ﬁrst four processing levels in the LRM framework all mediate perceptual processes underly- ing speech and language recognition in preparation for upcoming vocal productions (e.g., reading, picture naming). These levels of processing have been well investigated and were summa- rized in a meta-analysis of neuroimaging, electrophysiology and neuro-stimulation studies of speech and language (Indefrey and February 2015 \| Volume 9 \| Article 97 \| 1 www.frontiersin.org www.frontiersin.org ECoG representations of segmental speech features Lotte et al. greatly contributed to our understanding of the motor cor- tical representations of isolated syllable production; however, in typical speech, syllable production is rarely performed in isolation. Here, we generalize and improve upon these results by investigating articulation as it occurs during continuous, ﬂu- ent speech. 1. INTRODUCTION In contrast, electroencephalography (EEG) and mag- netoencephalography (MEG) can readily detect neurological sig- nals at these temporal scales, but cannot precisely ascribe their source to a particular location. In addition, EEG, MEG, and fMRI are all susceptible to electrical and/or movement artifacts created by speech articulation, and thus are typically used to investigate neurological activity prior to articulation or speech perception. p p g In our experiments, we asked subjects to perform an out- loud speech production task. We recorded the subjects’ acoustic output with a microphone and ECoG from widespread peri- sylvian areas that included locations with known involvement in the planning, execution and perception of speech. For each subject, we then converted the subject’s acoustic output into speech feature categories at the phonetic level (given in Table 2) and applied machine learning techniques to identify differential brain activity resulting from the production of speciﬁc speech features. The features used in our work were: place of articu- lation, manner of articulation, voicing status and phonological category of consonant or vowel. These techniques allowed us to investigate the topographical as well as temporal distribu- tions of brain activity that differentiates each type of speech feature amongst other features, which may temporally overlap in continuous speech. The analysis techniques used in our study can also be used to predict the occurrence of a speech feature from the ECoG signals. Therefore, our study provides important insights into the coordination of individual articulatory neuro- motor processes as they are sequenced together for production of ﬂuent speech output, and should provide an important basis for future development of a brain-to-text brain-computer interface (BCI). Electrical signals recorded directly from the cortical surface [electrocorticography (ECoG)] have recently begun to attract increasing attention for basic and translational neuroscience research, because they allow for examination of the precise spatio- temporal evolution of neurological processes associated with complex behaviors, including speech output. Speciﬁcally, ECoG has been used to investigate neurological activity during a num- ber of tasks including linguistic processing (Towle et al., 2008; Edwards et al., 2010), speech perception and feedback process- ing (Crone et al., 2001; Chang et al., 2010; Pei et al., 2011a,b; Pasley et al., 2012), as well as articulation of phonemes, syllables, and words (Blakely et al., 2008; Kellis et al., 2010; Leuthardt et al., 2012; Bouchard et al., 2013; Riès et al., 2013; Mugler et al., 2014). 1. INTRODUCTION One major difference between isolated production of speech sounds and continuous speech is the presence and degree of coarticulation, or the inﬂuence of past and future speech requirements over current productions (Hardcastle and Hewlett, 1999). The two varieties of coarticulation include: (1) carry-over, in which upcoming speech productions are based on the vocal tract conﬁgurations of past utterances; and (2) anticipatory, in which the production of current speech sounds is altered based on expected requirements of future sounds. The extent to which segmental and phonological boundaries inﬂuence the degree of coarticulation (Recasens, 1999) is currently subject of debate (e.g., whether a boundary facilitates or inhibits coarticulation). In our study, we assume coarticulation is occurring as participants pro- duce speech, and our results are based solely on the amount of speech information present in the ECoG signal. Levelt, 2004), and more recently by Riès et al. (2013). The ﬁnal two stages, phonetic encoding and articulation (of articulatory scores), describe the motor aspects of vocal communication and are the focus of the present study. According to the LRM frame- work, the phonetic encoding stage translates a phonological word (from the previous phonological encoding stage) into an artic- ulatory score, which can be processed and transmitted to the articulatory musculature for speech motor output. Levelt, 2004), and more recently by Riès et al. (2013). The ﬁnal two stages, phonetic encoding and articulation (of articulatory scores), describe the motor aspects of vocal communication and are the focus of the present study. According to the LRM frame- work, the phonetic encoding stage translates a phonological word (from the previous phonological encoding stage) into an artic- ulatory score, which can be processed and transmitted to the articulatory musculature for speech motor output. The precise nature by which the brain realizes these pho- netic encoding and articulation functions are still unknown. One possible explanation for this lack of understanding stems from the difﬁculty in measuring the neurological processes involved in the planning and production of speech. Indefrey and Levelt estimate a total speech-language processing time of approxi- mately only 600 ms (not including articulation) from beginning to end, with individual durations of approximately 100–200 ms for each processing component in their model (Indefrey and Levelt, 2004). Functional magnetic resonance imaging (fMRI), which is the primary neuroimaging technique used in speech neuroscience, cannot resolve brain activity at that temporal resolution. Frontiers in Human Neuroscience 2.3. SIGNAL PROCESSING AND ANALYSIS The goal of our study was to identify those locations or times in which differential ECoG activity was found between overtly produced speech utterances based on articulatory-acoustic and phonological features (e.g., segmental features) of phonemes1. In this work, we used a vowel vs. consonant contrast as the primary phonological discriminatory dimension. In addition, we examined the articulatory-acoustic dimension by testing the manner (e.g., voicing quality: obstruent vs. sonorant) and the place (e.g., location of articulatory closure or constriction) of speech articulation, and voicing (e.g., quasiperiodic oscillations of the vocal folds: voiced vs. voiceless). The place features are primarily used to characterize consonant sounds, while the man- ner and voicing features can be used in both consonant and vowel descriptions. We conducted an analysis of a feature rep- resenting the tongue conﬁgurations involved in the produc- tion of vowel sounds (e.g., height & frontness within the oral cavity); however, it did not reveal different patterns of spa- tiotemporal activations and will not be discussed in subsequent sections. Grid locations were veriﬁed in each subject using a co- registration method that included pre-operative structural mag- netic resonance (MR) imaging and post-operative computed tomography (CT) imaging (Kubanek and Schalk, 2014). We then used Curry software (Compumedics, Charlotte, NC) to extract three-dimensional cortical models of individual subjects, to co- register the MR and CT images, and to extract electrode loca- tions. Electrode locations are shown for each subject in Figure 1. Electrode locations were further assigned to cortical lobe using the Talairach Daemon (http://www.talairach.org, Lancaster et al., 2000). ECoG signals were recorded at the bedside using eight 16- channel g.USBamp biosignal acquisition devices (g.tec, Graz, Austria) at a sampling rate of 1200 Hz, and stored for further analyses. Electrode contacts distant from epileptic foci and areas of interest were used for reference and ground and any channels with obvious electrical or mechanical artifacts removed. The total number of electrodes used per subject is listed in Table 1. 1As deﬁned by the International Phonetic Association, a phoneme is “the smallest segmental unit of sound employed to form meaningful contrasts between utterances” (International Phonetic Association, 1999). Table 1 \| Clinical proﬁles of participants. 2.1. HUMAN SUBJECTS AND DATA COLLECTION The seven subjects who participated in this study were patients with intractable epilepsy at Albany Medical Center. Subjects underwent temporary placement of subdural electrode arrays to localize seizure foci prior to surgical resection of epileptic tissue. The seven subjects who participated in this study were patients with intractable epilepsy at Albany Medical Center. Subjects underwent temporary placement of subdural electrode arrays to localize seizure foci prior to surgical resection of epileptic tissue. All gave informed consent to participate in the study, which was approved by the Institutional Review Boards of the hospital, had performance IQs of at least 85, and were mentally, visually and physically capable of performing the task. Table 1 summarizes the subjects’ clinical proﬁles. All gave informed consent to participate in the study, which was approved by the Institutional Review Boards of the hospital, had performance IQs of at least 85, and were mentally, visually and physically capable of performing the task. Table 1 summarizes the subjects’ clinical proﬁles. The implanted electrode grids (Ad-Tech Medical Corp., Racine, WI) consisted of platinum-iridium electrodes (4 mm in diameter, 2.3 mm exposed) that were embedded in silicon and spaced at an inter-electrode distance of 1 cm. Subject G had implanted electrodes with 6 mm grid spacing (PMT Corp, Chanhassen, MN). All subjects received electrode grid implan- tations over the left hemisphere, though the total number of electrodes implanted was different for each subject. Grid place- ment and duration of ECoG monitoring were based solely on the requirements of the clinical evaluation without any consideration of this study. 1. INTRODUCTION In the present study, we apply machine learning techniques to evaluate the neurological activity during speech production based on segmental features (i.e., phonology, and articulatory-acoustic descriptors) and the resulting ECoG signals. By analyzing these features, rather than phonemes, syllables, or words, we are able to identify a low-dimensional and invariant basis by which to inter- pret neural activity related to overt speech production that can be upscaled to more complex vocalizations. The results of our analyses revealed a broad network involv- ing fronto-motor and temporal cortices that were active during the preparation, execution and feedback monitoring of place of articulation. In contrast, ECoG responses labeled by manner of articulation involved a widespread auditory cortical network that was active near the start of speech onset and persisting through- out the feedback monitoring process. Analysis of voicing status largely mirrored the manner of articulation results suggesting that the production of different manners of articulation and voicing involve large auditory cortical networks for processing for proper speech motor control, while place of articulation more equally weights processing at all three stages of production. Interestingly, our analysis of both the manner and voicing conditions included a focal motor response that likely reﬂects speciﬁc differences in A recent ECoG study (Bouchard et al., 2013) employed such an articulation-based approach in which subjects were required to produce isolated CV syllables. The authors observed both a topographic and temporal organization of ECoG signals over the speech-motor cortex related to speech articulation. Speciﬁcally, their results showed that the production of isolated syllables resulted in differential neurological activity clustered by articu- latory feature (e.g., lip and tongue movements). These ﬁndings February 2015 \| Volume 9 \| Article 97 \| 2 www.frontiersin.org www.frontiersin.org www.frontiersin.org ECoG representations of segmental speech features Lotte et al. 2.2. EXPERIMENTAL PARADIGM the motor control of voicing (e.g., voiced vs. voiceless produc- tion). We elaborate on these results and their interpretation in the sections that follow. In this study, subjects were asked to perform an overt speech pro- duction task in which stimuli consisted of well-known political speeches or nursery rhymes ranging between 109 and 411 words in length. The stimulus text was presented visually and scrolled across a computer screen from the right to the left at a constant rate and subjects repeated each word as it appeared on the screen. The rate was set for each subject to be appropriate for the sub- ject’s level of attentiveness, cognitive, and comprehension abilities (see Table 1). The computer screen was placed approximately 1 m from the subjects. A single experimental run consisted of reading an entire stimulus passage, and subjects completed between 2–4 runs. All subjects completed the experiment in a single session except for Subject D, who required two sessions. Data collec- tion from the g.USBamp acquisition devices, as well as control of the experimental paradigm were accomplished simultaneously using BCI2000 software (Schalk et al., 2004; Mellinger and Schalk, 2010). A schematic illustrating the experimental setup is shown in (Figure 2). February 2015 \| Volume 9 \| Article 97 \| 3 2.3. SIGNAL PROCESSING AND ANALYSIS Subject Age Sex Handedness Performance IQ Verbal IQ Seizure focus # Electrodes # Words A 29 F R 136 118 Left temporal 96 278 B 30 M R 90 64 Left temporal 83 109 C 29 F R 90 91 Left temporal 101 283 D 19 M R 85 87 Left frontal 84 411 E 26 F R 117 106 Left temporal 109 411 F 56 M R 87 82 Left temporal 97 411 G 29 F R 95 111 Left temporal 112 411 Frontiers in Human Neuroscience www.frontiersin.org February 2015 \| Volume 9 \| Article 97 \| 3 Table 1 \| Clinical proﬁles of participants. February 2015 \| Volume 9 \| Article 97 \| 3 ECoG representations of segmental speech features Lotte et al. FIGURE 1 \| Locations of implanted grids on individual subject cortical models based on co-registered pre-op MR and post-op CT data. The bottom right ﬁgure shows the electrode locations projected on an average brain for those four subjects (B, E, F, and G) that passed initial screening (subjects A, C, and D were not included, see Section 2.3.5). Each subject’s electrodes are represented with a different color. FIGURE 2 \| Experimental setup. brain for those four subjects (B, E, F, and G) that passed initial screening (subjects A, C, and D were not included, see Section 2.3.5). Each subject’s electrodes are represented with a different color. brain for those four subjects (B, E, F, and G) that passed initial screening (subjects A, C, and D were not included, see Section 2.3.5). Each subject’s electrodes are represented with a different color. FIGURE 1 \| Locations of implanted grids on individual subject cortical models based on co-registered pre-op MR and post-op CT data. The bottom right ﬁgure shows the electrode locations projected on an average brain for those four subjects (B, E, F, and G) that passed initial screening (subjects A, C, and D were not included, see Section 2.3.5). Each subject’s electrodes are represented with a different color. FIGURE 2 \| Experimental setup. FIGURE 2 \| Experimental setup. FIGURE 2 \| Experimental setup. 2.3.1. Articulatory-acoustic feature descriptions Frontiers in Human Neuroscience 2.3.1. Articulatory-acoustic feature descriptions voiced (with only a few exceptions) while obstruents have voiced and voiceless pairs (e.g., the bilabial pair “b” [voiced] and “p” [voiceless]). y p The articulatory features used in the present study generally char- acterize the vocal tract movements and conﬁgurations required for speech production. The place of articulation deﬁnes a location where speech articulators either close or constrict the vocal tract. In our analysis, high-level descriptions of place of articulation broadly describe the closure of the lips (labial) and the location in the oral cavity where the tongue contacts or approaches the hard and soft palates (coronal and dorsal) (Hall, 2007). The man- ner of articulation describes the relative closure of the vocal tract and resultant airﬂow path during phonation; it can be coarsely grouped into obstruents (those articulations that impede air- ﬂow in the vocal tract) and sonorants (those which maintain an open vocal tract) (Hall, 2007). The voicing feature indicates whether the vocal folds are active and oscillating during produc- tion of speech sounds. Speech sounds are classiﬁed as “voiced” if the vocal folds are oscillating and “voiceless” if they are not. All sonorant sounds, including all vowels, in English are considered Both the place and manner of articulation can be speciﬁed at increasingly reﬁned levels. For place, some examples of the labial feature includes bilabials (“b”) and labiodentals (“v”), an exam- ple of a coronal includes alveolars (“d” in “dog”) and palatals, and the dorsal group includes consonants with contact on the velum or soft palate (“g” in “good”). Additionally, the dorsal group can be used to describe the relative movements of all the vowels, though not their speciﬁc conﬁgurations. These additional place descriptors can further reﬁne the locations of the hard and soft palates contacted by the tongue and vice versa as well (e.g., they describe the portions of the tongue used to contact the palate). The manner of articulation can also be described with ﬁner lev- els of detail, with examples of the obstruent category including features for stops (“b” in “boy”), fricatives (“v” in “vast”), and affricates (“ch” in “chest”) while the sonorant category contains February 2015 \| Volume 9 \| Article 97 \| 4 www.frontiersin.org www.frontiersin.org www.frontiersin.org ECoG representations of segmental speech features Lotte et al. assigned in a binary fashion in which “+” indicated the pres- ence of a feature, and “−” the absence. 2.3.1. Articulatory-acoustic feature descriptions Importantly, while the features were coded as binary, any one phoneme may code for multiple combinations of features (e.g., consonant+, obstruent+, labial+ and voicing+ for the “b” sound). In other words, a par- ticular phonemic feature was assigned a value (“+” or “−”) for each phoneme. Overall, we identiﬁed 33 different phonemes with 1226–4872 combined occurrences per subject. Each phoneme was deﬁned by a particular onset and offset time that was used for sub- sequent neurophysiological analyses. An example of audio signal transcription and feature labeling (for the feature: vowel) is given in Figure 3 along with synchronized ECoG recordings (gamma band power) at two electrode sites. the features for approximants (“l” in “less”) and nasals (“n” in “nine”). These additional levels of description characterize spe- ciﬁc differences in airﬂow resulting from speech production. To simplify the analysis and provide sufﬁcient data for estimation of our machine learning models, we concentrated on the high-level categorical groupings: obstruent vs. sonorant for manner of artic- ulation, and labial vs. coronal vs. dorsal for place of articulation. A summary of the phonetic feature descriptions used in this study can be found in Table 2. 2.3.3. ECoG segments extraction and labeling We analyzed event-related changes in 700 ms ECoG epochs aligned to phoneme acoustic onset. To do this, we ﬁrst high-pass ﬁltered the continuous ECoG recordings using a cutoff frequency of 0.5 Hz and a forward-backward Butterworth ﬁlter of order 4 to remove DC signal components (Matlab functions filtfilt and butter). The data were then notch-ﬁltered at 120 Hz using a forward-backward inﬁnite impulse response (IIR) notch ﬁlter with a Q-factor of 35 (q = ω0/bw, where ω0 = 120 and q = 35) A summary of all phoneme transcriptions and data features used in this study is provided in Table 2. Each speech feature was Table 2 \| Features and frequencies observed in the speech stimuli. Place of articulation Manner of articulation Feature Frequency (%) Phonemes Feature Frequency (%) Phonemes Labial 22.9 /b p f v m w/ Obstruent 59.1 /b p g k d t f v/ /tS dZ D T s z S Z/ Coronal 78.0 /t d T D s z S Z n/ /tS dZ r l j/ Sonorant 37.5 /i I E æ A @ u U/ /3~ aI eI aU oU OI/ /w j r l m n N/ Dorsal 12.4 /k g w/ Phonological Voicing Feature Frequency (%) # Feature Frequency (%) Phonemes Consonant 60.8 24 Voiced 78.0 /i I E æ A @ u U/ /3~ aI eI aU oU OI/ /w j r l m n N/ /b d g v S Z dZ/ Vowel 39.2 15 Voiceless 22.0 /p t k f s z tS/ Frontiers in Human Neuroscience www.frontiersin.org February 2015 \| Volume 9 \| Article 97 \| 5 Table 2 \| Features and frequencies observed in the speech stimuli. 2.3.2. Speech segmentation into phonemes We ﬁrst segmented the acoustic speech signals into individual phonemes. This segmentation served to (1) separate each individ- ual spoken word and (2) identify and temporally locate phonemes within each word. Our segmentation procedure obtained pho- netic transcriptions using a semi-automated algorithm that ﬁrst isolated the spoken words from silence followed by identiﬁcation of constituent phonemes. The onset and termination of spo- ken words were manually located in the audio signal waveforms. Initial manual segmentation of word boundaries was necessary for accurate speech analysis, and was often completed with min- imal effort. Following word segmentation, phonemes were auto- matically labeled and aligned to the audio signal, using a Hidden Markov Model (HMM) classiﬁer with Mel-Frequency Cepstrum Coefﬁcients (MFCC) and their ﬁrst and second derivatives as fea- tures (Rabiner and Juang, 1993). The phonetic transcription and alignment was performed using the HMM ToolKit (HTK) (Young et al., 2006). Our rationale for automated phonetic transcription was to minimize human errors and provide an objective solu- tion for a fair comparison between participants. Each phoneme was then classiﬁed as (1) a consonant or a vowel [phonologi- cal], (2) an obstruent or sonorant [manner], (3) according to vocal tract contacts or constrictions [place] and (4) voicing status [voicing]. The automatic speech recognition system described above was adapted from the original implementation to achieve robust and accurate speaker-dependent classiﬁcation for use with all of our study participants. The classiﬁer was ﬁrst trained on an “ideal” source based on a triphone acoustic model to establish a baseline. Then, the classiﬁer was adapted to account for each partici- pant’s individual speech acoustic characteristics using the speech recorded from each subject, creating a speaker-dependent recog- nition and phonetic transcription system. The speaker-dependent model outperformed the speaker-independent model in terms of producing more accurate phoneme boundaries. All auto- matic phoneme alignments were visually checked by a speech recognition expert who conﬁrmed their quality. 2.3.3. ECoG segments extraction and labeling Place of articulation Manner of articulation Feature Frequency (%) Phonemes Feature Frequency (%) Phonemes Labial 22.9 /b p f v m w/ Obstruent 59.1 /b p g k d t f v/ /tS dZ D T s z S Z/ Coronal 78.0 /t d T D s z S Z n/ /tS dZ r l j/ Sonorant 37.5 /i I E æ A @ u U/ /3~ aI eI aU oU OI/ /w j r l m n N/ Dorsal 12.4 /k g w/ Phonological Voicing Feature Frequency (%) # Feature Frequency (%) Phonemes Consonant 60.8 24 Voiced 78.0 /i I E æ A @ u U/ /3~ aI eI aU oU OI/ /w j r l m n N/ /b d g v S Z dZ/ Vowel 39.2 15 Voiceless 22.0 /p t k f s z tS/ Frontiers in Human Neuroscience www.frontiersin.org February 2015 \| Volume 9 \| Article 97 \| 5 Table 2 \| Features and frequencies observed in the speech stimuli. Frontiers in Human Neuroscience February 2015 \| Volume 9 \| Article 97 \| 5 ECoG representations of segmental speech features Lotte et al. FIGURE 3 \| Example of the ECoG gamma envelope from the two electrodes circled in green and blue, for the production and perception of the words “abolish all.” The transcription of these two words into phonemes is all also provided, together with the corresponding class label for the articulatory feature “vowel” (“+”: the phoneme is a vowel, “−”: the phoneme is a consonant). phonemes is all also provided, together with the corresponding class label for the articulatory feature “vowel” (“+”: the phoneme is a vowel, “−”: the phoneme is a consonant). FIGURE 3 \| Example of the ECoG gamma envelope from the two electrodes circled in green and blue, for the production and perception of the words “abolish all.” The transcription of these two words into 2. Preprocess ECoG gamma band power (as in Section 2.3.3). (Antoniou, 1993) to remove the power line harmonics (ﬁrst har- monic) interference. Note that we did not ﬁlter the signals at the fundamental frequency of the power line (60 Hz) nor its other harmonics (180, 240 Hz, etc.) since our analysis only involved the gamma band (70–170 Hz) of the ECoG signals. Following ﬁltering, the ECoG signals were re-referenced to the common average reference (CAR), separately for each grid of implanted electrodes2. 2Most subjects had more than one ECoG grid implanted; therefore, the electrodes from each grid were re-referenced to the grid average. 2.3.3. ECoG segments extraction and labeling Finally, the ECoG gamma band power was obtained by applying a bandpass ﬁlter in the range of 70–170 Hz using a fourth order forward-backward Butterworth ﬁlter, squaring the result and log-transforming the signal. 3. Choose analysis features based on the number of ECoG elec- trodes, and reduce feature dimensionality according to the minimal Redundancy Maximal Relevance (mRMR) feature selection procedure (Peng et al., 2005). 4. Train and apply a regularized linear discriminant analysis (LDA) classiﬁer (Lotte et al., 2007) for distinguishing selected features using 5 fold cross-validation for each subject and run. Note that feature selection was performed, for each fold of the cross-validation, on the training data only. 5. Evaluate classiﬁer using receiver operating characteristics (ROC) curves, and obtain the area under the curve (AUC) as the primary performance measure. After preprocessing the recorded ECoG signals, we extracted a 700 ms window of data from the continuous recording. This window was aligned to the onset of each phoneme identiﬁed by the semi-automated phoneme transcription procedure described above. Each window was centered on the phoneme onset, and thus consisted of a 350 ms pre-phoneme interval and a 350 ms post-phoneme interval, which provides sufﬁcient opportunity to examine the neurological processing per phoneme. Each win- dow was tagged with the phoneme’s feature vector (i.e., “+” or “−” deﬁnition for each phonemic feature) for subsequent classiﬁcation / discrimination analysis. LDA regularization was achieved using covariance matrix shrink- age according to the Ledoit and Wolf method for automatically estimating large dimensional covariance matrices from small data observations (Ledoit and Wolf, 2004). Regularized LDA using this technique has been previously used in brain-machine interfacing experiments where data and feature dimensionality are consis- tently problematic (Lotte and Guan, 2010; Blankertz et al., 2011). According to our cross-validation procedure, the data were split into ﬁve non-overlapping subsets, four of which were used for LDA training and feature selection and the remaining, mutually- exclusive data set, used for testing. The training and testing procedures were repeated ﬁve times, once for each mutually exclusive validation set, and the performance was averaged over all test-set results. Note, classiﬁer training and feature selection were performed only on the training part of each cross-validation fold. 2.3.5. Subject screening and inclusion (1) As a screening measure, we ﬁrst determined which of the sub- jects produced ECoG signals that were different between spoken words and silence. Subjects whose classiﬁcation results exceeded our threshold (see below for details) were analyzed further for the speech feature analysis. According to the classiﬁcation pro- cedure described in Section 2.3.4, we ﬁrst manually obtained the boundaries of all words from the acoustic signal and extracted ECoG gamma band power from a 700 ms window centered on each word. We then obtained an equal number of ECoG seg- ments taken from 700 ms windows of silence and labeled the segments as “speech” or “silence.” For each electrode, the pre- processed 700 ms ECoG signal was segmented in time using 50 ms long windows with 25 ms overlap based on the parameters from prior studies (Pei et al., 2011a,b). This procedure resulted in an initial set of 27 gamma-band features per electrode (between 83 and 112 electrodes per subject), which were taken from cortical areas covering the perisylvian and Rolandic cortices (e.g., primary motor, premotor, auditory and somatosensory cortices; Broca’s and Wernicke’s areas). We then used the mRMR procedure to reduce the feature dimension by selecting 50 features from the larger data set. Last, we obtained the ROC curve and set a thresh- old of AUC > 0.8 for inclusion in the remainder of the speech feature analysis. An AUC of 0.5 represents chance performance, we therefore utilized a higher threshold for use as a screening criterion. where log denotes the natural logarithm. These activation indices for each electrode channel were accumulated across subjects and mapped onto a template brain (Montreal Neurological Institute [MNI]; http://www.bic.mni.mcgill.ca) using in-house Matlab routines (Kubanek and Schalk, 2014). where log denotes the natural logarithm. These activation indices for each electrode channel were accumulated across subjects and mapped onto a template brain (Montreal Neurological Institute [MNI]; http://www.bic.mni.mcgill.ca) using in-house Matlab routines (Kubanek and Schalk, 2014). 2.3.6.2. Temporal dynamics analysis. The temporal analysis of speech features over the duration of each data segment involved similar processing steps used in the spatial analysis. For each sub- ject, we ﬁrst limited the temporal analysis to ECoG electrode channels with statistically signiﬁcant activation indices found in the spatial topography analysis. 2.3.4. Classiﬁcation analysis technique In the following sections, we describe the method used to evaluate the spatial and temporal patterns of neurological activity involved in speech production. Speciﬁcally, we employed a classiﬁcation analysis to determine which brain regions differ in their patterns of activity during the production of speech that varies by place of articulation, manner of articulation, voicing and phonological category of consonant or vowel (Section 2.3.6). We include also a classiﬁcation analysis of brain activity during active speaking vs. silence (Section 2.3.5). The same procedure was used for all classiﬁcation analyses, and is summarized as follows: Additionally, we chose area under the ROC curve as the measure of performance since it is speciﬁcally designed for unbal- anced binary classiﬁcation problems (Fawcett, 2006). In our study, the number of phonemes labeled “+” for a speech fea- ture was not necessarily the same as the number of phonemes labeled “−,” therefore the classiﬁcation problem was unbalanced. The “+” class was used as the positive class for ROC curves computation. Statistical signiﬁcance of the obtained AUC values was determined using the Hanley and McNeil formula for esti- mating standard error (Hanley and McNeil, 1983). The resulting p-value was then corrected for multiple comparisons (number of 1. Process and segment speech signal for features of interest (e.g., speech vs. silence, place, manner and voicing features, phonological features). 2Most subjects had more than one ECoG grid implanted; therefore, the electrodes from each grid were re-referenced to the grid average. February 2015 \| Volume 9 \| Article 97 \| 6 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org ECoG representations of segmental speech features Lotte et al. subjects × number of ECoG electrodes per subject) using the false discovery rate (FDR) approach (Noble, 2009). index” that was proportional to the AUC p-value for each tested feature. The activation index (AI) was deﬁned as: ψ(p) = −log(p) p < 0.01 0 otherwise (1) 2.3.6. Classiﬁcation of articulatory features Determination of the differential neurological activity used in the production of each articulatory-acoustic and phonological features (described in Section 2.3.1) was split into separate anal- yses of spatial topography and temporal dynamics. In the spatial topography analysis, we projected the results onto the cortical sur- face and plotted the results over time for the temporal dynamics analysis. In these two procedures, the spatial analysis considered ECoG activity at each location throughout each windowed epoch; the temporal analysis considered ECoG activity at a particular time but across all locations. 2.3.5. Subject screening and inclusion For this analysis, we ﬁrst re- estimated the ECoG gamma band power using 50 ms time bins, but with 40 ms overlaps (10 ms steps) for use in the LDA proce- dure. The change in overlap was used to visualize and analyze the activation index time course with a higher resolution, such res- olution is neither needed nor desired for the spatial topography analysis. The same speech features and phonetic boundaries used in the spatial analysis were used here as well. Also in this anal- ysis, dimension reduction and regularization were not required since there was only one data feature (time-binned ECoG band power) per classiﬁcation attempt. The average AUC was then used to compute a signiﬁcance p-value, corrected for multiple comparisons (subjects, time bins and electrodes with statistically signiﬁcant activation indices in the spatial topography analysis) using the FDR, and transformed into an activation index. The temporal proﬁles of the activation indices were averaged across subjects and over all electrodes per speech feature to represent the gross cortical processes involved in the discrimination of speech articulation features. Frontiers in Human Neuroscience 3.1. SPEECH vs. SILENCE We employed a functional screening criteria based on classiﬁca- tion results for a speech vs. silence discrimination analysis. These results are summarized in Table 3. Those subjects that did not have neural responses that consistently responded to the task, and thus had signals that could differentiate between speech and silence, were excluded from the remainder of the speech feature analysis. Recall, an AUC value of 0.5 represents chance discrimi- nation, while a value of 1.0 indicates perfect discrimination. None of our analyses resulted in AUC values less than 0.5, indicating that all classiﬁcations were above chance levels. However, as illus- trated in Table 3, our analysis was not able to well-differentiate the neural activation patterns for the speech vs. silence contrast for subjects A, C, and D using our higher screening threshold (AUC < 0.8), which would lead to similarly poor results in any subsequent analyses of articulatory and phonological features. In contrast, the analysis for subjects B, E, F, and G resulted in rela- tively good differentiation between speech and silence (AUC > 0.8). Thus, we included only data from these subjects in the 2.3.6.1. Spatial topography analysis. Using the classiﬁcation procedure described in Section 2.3.4 as a guide, we ﬁrst obtained the boundaries of all phonemes in the acoustic signal (see Section 2.3.2), extracted the ECoG gamma band power from a 700 ms window centered on the onset of each phoneme, and segmented it in time using 50 ms long windows (25 ms overlap). We then used the mRMR procedure to select 10 time segments per phoneme and electrode to minimize the effects of overﬁtting while training the regularized LDA classiﬁer. A new classiﬁer was trained on each of the speech features to discriminate between the “+” and “−” category members. To analyze the three levels place of articula- tion features, we computed three binary comparisons: labial+ vs. labial–, coronal+ vs. coronal−and dorsal+ vs. dorsal−. All other features contained only two levels, therefore, only a single binary comparison is needed for each. We then computed an “activation February 2015 \| Volume 9 \| Article 97 \| 7 www.frontiersin.org www.frontiersin.org www.frontiersin.org ECoG representations of segmental speech features Lotte et al. condition at −185, −75, and +100 ms, the coronal condition at −195, −85, and +100 ms, and the dorsal condition at −165 and +115 ms. remainder of our study. 3.2.1. Place of articulation We analyzed ECoG recordings to identify differential neural activ- ity for three place of articulatory features: labial, coronal, and dorsal representing vocal-tract closures at the lips (labial), tongue tip and blade (coronal), and tongue dorsum (dorsal). We then used statistically signiﬁcant, above-chance LDA classiﬁcations (AUC > 0.5) as a measure of differential neurological represen- tations of each speech feature. We generally found statistically signiﬁcant responses across the sensorimotor speech production network and auditory feedback processing regions (see left col- umn Figure 4). The responses superior to the Sylvian ﬁssure are distributed over the primary motor and somatosensory cor- tices (sensorimotor cortex for speech), while the responses in the temporal lobe are found in perisylvian auditory cortex, partic- ularly in the posterior aspects of the superior temporal gyrus (e.g., Wernicke’s area). The coronal feature resulted in the largest topographical montage of statistically signiﬁcant ECoG electrodes contributing to differentiation of place of articulation (N = 19 of 401 electrodes), followed by the labial (N = 9) and dorsal (N = 3) features. A summary of these results is found in Table 4. The spatial topography of electrodes with differential activ- ity patterns between the two manner categories are shown in Figure 5, left column. This analysis revealed statistically sig- niﬁcant perisylvian auditory cortex and sensorimotor cortex FIGURE 4 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “place” category levels: labial, coronal, and dorsal. In the temporal dimension group analysis, we found the latency of peak AI for all three place conditions near the onset of phoneme alignment at 0 ms (see Figure 4, right column and summarized in Table 4). Speciﬁcally, the labial condition is char- acterized by an overall difference from all other features that rose markedly to a peak response at +25 ms (with 21.24 peak activation index) and persisted well afterward. The peak activa- tion index for the coronal condition was 13.42 at +35 ms latency and the dorsal condition was 6.25 at +45 ms latency. In general, both the labial and coronal temporal proﬁles indicated prolonged duration of statistically signiﬁcant activation indices preceding and following peak response near 25–35 ms while the dorsal con- dition was much more narrow in its response. 3.1. SPEECH vs. SILENCE The resulting combined electrode loca- tions for these four subjects can be found in the bottom right of Figure 1. 3.2.2. Manner of articulation Topographical cortical mappings and temporal proﬁles reported here reﬂect electrodes, grouped over all four subjects, with sta- tistically signiﬁcant differences in ECoG recordings between our speech features of interest. In our method, each discrimination is along a binary feature dimension and represents a comparison of neural patterns of activation between pairs of speech features. The analysis of place of articulation is oriented toward the artic- ulations and points-of-contact in the oral portion of the upper vocal tract. In contrast, manner of articulation, which describes airﬂow resulting from constriction or closure (release) is oriented generally as the muscular activation of the entire upper vocal tract (larynx, velum and oral structures). In typical deﬁnitions of man- ner of articulation, categorical features are used to describe the overall airﬂow. In the present analysis, we follow this conven- tion and examined two main classes of manner: obstruents and sonorants. 4. DISCUSSION 4.1. GENERAL COMMENTS In contrast to both the manner and place features, voicing refers to only one articulatory structure, the larynx, or more speciﬁcally, the vocal folds. The spatial topography of electrodes (left column, Figure 6) with differential patterns of activity between the voiced and voiceless classes of phonemes is concentrated in the perisyl- vian auditory and motor cortex, with additional activation of the ventral motor cortex. In our analysis, 12 electrodes contributed to differentiation of phonemes along the voicing dimension. The temporal proﬁle of these activations (right column, Figure 6) indicate a peak statistical difference at +95 ms with an activation index of 11.45. There was a smaller local peak just prior to vocal- ization onset at −25 ms. These results are summarized in Table 6. In this paper, we identiﬁed patterns of cortical topographies and temporal dynamics involved in speech production based on segmental articulatory-acoustic and phonological character- istics. To do this, we used a classiﬁcation analysis to identify spatial or temporal neurological activity that best discriminated between common sets of articulatory and phonological fea- tures of continuous speech production. Some recent studies of speech production using ECoG and intracortical microelec- trode recordings have also examined phonetic content (Blakely et al., 2008), and articulatory-acoustic features (Brumberg et al., FIGURE 5 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “manner” category levels: obstruent and sonorant. Table 5 \| Summary of results for manner of articulation over all sampled electrodes. Manner # Electrodes Peak AI Peak latency Obstruent 10 18.26 85 ms Sonorant 11 28.58 85 ms FIGURE 6 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “voicing” (i.e., voiced vs. voiceless) and “phonological” (i.e., consonant vs. vowel) categories. Table 6 \| Summary of results for voicing and phonological category (vowels only) over all sampled electrodes. 3.2.4. Vowel vs. consonant We examined the vowel vs. consonant contrast to determine whether differences existed in neural activation patterns between production of sounds varying in phonological class. The spa- tial topography and temporal dynamics representing differences between these two classes were represented by a large region of auditory cortex and a more focal region of sensorimotor cortex. The temporal patterns of neural activation had peak statistical dif- ference +95 ms, but appear to also show moderate differentiation at speech-leading intervals, with a local maxima at −105 ms as shown in Figure 6 (right column). A summary of these results can be found in Table 6. The temporal proﬁle results (right column, Figure 5) indi- cate very limited differences between manner categories prior to phoneme onset (speech-leading latencies with negative intervals) and greater differences at speech-following latencies (positive intervals). Speciﬁcally, the peak statistical signiﬁcance for differ- entiating manner features from each other at +85 ms for both obstruents and sonorants. These differences are largely present during the entire post-onset speech period. These results are summarized in Table 5. 3.2.1. Place of articulation We should note that this may be due to the relatively few sounds with constriction or closure of the tongue along soft palate compared to those in the anterior portions of the oral cavity. Furthermore, each of the three place conditions had multiple local maxima throughout the anal- ysis window. Speciﬁcally, local maxima were found for the labial FIGURE 4 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “place” category levels: labial, coronal, and dorsal. FIGURE 4 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “place” category levels: labial, coronal, and dorsal. Table 4 \| Summary of results for place of articulation over all sampled electrodes. Place # Electrodes Peak AI Peak latency Local maxima Labial 9 21.24 25 ms −185, −75 , +105 ms Coronal 19 13.42 35 ms −195, −85, +95 ms Dorsal 3 6.25 45 ms −165, +115 ms tiersin.org February 2015 \| Volume 9 \| Article 97 \| 8 Table 4 \| Summary of results for place of articulation over all sampled electrodes. Table 3 \| AUC cross-validation performances obtained for each subject to classify “spoken word” vs. “silence” ECoG segments. Subject A B C D E F G AUC 0.57 0.81 0.51 0.68 0.91 0.87 0.91 electrodes. Place # Electrodes Peak AI Peak latency Local maxima Labial 9 21.24 25 ms −185, −75 , +105 ms Coronal 19 13.42 35 ms −195, −85, +95 ms Dorsal 3 6.25 45 ms −165, +115 ms Frontiers in Human Neuroscience www.frontiersin.org February 2015 \| Volume 9 \| Article 97 \| 8 Table 3 \| AUC cross-validation performances obtained for each subject to classify “spoken word” vs. “silence” ECoG segments. February 2015 \| Volume 9 \| Article 97 \| 8 Frontiers in Human Neuroscience www.frontiersin.org ECoG representations of segmental speech features Lotte et al. response contributing to differentiation of the obstruent (N = 10 electrodes) and sonorant (N = 11 electrodes) features. 4. DISCUSSION 4.1. GENERAL COMMENTS Manner # Electrodes Peak AI Peak latency Local maxima Voicing 12 11.45 95 ms –25 ms Vowels 8 21.80 95 ms –105 ms Frontiers in Human Neuroscience www.frontiersin.org February 2015 \| Volume 9 \| Article 97 \| 9 FIGURE 5 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “manner” category levels: obstruent and sonorant. FIGURE 6 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “voicing” (i.e., voiced vs. voiceless) and “phonological” (i.e., consonant vs. vowel) categories. FIGURE 5 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “manner” category levels: obstruent and sonorant. FIGURE 5 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “manner” category levels: obstruent and sonorant. FIGURE 6 \| The spatial topography and temporal dynamics are shown in the left and right columns, respectively, for electrode locations with signiﬁcant machine learning classiﬁcation for the “voicing” (i.e., voiced vs. voiceless) and “phonological” (i.e., consonant vs. vowel) categories. Table 5 \| Summary of results for manner of articulation over all sampled electrodes. Manner # Electrodes Peak AI Peak latency Obstruent 10 18.26 85 ms Sonorant 11 28.58 85 ms Table 6 \| Summary of results for voicing and phonological category (vowels only) over all sampled electrodes. Manner # Electrodes Peak AI Peak latency Local maxima Voicing 12 11.45 95 ms –25 ms Vowels 8 21.80 95 ms –105 ms Frontiers in Human Neuroscience www.frontiersin.org February 2015 \| Volume 9 \| Article 97 \| 9 Table 5 \| Summary of results for manner of articulation over all sampled electrodes. Table 5 \| Summary of results for manner of articulation over all sampled electrodes. Table 6 \| Summary of results for voicing and phonological category (vowels only) over all sampled electrodes. Table 6 \| Summary of results for voicing and phonological category (vowels only) over all sampled electrodes. ECoG representations of segmental speech features Lotte et al. 2011; Bouchard et al., 2013). 4. DISCUSSION 4.1. GENERAL COMMENTS Importantly, our task and analy- ses differ from these earlier attempts by ﬁrst considering ﬂuent, continuous speech production of whole sentences and para- graphs, which is more natural than isolated utterances and may account for effects of coarticulation. Second, our signal record- ings come from a much larger area of the cortical surface, which enabled us to investigate all of the lateral (perisylvian) regions involved in the motor, perceptual and planning neuro- logical processing components of speech production. Last, our analysis focuses on the determination of the neurological activ- ity that differentiates speech segments (e.g., phonemes) from one another based on their phonological and articulatory fea- tures. articulators as well as tactile (for closure) and proprioceptive (for constriction) somatosensation. In our analysis, we used the place features labial, coronal and dorsal for discriminating ECoG responses as a result of speech articulation. The sensorimotor interpretation for the labial feature refers to closure of the lips, either against each other (bilabial) or of the lower lip against the maxillary teeth (labiodental), both result from the movement of the lip(s) and/or jaw. Similarly, the sensory interpretation for the feature coronal refers to closures occurring between the tongue, maxilla and hard palate, while the motor interpretation refers to muscular involvement of the tongue tip, tongue body and anterior portions of the tongue body as they contact the teeth (dentals), alveolar ridge (alveolars) and hard palate (palatals). Finally, the sensorimotor interpretation of the dorsal feature refers to a ver- tical and posterior movement of the tongue dorsum for closure against the soft palate, or velum, resulting in the class of velar sounds. Additionally, vowel sounds can be included in the dorsal feature owing to the motor execution requirements of the tongue, but they are not included in any other place category (Hall, 2007), and we do not include them here. The continuous speaking task is doubly advantageous as it allows for acquisition of a large amount of phoneme data in a short amount of time, which is imperative when interacting with patients with an ECoG implant. We are also able to analyze simultaneously overlapping processes of phonological process- ing, execution of articulatory plans and monitoring of acoustic feedback in a manner. 4. DISCUSSION 4.1. GENERAL COMMENTS Our technique of machine learning classi- ﬁcation for discrimination of speech features via ECoG recordings enable direct inference of the neurological structures and dynam- ics that dissociate production of phonemes with varying phono- logical and articulatory characteristics. We discuss the major implications of our results along these themes in the following sections. In general, the neurological structures and dynamics revealed in our study overlapped with many of our expecta- tions (Penﬁeld and Roberts, 1959; Bouchard et al., 2013), but our speciﬁc analyses identiﬁed some striking differences from prior work. Our analysis revealed a network of neurological structures typ- ically involved in speech motor control with auditory feedback exhibiting patterns of ECoG recordings between three top-level place of articulation categories (labial, coronal, dorsal). These regions included speech sensorimotor cortices, premotor cortex, auditory cortex and Wernicke’s area. The combined contribu- tions of all electrodes over the 700 ms time window place cat- egory discrimination indicates a primary role in instantaneous motor execution and sensory processing as evidenced by peak statistically signiﬁcant responses near zero ms latency relative to speech output. These networks are also likely involved in plan- ning and feedback processing as shown by statistically signiﬁcant responses with local AI maxima at speech leading latencies (−300 to 0 ms) and speech lagging latencies (0–300 ms), respectively. The topography over the primary motor and somatosensory cor- tices in Figure 4 provide neurophysiological evidence to support this intuitive interpretation. Further, the presence of overlap- ping sensorimotor locations (deﬁned by electrode placements) suggests the primary motor, premotor and somatosensory cor- tices are all differentially active across various conﬁgurations of the lips and tongue used in speech. The spatial topography also includes perisylvian auditory regions for all feature categories. We interpret these results as representing both prediction of sensory consequences as well as self-perception of vocalized output (e.g., efference motor copy Houde et al., 2002), evidenced by signiﬁcant contributions preceding and following speech onset, respectively. Like the motor production results, the overlapping auditory cor- tical responses between conditions indicate that phonemes yield differential ECoG signals during auditory feedback (cf. Pasley et al., 2012). 4.2.2. Manner of articulation Like place, the manner of articulation also results from muscular contraction of the vocal tract, but is used to describe the quality of vocal airﬂow during speech production. In the present anal- ysis, we focus on two major feature descriptions of phonemes: obstruent and sonorant. Obstruent sounds are characterized by 4.2. MOTOR AND SENSORY PROCESSING Speech articulation is composed of at least two “ﬁrst-order" pro- cesses: motor control and sensory (i.e., acoustic) feedback, whose functionality is typically reﬂected by neural activation of the pre- central gyrus and superior temporal gyrus, respectively. Though both types of processes are certainly involved in speech produc- tion, the relative timing of neural activations, before or following speech, can help to determine whether processing is related to planning and execution of speech sounds (speech-leading) or feedback maintenance (speech-lagging). The design of our analysis procedures allowed us to simultane- ously analyze neural recordings of continuous speech production from two separate perspectives. In the place and voicing analyses, we examine the contribution of neural signals to speciﬁc artic- ulatory gestures (just the larynx in the case of voicing), while in the manner analysis, the motor response is not differentiated. Without examining both, we would have limited the explana- tory potential of the recorded data and miss the observation of a dual-role played by sensory cortex (receptive cortex) in speech production. These results are described in more detail in the following sections. Frontiers in Human Neuroscience 4.2.1. Place of articulation Place of articulation is easily interpreted along motor and somatosensory dimensions. The placement of a vocal tract clo- sure or constriction necessarily involves movement of the speech February 2015 \| Volume 9 \| Article 97 \| 10 Frontiers in Human Neuroscience Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org ECoG representations of segmental speech features Lotte et al. and voicing analyses indicates that there is less overall differen- tial sensorimotor activation between the production of obstruent and sonorant phonemes and those with and without voicing. Interestingly, the location of the manner and voicing sensorimo- tor response is similar to a region recently proposed to represent laryngeal muscular activation during phonation (Brown et al., 2008; Simonyan and Horwitz, 2011). The larynx, with the respi- ratory system, is critical for phonation and generation of acoustic signals in the vocal tract. Our result supports the hypothesis that this region is involved in the planning and execution of laryn- geal movements used to separately produce voiced and voiceless speech. That a putative neural correlate of laryngeal excitation may be useful for discrimination of obstruents from sonorants potentially implicates a fundamental role of the larynx for plan- ning and executing different manners of articulation as well. Last, recent evidence has also shown this region responds to auditory processing during perception of music (Potes et al., 2012). These combined observations suggest that portions of the motor cor- tex may be involved in both motor and auditory processing. With the limited number of subjects meeting our screening criteria, we were unfortunately unable to complete a combined spatio- temporal analysis with the statistical power necessary to precisely determine the role of the sensorimotor activity. Future work with an increased sample size will be required to fully investigate these effects. a blockage of the oral cavity that prohibits sustained voicing, while sonorants facilitate sustained voicing through a relatively open vocal tract. Obstruents include stops (/b/), fricatives (/f/) and affricates (/tS/) while sonorants include nasals (/m/), liquids (/l/), glides (/w/) and vowels. It is possible to examine neuro- logical responses to each of the manner subtypes. However, for this analysis we chose to focus on the top-level categories to boost the feature sample size given our phoneme data taken from continuous speaking of paragraph scripts. 4.3. EXAMINING PHONOLOGICAL DISCRIMINATION 4.3. EXAMINING PHONOLOGICAL DISCRIMINATION We last examined differences in ECoG recordings between pro- duction of consonants and vowels. The category of vowel vs. consonant is mutually exclusive and binary. As seen in Figure 6 (bottom), portions of the speech production and auditory feed- back processing networks are differentially active for production of consonants vs. production of vowels, with similar spatial topography as observed in the analysis of manner and voicing. The similarity between these and our previous manner and voic- ing results is not surprising, as the consonant-vowel, obstruent- sonorant and voiced-voiceless classes encompass nearly the same distribution of phonemes. The main difference between the two features is that certain sonorants are included as consonants, but not obstruents (e.g., nasals, liquids and glides); similarly, some consonants are included in the voiced category largely consist- ing of vowels. The consonant-vowel contrast is represented by a primary peak in statistically signiﬁcant differences in activation indices at +95 ms, with a secondary increase in the range −110 to 0 ms relative to onset of speech output. This bimodal response is different than the observed response for manner and place, and likely reﬂects the complex motor-sensory dynamics involved in the production of all speech sounds, which are particularly inter- twined when considering a higher level, phonological concept. In contrast, the manner feature appears to be solely determined by neural analysis of resulting auditory streams. 4.2.4. Summary of acoustic-articulatory features The sensorimotor contribution for discriminating manner of articulation and voicing is subdued and focal compared to responses in the place of articulation analysis. According to the analysis of place, widespread activity over the precentral gyrus was likely related to discriminating the three classes of articula- tion according to different lip, jaw and tongue conﬁgurations. In contrast, the focal sensorimotor response observed in the manner Frontiers in Human Neuroscience 4.2.3. Voicing V i i ﬂ Voicing reﬂects both the laryngeal muscular contractions needed to conﬁgure the larynx for phonation as well as the acoustic per- ception of phonated speech (i.e., contains vocal fold oscillation). The voicing feature is separated into just two classes, voiced and voiceless, and therefore can be represented in our analysis by a single voicing feature. All of the sonorant sounds used in this analysis are included in the [voiced] feature, as are those obstru- ents that are produced with vocal fold oscillation (e.g., /b/ and /v/). The remaining obstruents are included in the [voiceless] feature. The spatial topography analysis revealed a network of perisyl- vian regions extending into both the motor and auditory cortices, and was similar to the patterns found in the manner condition analysis. The peak response occurred at 95 ms post-vocalization, which suggests that this network is primarily involved in the acoustic perception of voicing in self-produced speech. There is, however, a small pre-vocalization response at −25 ms that may be interpreted as involved in the preparation or execution of laryn- geal commands for initiating (voiced), or preventing (voiceless) vocal fold oscillation. 4.2.1. Place of articulation The spatial topography and temporal dynamics of statistically signiﬁcant differences in neural activity between manner features revealed a network involving the premotor cortex, auditory cor- tex and the posterior superior temporal gyrus (i.e., Wernicke’s area) for obstruent and sonorant features. The perisylvian audi- tory regions were activated to a larger spatial extent compared to the more focal premotor contribution. The temporal dynamics reach peak levels between 65–145 ms following acoustic output of the phoneme and persists throughout the speech production win- dow (up to 300 ms). These observations of spatial and temporal results have three implications: (1) motor and sensory processes are involved in the production of requisite airﬂow for different classes of phonemes (obstruents and sonorants), (2) that dis- criminating auditory feedback of manner is represented over a relatively large region of perisylvian auditory cortex, and (3) the differences in motor production of manner is represented by a focal region of motor cortex. 4.4. MORE FEATURES h d In the present study, we examined differential neural represen- tations of high-level articulatory-acoustic (place and manner of articulation) and phonological characteristics during speech pro- duction. In particular, we focused on the places: labial, coronal February 2015 \| Volume 9 \| Article 97 \| 11 www.frontiersin.org www.frontiersin.org ECoG representations of segmental speech features Lotte et al. and dorsal, the manners: obstruent and sonorant and voicing: voiced and voiceless. As noted previously, the place and manner factors have additional sublevels of increasing reﬁnement (e.g., bilabial and labiodental for place; nasal and fricative for manner). With the present sample size, and the limited amount of time available with each patient, we were not able to examine these additional features. For those factors that we did test, but did not report (e.g., vowel tongue position), we believe that movements of the tongue for vowels are so common to all production attempts that there were no differentially distinguishing features in the ECoG recordings. For results such as these, prior investigations of the overall neural activations found during speech production adequately describe these processes. Future studies with addi- tional subjects and stimuli may help to pick up where this study leaves off. In particular, new studies may optimize speech stimuli selected for representation of as many phonemes and articula- tions as possible, while maintaining low user effort requirements. In addition, it is possible that the electrode size and spacing in this study was too coarse to disambiguate the ﬁne distinctions between all possible features of speech articulation (Blakely et al., 2008; Bouchard et al., 2013). Advances in micro-ECoG (Blakely et al., 2008; Kellis et al., 2010) and additional studies employ- ing such preparations should be able to more comprehensively investigate additional features. sequences have been alternately proposed in the literature to arise from phonetic transcriptions from phonological representa- tions (e.g., phonemes and syllables) (Levelt et al., 1999; Indefrey and Levelt, 2004; Guenther et al., 2006), or theorized as the basis for speech planning and production (e.g., gestural scores) (Saltzman and Munhall, 1989). The present study brings us closer to resolving this debate by ﬁrst determining whether fundamental articulatory features are identiﬁable from electrocorticographic recordings in human subjects. The shift toward articulation changes the paradigm of functional neural analysis toward under- standing invariant motor outputs of language and away from abstract representations of speech motor control (e.g., phonemes, syllables and words). REFERENCES Antoniou, A. (1993). Digital Filters, 2nd Edn. New York, NY: McGraw Hill. Blakely, T., Miller, K. J., Rao, R. P. N., Holmes, M. D., and Ojemann, J. G. (2008). Localization and classiﬁcation of phonemes using high spatial resolution elec- trocorticography (ECoG) grids. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2008, 4964–4967. doi: 10.1109/IEMBS.2008.4650328 Blankertz, B., Lemm, S., Treder, M., Haufe, S., and Müller, K.-R. (2011). Single- trial analysis and classiﬁcation of ERP components–a tutorial. Neuroimage 56, 814–825. doi: 10.1016/j.neuroimage.2010.06.048 Bouchard, K. E., Mesgarani, N., Johnson, K., and Chang, E. F. (2013). Functional organization of human sensorimotor cortex for speech articulation. Nature 495, 327–332. doi: 10.1038/nature11911 Brown, S., Ngan, E., and Liotti, M. (2008). A larynx area in the human motor cortex. Cereb. Cortex 18, 837–845. doi: 10.1093/cercor/bhm131 Brumberg, J. S., Wright, E. J., Andreasen, D. S., Guenther, F. H., and Kennedy, P. R. Brumberg, J. S., Wright, E. J., Andreasen, D. S., Guenther, F. H., and Kennedy, P. R. (2011). Classiﬁcation of intended phoneme production from chronic intracor- tical microelectrode recordings in speech-motor cortex. Front. Neurosci. 5:65. doi: 10.3389/fnins.2011.00065 Chang, E. F., Rieger, J. W., Johnson, K., Berger, M. S., Barbaro, N. M., and Knight, R. T. (2010). Categorical speech representation in human superior temporal gyrus. Nat. Neurosci. 13, 1428–1432. doi: 10.1038/nn.2641 Crone, N. E., Boatman, D., Gordon, B., and Hao, L. (2001). Induced electrocor- ticographic gamma activity during auditory perception. Clin. Neurophysiol. 112, 565–582. doi: 10.1016/S1388-2457(00)00545-9 Edwards, E., Nagarajan, S. S., Dalal, S. S., Canolty, R. T., Kirsch, H. E., Barbaro, N. M., et al. (2010). Spatiotemporal imaging of cortical activation during verb generation and picture naming. Neuroimage 50, 291–301. doi: 10.1016/j.neuroimage.2009.12.035 AUTHOR CONTRIBUTIONS GS designed the research. AR, PB and AG conducted the research protocol. FL and CG contributed data analysis. JB and GS contributed to the interpretation of the results. FL, JB and GS wrote the paper. Frontiers in Human Neuroscience ACKNOWLEDGMENTS This work was supported by grants from the US Army Research Ofﬁce (W911NF-07-1-0415, W911NF-08-1-0216, W911NF-12- 1-0109), the NIH/NIBIB (EB006356 and EB000856) and the NIH/NIDCD (R03-DC011304). The authors would like to thank Dr. Cheung Chi Leung, Dr. Bin Ma, Christian Herff and Dr. Haihong Zhang for their help in the segmentation of the audio signal. 4.5. POTENTIAL APPLICATIONS FOR BRAIN-COMPUTER INTERFACING Another guiding principle of this work concerned potential appli- cation to a neural speech prosthesis, which can interpret brain activity for generating speech output, or a “brain-to-text” device. Our techniques are directly applicable to a motor-speech brain- computer interface (BCI) as the major observations were all based on machine learning classiﬁcation of speech sounds, which is alternately known as prediction or decoding. Martin and col- leagues (Martin et al., 2014) have recently developed a similar method that attempts to predict actual speech acoustic output from recorded ECoG signals. Our work is distinguished from the Martin et al. technique by the adoption of articulatory gestures as the classiﬁcation basis as opposed to direct acoustic predic- tion. However, both methods are advantageous as they limit the required classiﬁcation dictionary (cf. thousands of words needed for word prediction vs. a dozen of articulatory features or acoustic bases) and offer a generative means for word and sentence pre- diction. In other words, by classifying or predicting a small set of place, manner, voicing and phonological features, it is possi- ble to represent any phoneme, combinations of phonemes (i.e., syllables, words), or even sentences. By considering continuous speech, our methods are also capable of keeping pace with speak- ing rates observed during natural communication, which would be a marked advancement in the ﬁeld of augmentative and alter- native communication as well as brain-computer interfacing. In contrast, classifying individual discrete words from brain signals would require a prohibitively large data set to select the correct word from the thousands of words used in language. 4.4. MORE FEATURES h d The combined analysis of motor sequences and phonological representations will provide the requisite means for conﬁrming or rejecting these two different theories of speech production. ECoG representations of segmental speech features ECoG representations of segmental speech features Lotte et al. Guenther, F. H., Ghosh, S. S., and Tourville, J. A. (2006). Neural modeling and imaging of the cortical interactions underlying syllable production. Brain Lang. 96, 280–301. doi: 10.1016/j.patrec.2005.10.010 Pasley, B. N., David, S. V., Mesgarani, N., Flinker, A., Shamma, S. A., Crone, N. E., et al. (2012). Reconstructing speech from human auditory cortex. PLoS Biol. 10:e1001251. doi: 10.1371/journal.pbio.1001251 j Hall, T. A. (2007). “Segmental features,” in The Cambridge Handbook of Phonology, ed P. de Lacy (New York, NY: Cambridge University Press), 324. j Pei, X., Barbour, D. L., Leuthardt, E. C., and Schalk, G. (2011a). 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Kellis, S., Miller, K., Thomson, K., Brown, R., House, P., and Greger, B. (2010). ECoG representations of segmental speech features Decoding spoken words using local ﬁeld potentials recorded from the cortical surface. J. Neural Eng. 7:056007. doi: 10.1088/1741-2560/7/5/056007 Recasens, D. (1999). “Lingual coarticulation,” in Coarticulation: Theory, Data and Techniques, eds W. J. Hardcastle and N. Hewlett (Cambridge University Press), 80–104. Kubanek, J., and Schalk, G. (2014). NeuralAct: a tool to visualize electrocortical (ECoG) activity on a three-dimensional model of the cortex. Neuroinformatics doi: 10.1007/s12021-014-9252-3. [Epub ahead of print]. Riès, S., Janssen, N., Burle, B., and Alario, F.-X. (2013). Response-locked brain dynamics of word production. PloS ONE 8:e58197. doi: 10.1371/jour- nal.pone.0058197 Lancaster, J. L., Woldorff, M. G., Parsons, L. M., Liotti, M., Freitas, C. S., Rainey, L., et al. (2000). Automated Talairach atlas labels for func- tional brain mapping. Hum. Brain Mapp. 10, 120–131. doi: 10.1002/1097- 0193(200007)10:3<120::AID-HBM30>3.0.CO;2-8 Saltzman, E. L., and Munhall, K. G. (1989). A dynamical Approach to gestural patterning in speech production. Ecol. Psychol. 1, 333–382. doi: 10.1207/s15326969eco0104/2 Schalk, G., McFarland, D. J., Hinterberger, T., Birbaumer, N., and Wolpaw, J. R. (2004). BCI2000: a general-purpose brain-computer interface (BCI) system. IEEE Trans. Biomed. Eng. 51, 1034–1043. doi: 10.1109/TBME.2004.827072 Ledoit, O., and Wolf, M. (2004). A well-conditioned estimator for large- dimensional covariance matrices. J. Multivariate Anal. 88, 365–411. doi: 10.1016/S0047-259X(03)00096-4 Leuthardt, E. C., Pei, X.-M., Breshears, J., Gaona, C., Sharma, M., Freudenberg, Z., et al. (2012). Temporal evolution of gamma activity in human cortex dur- ing an overt and covert word repetition task. Front. Hum. Neurosci. 6:99. doi: 10.3389/fnhum.2012.00099 Simonyan, K., and Horwitz, B. (2011). Laryngeal motor cortex and control of speech in humans. Neuroscientist 17, 197–208. doi: 10.1177/1073858410386727 Towle, V. L., Yoon, H. A., Castelle, M., Edgar, J. C., Biassou, N. M., Frim, D. M., et al. (2008). ECoG gamma activity during a language task: differenti- ating expressive and receptive speech areas. Brain 131(Pt 8), 2013–2027. doi: 10.1093/brain/awn147 Levelt, W. J. M., Roelofs, A., and Meyer, A. S. (1999). A theory of lexical access in speech production. Behav. Brain Sci. 22, 1–75. doi: 10.1017/S0140525X99001776 Young, S., Evermann, G., Gales, M., Hain, T., Kershaw, D., Liu, X., et al. (2006). The HTK Book (for HTK Version 3.4). Cambridge University Engineering Department. Lotte, F., Congedo, M., Lécuyer, A., Lamarche, F., and Arnaldi, B. (2007). A review of classiﬁcation algorithms for EEG-based brain-computer interfaces. J. Neural Eng. 4, R1–R13. Noble, W. (2009). How does multiple testing correction work? Nat. Biotechnol. 27, 1135–1137. doi: 10.1038/nbt1209-1135 5. CONCLUSIONS In the present study, we examined speech production in the human brain as a sequence of articulatory movements. These Fawcett, T. (2006). An introduction to ROC analysis. Pattern Recognit. Lett. 27, 861– 874. doi: 10.1016/j.patrec.2005.10.010 Frontiers in Human Neuroscience February 2015 \| Volume 9 \| Article 97 \| 12 www.frontiersin.org 12 ECoG representations of segmental speech features doi: 10.1088/1741-2560/4/2/R01 Conﬂict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Lotte, F., and Guan, C. (2010). “Learning from other subjects helps reduc- ing brain-computer interface calibration time,” in International Conference on Audio, Speech and Signal Processing (ICASSP’2010), 614–617. doi: 10.1109/ICASSP.2010.5495183 Received: 18 September 2014; accepted: 06 February 2015; published online: 24 February 2015. Citation: Lotte F, Brumberg JS, Brunner P, Gunduz A, Ritaccio AL, Guan C and Schalk G (2015) Electrocorticographic representations of segmental features in continuous speech. Front. Hum. Neurosci. 9:97. doi: 10.3389/fnhum.2015.00097 This article was submitted to the journal Frontiers in Human Neuroscience. Copyright © 2015 Lotte, Brumberg, Brunner, Gunduz, Ritaccio, Guan and Schalk. 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. Received: 18 September 2014; accepted: 06 February 2015; published online: 24 February 2015. Martin, S., Brunner, P., Holdgraf, C., Heinze, H.-J., Crone, N. E., Rieger, J., et al. (2014). Decoding spectrotemporal features of overt and covert speech from the human cortex. Front. Neuroengineering 7:14. doi: 10.3389/fneng.2014.00014 Citation: Lotte F, Brumberg JS, Brunner P, Gunduz A, Ritaccio AL, Guan C and Schalk G (2015) Electrocorticographic representations of segmental features in continuous speech. Front. Hum. Neurosci. 9:97. doi: 10.3389/fnhum.2015.00097 Mellinger, J., and Schalk, G. (2010). “Brain-computer interfaces,” in Brain- Computer Interfaces, eds B. Graimann, G. Pfurtscheller, and B. Allison (Berlin;Heidelberg: Springer Berlin Heidelberg), 259–279. This article was submitted to the journal Frontiers in Human Neuroscience. Mugler, E. M., Patton, J. L., Flint, R. D., Wright, Z. A., Schuele, S. U., Rosenow, J., et al. (2014). Direct classiﬁcation of all American english phonemes using signals from functional speech motor cortex. J. Neural Eng. 11:035015. doi: 10.1088/1741-2560/11/3/035015 Copyright © 2015 Lotte, Brumberg, Brunner, Gunduz, Ritaccio, Guan and Schalk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). ECoG representations of segmental speech features 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. Noble, W. (2009). How does multiple testing correction work? Nat. Biotechnol. 27, 1135–1137. doi: 10.1038/nbt1209-1135 February 2015 \| Volume 9 \| Article 97 \| 13 Frontiers in Human Neuroscience www.frontiersin.org
https://openalex.org/W3092528480	https://zenodo.org/record/3872869/files/Lodola%20et%20al.%20-%202019%20-%20Conjugated%20polymers%20optically%20regulate%20the%20fate%20of.pdf	English	null	Conjugated polymers optically regulate the fate of endothelial colony forming cells	Vascular pharmacology	2,020	cc-by	15,676	INTRODUCTION I would be key to regenerative medicine and drug screening. The pres- ently dominant approaches to reliably regulate stem and progenitor cell fate for regenerative purposes mainly rely on the use of chemical cues. However, irreversibility and lack of spatial selectivity represent important limitations of these methods. Whenever targeting in vivo applications, one must face the major, unsolved problem of diffusion of neurotrophic molecules by the conventional intravenous or oral routes. In addition, the therapeutic outcome of autologous cell-based therapy is often impaired by low engraftment, survival, and poor inte- gration of stem cells within the environment of the targeted tissue. Other stimuli, mainly consisting of mechanical and electrical cues, were recently reported to have some notable effects, and recent advances in nanotechnology and material science enabled versatile, robust, and larger-scale modulation of the cell fate. In particular, carbon-based materials and conjugated polymers led to interesting results (18). However, their distinctive visible light absorption was never exploited in optically driven techniques. In recent years, organic semiconductors have emerged as highly promising materials in biotechnology, thanks to several key-enabling features. Differently from silicon-based electronics, they support both electronic and ionic charge transport (1); they can be easily functiona- lized with specific excitation and sensing capabilities (2–4); and they are solution processable, soft, and conformable (5). They are highly biocompatible, being suitable for in vivo implantation and long-term operation, as recently reported for many different applications, in- cluding electrocorticography, precise delivery of neurotransmitters, electrocardiography, deep brain stimulation, and spinal cord injury (6–9). An important, distinctive feature of organic semiconductors is their sensitivity to the visible and near-infrared light. Recently, our and other groups have exploited it for optical modulation of cell electrophysiological activity, by using conjugated polymers and or- ganic molecules as exogenous light-sensitive actuators (10–12). Interesting applications have been reported in the field of artificial vi- sual prosthesis (5), photothermal excitation or inhibition of cellular activity (13, 14), and modulation of animal behavior (15). Use of light actuation has been proposed either by viral transfer of light-sensitive proteins, by optogenetics tools, or by absorption of endogenously expressed light-sensitive moieties, based on low light–level therapies (19–21). In the first case, interesting results were obtained (22); however, this approach bears all the drawbacks related to the need for viral gene transfer. Conjugated polymers optically regulate the fate of endothelial colony-forming cells F. Lodola1†, V. Rosti2†, G. Tullii1,3, A. Desii1‡§, L. Tapella4, P. Catarsi2, D. Lim4, F. Moccia5\|\|, M. R. Antognazza1\|\| The control of stem and progenitor cell fate is emerging as a compelling urgency for regenerative medicine. Here, we propose a innovative strategy to gain optical control of endothelial colony-forming cell fate, which represents the only known truly endothelial precursor showing robust in vitro proliferation and overwhelming vessel formation in vivo. We combine conjugated polymers, used as photo-actuators, with the advantages offered by optical stimulation over current electromechanical and chemical stimulation approaches. Light mod- ulation provides unprecedented spatial and temporal resolution, permitting at the same time lower invasiveness and higher selectivity. We demonstrate that polymer-mediated optical excitation induces a robust enhancement of proliferation and lumen formation in vitro. We identify the underlying biophysical pathway as due to light-induced activation of TRPV1 channel. Altogether, our results represent an effective way to induce angiogenesis in vitro, which represents the proof of principle to improve the outcome of autologous cell-based therapy in vivo. A P P L I ED S C I E N C E S A N D EN G I N E E R I N G Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to originalU.S.Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E INTRODUCTION I Intracellular Ca2+ signals play a crucial role in stimulating ECFC proliferation and tubulogenesis by promoting the nuclear trans- location of the Ca2+-sensitive nuclear transcription factor kB (NF-kB) (26–28). It has, therefore, been suggested that intracellular Ca2+ signaling could be targeted to boost the regenerative potential of autol- ogous ECFCs for regenerative purposes (29). For the above-mentioned reasons, ECFCs represent a valuable test bed model for assessing the possibility to exploit the visible light sensitivity of conjugated polymers to gain touchless, optical modulation of cell proliferation and function. y g ECFC proliferation on polymer substrates has been primarily assessed in dark conditions at three different time points, namely, 24, 48, and 72 hours after plating (Fig. 1B). Polymer-coated samples, while showing from the very beginning a slightly lower number of cells as compared with control substrates, exhibit a proliferation rate fully similar to cells plated on glass substrates (slope of the linear fitting is 0.034 ± 0.003, R2 = 0.99 and 0.034 ± 0.005, R2 = 0.96 for control and P3HT polymer samples, respectively). g p p In this framework, we demonstrate that polymer-mediated optical excitation during the first steps of ECFC growth leads to a robust en- hancement of both proliferation and tubulogenesis through the optical modulation of the Ca2+-permeable transient receptorpotential vanilloid 1 (TRPV1) channel and NF-kB–mediated gene expression. Our results represent, to the best of our knowledge, the first report on the use of polymer photoexcitation for the in vitro modulation of ECFC fate and function, thereby representing the proof of principle to obtain di- rect control of progenitor cell fate. p y p p y Once assessed that the P3HT polymer surface represents a nicely biocompatible substrate for ECFC seeding and proliferation in the dark, we moved to investigate the effect of polymer photoexcitation. In more detail, to evaluate the effect of optical stimulation on cell proliferation and network formation, we continuously shined light for the whole temporal window required for cell growth, and we rea- lized an ad hoc system suitable for operation within the cell incuba- tor. The experimental configuration and the excitation protocol are schematically represented in Fig. 1C. Optical excitation is provided by a light-emitting diode (LED) source, with maximum emission wavelength at 525 nm, incident from the substrate side. RESULTS Realization of poly(3-hexyl-thiophene) polymer/ECFC culture interfaces, viability assay, and optical excitation protocol INTRODUCTION I Photobiomodulation led to interest- ing outputs as well, but overall efficiency is hampered by the limited absorption of light-responsive molecules endogenously expressed in living cells. In this framework, the opportunity to use polymer-based photo- transduction mechanisms to regulate the very early stages of living cell development has been very scarcely considered (16, 17). The pos- sibility to selectively and precisely regulate a number of cell pro- cesses, such as adhesion, differentiation, proliferation, and migration, In this work, we propose to couple the use of conjugated polymers with visible light excitation to gain optical control of cell fate. We focus our attention on endothelial progenitor cells (EPCs) and, in particular, on endothelial colony-forming cells (ECFCs), which are currently considered the bona fide best surrogate of EPCs (23). ECFCs are mobilized from the bone marrow and vascular stem cell niche to reconstruct the vascular network destroyed by an ischemic insult and to restore local blood perfusion (24). ECFCs may be easily harvested from peripheral blood, display robust clonogenic potential, exhibit tube-forming capacity in vitro, and generate vessel-like struc- tures in vivo (24, 25), thereby representing a promising candidate for autologous cell-based therapy of ischemic disorders (24). Manipulating 1Center for Nano Science and Technology @PoliMi, Istituto Italiano di Tecnologia, via Pascoli 70/3, 20133 Milano, Italy. 2Center for the Study of Myelofibrosis, Laboratory of Biochemistry, Biotechnology and Advanced Diagnosis, IRCCS Policlinico San Matteo Foundation, Pavia, Italy. 3Department of Physics, Politecnico di Milano, Piazza L. Da Vinci 32,20133Milano,Italy. 4DepartmentofPharmaceuticalSciences,UniversitàdelPiemonte Orientale “Amedeo Avogadro,” Novara, Italy. 5Laboratory of General Physiology, Depart- ment of Biology and Biotechnology “Lazzaro Spallanzani,” University of Pavia, Pavia, Italy. Corresponding author. Email: francesco.lodola@iit.it (F.L.); mariarosa.antognazza@iit.it (M.R.A.) ‡Present address: Dipartimento di Ingegneria Meccanica e Aerospaziale, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy. §Present address: Smart Bio-Interfaces Italian Institute of Technology, Viale Rinaldo Piaggio 34, 56025 Pontedera (PI), Italy. §Present address: Smart Bio-Interfaces Italian Institute of Technology, Viale Rinaldo Piaggio 34, 56025 Pontedera (PI), Italy. 1 of 12 1 of 12 Lodola et al., Sci. Adv. Realization of poly(3-hexyl-thiophene) polymer/ECFC culture interfaces, viability assay, and optical excitation protocol on October 8, 2019 ances.sciencemag.org/ Figure 1A shows a sketch of the bio/polymer interface developed for obtaining optical control of ECFC proliferation and network for- mation, together with the polymer chemical structure and the optical absorption spectrum. The material of choice for light absorption and phototransduction is a workhorse organic semiconductor, widely used in photovoltaic and photodetection applications, namely, re- gioregular poly(3-hexyl-thiophene) (P3HT) (6). It is characterized by a broad optical absorption spectrum, in the blue-green visible region, peaking at 520 nm. P3HT outstanding biocompatibility properties have been reported in a number of different systems, both in vitro and in vivo, including astrocytes (30), primary neurons and brain slices (14), and invertebrate models of Hydra vulgaris (15). Chronical implantation y p The temporally precise and spatially localized measurement of the temperature variation upon polymer photoexcitation at the polymer/ cell interface (i.e., within the cell cleft) is not straightforward because Fig. 1. Polymer devices for optical stimulation of ECFC cultures. (A) P3HT polymer optical absorption spectrum. Insets show the chemical structure of the conjugated polymer and a sketch of the polymer device used for cell optical activation. ECFCs are cultured on top of P3HT thin films, deposited on glass substrates. (B) ECFC viability at fixed time points after plating (24, 48, and 72 hours). Cell cultures were kept in dark conditions at controlled temperature (37°C) and fixed CO2 levels (5%). No statistically significant difference was observed between the glass and polymer substrates at any fixed time point (unpaired Student’s t test). (C) Experimental setup and optical excitation protocol for evaluation of polymer-mediated cell photoexcitation effects on cell fate. Polymer and control samples are positioned within a sterilized, home-designed petri holder. Light scattering effects are completely screened. The geometry and the photoexcitation protocol have been implemented to minimize overheating effects and to keep the overall extracellular bath temperature fairly unaltered. Thirty-millisecond-long green light pulses are followed by 70 ms in dark condition. L d l t l S i Ad 2019 5 4620 27 S t b 2019 2 f 12 Fig. 1. Polymer devices for optical stimulation of ECFC cultures. (A) P3HT polymer optical absorption spectrum. Insets show the chemical structure of the conjugated polymer and a sketch of the polymer device used for cell optical activation. ECFCs are cultured on top of P3HT thin films, deposited on glass substrates. INTRODUCTION I The choice of the protocol, continuously administered to the cell cultures during early seeding and proliferation stages, has been mainly dictated by the need to avoid overheating effects, with possible negative out- comes on the overall cell culture viability. On the basis of these con- siderations, we opted for a protocol based on 30-ms excitation pulses, followed by a 70-ms dark condition, at a photoexcitation den- sity of 40 mW/cm2. The whole protocol is continuously repeated for a minimum of 4 up to 36 hours, depending on the type of functional assay, at controlled temperature (37°C) and CO2 levels (5%). INTRODUCTION I 2019;5:eaav4620 27 September 2019 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E the signaling pathways that drive ECFC proliferation, migration, differ- entiation, and tubulogenesis could represent a reliable strategy to im- prove the regenerative outcome of therapeutic angiogenesis in the harsh microenvironment of an ischemic tissue, such as the infarcted heart (24, 25). Intracellular Ca2+ signals play a crucial role in stimulating ECFC proliferation and tubulogenesis by promoting the nuclear trans- location of the Ca2+-sensitive nuclear transcription factor kB (NF-kB) (26–28). It has, therefore, been suggested that intracellular Ca2+ signaling could be targeted to boost the regenerative potential of autol- ogous ECFCs for regenerative purposes (29). For the above-mentioned reasons, ECFCs represent a valuable test bed model for assessing the possibility to exploit the visible light sensitivity of conjugated polymers to gain touchless, optical modulation of cell proliferation and function. In this framework, we demonstrate that polymer-mediated optical excitation during the first steps of ECFC growth leads to a robust en- hancement of both proliferation and tubulogenesis through the optical modulation of the Ca2+-permeable transient receptorpotential vanilloid 1 (TRPV1) channel and NF-kB–mediated gene expression. Our results represent, to the best of our knowledge, the first report on the use of polymer photoexcitation for the in vitro modulation of ECFC fate and function, thereby representing the proof of principle to obtain di- rect control of progenitor cell fate. of P3HT-based devices in the rat subretinal space did not show substan- tial inflammatory reactions up to 6 months in vivo (10). Here, polymer thin films (approximate thickness, 150 nm) have been deposited by spin coating on top of polished glass substrates, as detailed in Materials and Methods. Both polymer-coated and glass substrates have been ther- mally sterilized (120°C, 2 hours), coated with fibronectin, and, lastly, used as light-sensitive and control cell culturing substrates, respectively. ECFCs have been isolated from peripheral blood samples of human vo- lunteers and seeded on top of polymer and glass substrates. the signaling pathways that drive ECFC proliferation, migration, differ- entiation, and tubulogenesis could represent a reliable strategy to im- prove the regenerative outcome of therapeutic angiogenesis in the harsh microenvironment of an ischemic tissue, such as the infarcted heart (24, 25). Realization of poly(3-hexyl-thiophene) polymer/ECFC culture interfaces, viability assay, and optical excitation protocol (B) ECFC viability at fixed time points after plating (24, 48, and 72 hours). Cell cultures were kept in dark conditions at controlled temperature (37°C) and fixed CO2 levels (5%). No statistically significant difference was observed between the glass and polymer substrates at any fixed time point (unpaired Student’s t test). (C) Experimental setup and optical excitation protocol for evaluation of polymer-mediated cell photoexcitation effects on cell fate. Polymer and control samples are positioned within a sterilized, home-designed petri holder. Light scattering effects are completely screened. The geometry and the photoexcitation protocol have been implemented to minimize overheating effects and to keep the overall extracellular bath temperature fairly unaltered. Thirty-millisecond-long green light pulses are followed by 70 ms in dark condition. Lodola et al., Sci. Adv. 2019;5:eaav4620 27 September 2019 2 of 12 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E S C I E N C E A D V A N C E S but kept in dark conditions for the whole duration of the experiment (+158% versus P3HT dark; P < 0.05). No statistically significant difference in proliferation was observed among cells seeded on glass, whether they were subjected to optical excitation or not (Fig. 2A). it requires the use of localized, submicrometer probes with a fast re- sponse time. However, according to the heat diffusion equation, we ex- pect that dissipation occurs within a few milliseconds, following exponential decrease dynamics (14). Moreover, we used the well- known method of the calibrated pipette (31) to characterize the tem- perature variation dynamics within the extracellular bath volume, defined by the cylinder with the base area corresponding to the light spot size and the height of about 1 mm. This choice is a good approx- imation of the overall volume occupied by a single ECFC cell; thus, it provides a realistic estimation of the average heating experienced by the cell (fig. S1A). We observe that temperature variation closely follows short optical pulse dynamics, reaching a maximum tempera- ture at the end of the 30-ms illumination period, quickly followed by an almost complete thermal relaxation to the basal temperature during the 70-ms-long dark period. Realization of poly(3-hexyl-thiophene) polymer/ECFC culture interfaces, viability assay, and optical excitation protocol We conclude that our polymer-based system provides a highly spatially and temporally resolved method for optical excitation, making it possible, in perspective, to selectively target single cells and even cell subcompartments. Upon prolonged illumination (hours), one should also consider possible overheating effects of the whole extracellular medium volume. The average tem- perature of the bath for the entire duration of the long-term experiment was measured by a thermocouple immersed in the medium. Data show that an equilibrium situation is established after 5 hours and that the absolute temperature of the bath is increased by about 1.5° (fig. S1B). The adopted prolonged excitation protocol does not negatively affect overall cell culture viability (see below). y j g Recent evidence demonstrated an interesting correlation between processes key to ECFC vascular regeneration, including proliferation and network formation, and activation of TRPV1 channels, which are expected to be endogenously expressed in ECFCs (32). In addi- tion, we recently reported that polymer photoexcitation leads to se- lective TRPV1 activation in transfected human embryonic kidney (HEK) cell models (33). Therefore, we were prompted to evaluate whether the increase in cell proliferation is distinctively determined by a polymer-mediated photoactivation of the TRPV1 channel. To this goal, we preliminarily checked the actual expression of the TRPV1 channel in the ECFC models by carrying out electrophysiology experiments in patch-clamp configuration. Methods and results are ex- tensively discussed in the Supplementary Materials (fig. S2 and related description). Briefly, the expression of the TRPV1 channel was con- firmed, as well as the capability to selectively excite its activity through localized polymer excitation at high optical power density. To establish whether the TRPV1 channel also has a role in the observed increase in cell proliferation upon polymer excitation, we performed the ex- periments under light illumination upon administration of a highly specific TRPV1 antagonist [capsazepine (CPZ), 10 mM], an aspecific TRPV channel inhibitor [ruthenium red (RR), 10 mM], and a selec- tive antagonist of a different temperature-sensitive channel, TRPV4, which is also endogenously expressed in ECFCs (RN-1734, 20 mM) (34) (Fig. 2B). TRPV1 inactivation by CPZ and RR results in a relative, strong reduction in cell proliferation by 51 and 30%, respectively, as compared with untreated cells. Conversely, in the case of RN-1734 treatment, the proliferation increase due to polymer photoexcitation is completely unaltered. Polymer optical excitation drives ECFC proliferation and in vitro tubulogenesis Figure 2 reports specific effects mediated by P3HT substrates and visible light stimulation on ECFC proliferation. ECFCs were plated in the presence of EGM-2 medium to facilitate the adhesion to the substrate. After 12 hours, the medium was switched to EBM-2 sup- plemented with 2% fetal bovine serum, and the cells were subjected to the long-term lighting protocol for 36 hours at controlled tem- perature (37°C) and CO2 levels (5%). Under these conditions, ECFCs seeded on P3HT and subjected to light stimulation undergo a significant increase in proliferation rate, as compared with the con- trol condition, i.e., to cells also seeded on P3HT polymer substrates y As mentioned earlier, intracellular Ca2+ signaling has been re- ported to drive ECFC proliferation (26, 28). To further investigate whether TRPV1-mediated extracellular Ca2+ entry mediates the proangiogenic response to light illumination, we pretreated ECFCs with [1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) BAPTA-AM] (30 mM), a membrane- permeable buffer of intracellular Ca2+ levels (26, 28). BAPTA-AM is Fig. 2. Polymer-mediated optical activation of TRPV1 stimulates proliferation in ECFCs. (A) Relative variation of the proliferation rate of ECFCs subjected to long- term optical excitation seeded on both bare glass and P3HT thin films, together with corresponding control samples kept in dark conditions. Cell proliferation was measured after 36 hours of culture in the presence of EBM-2 supplemented with 2% fetal calf serum. (B) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence (CTRL) and presence of 10 mM capsazepine (CPZ), 10 mM ruthenium red (RR), 20 mM RN-1734 (RN-1734), and 30 mM BAPTA-AM (BAPTA). The results are represented as the means ± standard error of the mean (SEM) of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way analysis of variance (ANOVA) coupled with Tukey (A) or Dunnett’s (B) post hoc test. P < 0.05. L d l l S i Ad 2019 5 4620 27 S b 2019 3 f 12 Fig. 2. Polymer-mediated optical activation of TRPV1 stimulates proliferation in ECFCs. (A) Relative variation of the proliferation rate of ECFCs subjected to long- term optical excitation seeded on both bare glass and P3HT thin films, together with corresponding control samples kept in dark conditions. Cell proliferation was measured after 36 hours of culture in the presence of EBM-2 supplemented with 2% fetal calf serum. Polymer optical excitation drives ECFC proliferation and in vitro tubulogenesis (B) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence (CTRL) and presence of 10 mM capsazepine (CPZ), 10 mM ruthenium red (RR), 20 mM RN-1734 (RN-1734), and 30 mM BAPTA-AM (BAPTA). The results are represented as the means ± standard error of the mean (SEM) of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way analysis of variance (ANOVA) coupled with Tukey (A) or Dunnett’s (B) post hoc test. P < 0.05. Lodola et al., Sci. Adv. 2019;5:eaav4620 27 September 2019 3 of 12 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E master segments (Fig. 3F), master junctions (Fig. 3G), and meshes (Fig. 3H). In all cases, a notable, statistically relevant difference is ob- served between cells subjected to polymer-mediated optical excitation and controls. Within the same considered temporal window, the com- bined use of polymer substrates and visible light stimuli does not lead to sizable toxicity effects or delays in cell proliferation. Conversely, it leads to enhanced cell proliferation (Fig. 2) and allows the achievement of the formation of a more extended and mature tubular network (Fig. 3). widely used to prevent the increase in intracellular Ca2+ concentration ([Ca2+]i) induced by extracellular stimuli and inhibits the downstream Ca2+-dependent processes. For instance, BAPTA-AM represents the most suitable tool to prevent the activation of Ca2+-sensitive decoders residing within tens of nanometers from the inner pore of plasmalem- mal Ca2+ channels (35). It was recently reported that, in the absence of Ca2+-mobilizing growth factors, it does not impair the low rate of ECFC growth(27). Here,however,BAPTA-AMclearly reducedthe light-driven proliferation increase, thus confirming that TRPV1 stimulates ECFCs through an increase in [Ca2+]i (Fig. 2B). ( g ) As evidenced for the proliferation rate, the TRPV1 channel activa- tion emerges to play also a fundamental role in tubulogenesis (Fig. 4). The TRPV1 pharmacological blockade with the specific inhibitor CPZ deterministically leads to a marked reduction in network formation (Fig. 4A). Upon CPZ administration, a statistically significant decrease in the relative variation of the number of master segments (Fig. 4E), master junctions (Fig. 4F), and meshes (Fig. 4G) is observed. In line with the results shown in Figs. Polymer optical excitation drives ECFC proliferation and in vitro tubulogenesis 2 and 3, RR administration resulted in a less marked but still sizable reduction in the tubular network (Fig. 4, B and E to G), probably due to the minor specificity toward TRPV1, while the protubular effect of light remained fully unaltered in the presence of the TRPV4inhibitor RN-1734(Fig. 4, Cand Eto G). Notably, the treatment with BAPTA-AM (30 mM), which affected ECFC proliferation, was able to prevent also in vitro tubulogenesis, thus corroborating the key role of intracellular Ca2+ signaling in the proangiogenic response to light illu- mination (Fig. 4, D and E to G). Control measurements carried out in dark conditions on polymer substrates upon the considered pharmaco- logical treatments do not show any relevant effect (fig. S4, A to C). Over- all, this evidence supports the notion that TRPV1 stimulates ECFC proliferation and network formation and demonstrates that optical ex- citation, properly mediated by biocompatible polymer substrates, pos- itively affects ECFC fate by spatially and temporally selective activation of the TRPV1 channel. g [ ]i ( g ) We further examined the physiological outcome of chronic light stimulation by carrying out a tube formation assay within an extra- cellular matrix protein-based scaffold, which is a surrogate of the basement membrane extracellular matrix. This assay recapitulates many steps of the angiogenic process, including adhesion, migration, protease activity, and tubule formation (27, 28). ECFCs were plated in the presence of EBM-2 medium supplemented with 2% fetal calf serum and subjected to the long-term lighting protocol for 8 hours at controlled levels of temperature and CO2. Control experiments carried out in dark conditions, either onto glass (see Fig. 3A for a representative optical image) or onto polymer substrates (Fig. 3C), as well as control experiments carried out upon photoexcitation of cells seeded on glass substrates (Fig. 3B), do not show remarkable differences. Conversely, ECFC cultures subjected to polymer-mediated optical excitation clearly tend to assemble into an extended bidimensional capillary-like network (Fig. 3D). Cell cultures were monitored up to 24 hours after illumina- tion onset, but results were comparable to observations reported here, after 8 hours of illumination. This qualitative observation is fully con- firmed by quantitative morphological analysis (27). As depicted in the sketch of Fig. 3E, we quantitatively evaluated the main features typical of the capillary-like network formation and, in particular, the number of Fig. 3. Light-induced photoexcitation stimulates tubulogenesis in ECFC cultures. Polymer optical excitation drives ECFC proliferation and in vitro tubulogenesis (A to D) Representative images of in vitro tubular networks of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT, as well as on corresponding control samples in dark conditions. Cultures were observed up to 24 hours, but their appearance did not substantially change after pictures were taken after 8-hour culture. Scale bars, 250 mm. (E) Sketch representing the main features typical of the capillary-like network that were considered for the topologic analysis. Number of master segments (F), master junctions (G), and meshes (H) analyzed in the different conditions. The results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Tukey post hoc test. P < 0.01 and P < 0.001. Lodola et al Sci Adv 2019;5:eaav4620 27 September 2019 4 of 12 Fig. 3. Light-induced photoexcitation stimulates tubulogenesis in ECFC cultures. (A to D) Representative images of in vitro tubular networks of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT, as well as on corresponding control samples in dark conditions. Cultures were observed up to 24 hours, but their appearance did not substantially change after pictures were taken after 8-hour culture. Scale bars, 250 mm. (E) Sketch representing the main features typical of the capillary-like network that were considered for the topologic analysis. Number of master segments (F), master junctions (G), and meshes (H) analyzed in the different conditions. The results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Tukey post hoc test. P < 0.01 and **P < 0.001. Fig. 3. Light-induced photoexcitation stimulates tubulogenesis in ECFC cultures. (A to D) Representative images of in vitro tubular networks of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT, as well as on corresponding control samples in dark conditions. Cultures were observed up to 24 hours, but their appearance did not substantially change after pictures were taken after 8-hour culture. Scale bars, 250 mm. (E) Sketch representing the main features typical of the capillary-like network that were considered for the topologic analysis. Number of master segments (F), master junctions (G), and meshes (H) analyzed in the different conditions. Phototransduction mechanism material of choice is a photoresist (MicroPosit S1813). S1813 thin films are realized by spin coating, and deposition parameters are op- timized to obtain optical absorbance values similar to the semi- conducting polymer samples at the considered excitation wavelength. The capability of photoresist substrates to sustain ECFC proliferation was successfully assessed in a control measurement, obtaining fully comparable results with respect to the P3HT substrates (Fig. 5A). The functional effect eventually driven by photoresist optical excitation on tubulogenesis was then investigated by using the same experimental conditions and analysis technique previously adopted for polymer and glass substrates (Fig. 5B). Data show that long-term photoresist ex- citation does not lead to sizable enhancement of the cellular network formation, thus pointing out that a purely photothermal effect does not play a major role in boosting the tubulogenesis process at variance with semiconducting polymer substrates. In a complementary experi- ment, we directly assessed the occurrence of photoelectrochemical reac- tions at the polymer/extracellular bath interface by measuring ROS production. We previously demonstrated that P3HT polymer thin films exposed to saline electrolytes sustain efficient light-triggered charge generation and charge transfer processes, giving rise to photoelectro- chemical reactions (38, 39). Moreover, we also reported that P3HT nanoparticles are efficiently internalized within the cytosol of secondary line cell models (HEK-293) and that their photoexcitation leads to the production of ROS and subsequent intracellular calcium modulation (15, 37). However, the actual capability to sustain photo- electrochemical reactions in the specific experimental conditions used in this work (polymer film deposition conditions, sterilization process, prolonged exposure to specific cellular growth medium in an incubating environment, prolonged exposure to a light excitation protocol, light wavelength, pulses duty cycle, and power density) was never assessed. In particular, direct measurement of intracellular ROS was never carried out in the presence of polymer thin films. We now turn our attention to elucidating the possible mechanisms leading to optically enhanced tubulogenesis, through TRPV1 channel activation, upon prolonged polymer excitation. We now turn our attention to elucidating the possible mechanisms leading to optically enhanced tubulogenesis, through TRPV1 channel activation, upon prolonged polymer excitation. Reliable optical modulation of the cell activity mediated by polymer photoexcitation has been reported in several, previous reports, both in vitro, at the level of single cells, and in vivo, at the level of the whole animal, as evidenced by behavioral studies on both invertebrate and vertebrate models. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Fig. 4. Light-induced TRPV1 activation promotes in vitro tubulogenesis in ECFC cultures. (A to D) Representative optical images of in vitro tubular network of ECFCs subjected to long-term optical excitation seeded either on bare glass or on P3HT thin films and treated respectively with CPZ (A), RR (B), RN-1734 (C), and BAPTA- AM (D). Scale bars, 250 mm. (E to G) Relative variation of number of master segments (E), master junctions (F), and meshes (G) of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence [control (CTRL)] and presence of 10 mM CPZ, 10 mM RR, 20 mM RN-1734 (RN-1734), and 30 mM BAPTA-AM (BAPTA). The results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Dunnett’s post hoc test. P < 0.05 and *P < 0.01. Fig. 4. Light-induced TRPV1 activation promotes in vitro tubulogenesis in ECFC cultures. (A to D) Representative optical images of in vitro tubular network of ECFCs subjected to long-term optical excitation seeded either on bare glass or on P3HT thin films and treated respectively with CPZ (A), RR (B), RN-1734 (C), and BAPTA- AM (D). Scale bars, 250 mm. (E to G) Relative variation of number of master segments (E), master junctions (F), and meshes (G) of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence [control (CTRL)] and presence of 10 mM CPZ, 10 mM RR, 20 mM RN-1734 (RN-1734), and 30 mM BAPTA-AM (BAPTA). The results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Dunnett’s post hoc test. P < 0.05 and P < 0.01. Polymer optical excitation drives ECFC proliferation and in vitro tubulogenesis The results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Tukey post hoc test. P < 0.01 and *P < 0.001. Lodola et al., Sci. Adv. 2019;5:eaav4620 27 September 2019 4 of 12 4 of 12 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Lodola et al., Sci. Adv. 2019;5:eaav4620 27 September 2019 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Fig. 5. Phototransduction mechanisms. (A) An electrically insulating, thermally conducting material (photoresist) is successfully used as an ECFC seeding substrate. (B) Photoresist long-term photoexcitation does not lead to sizable enhancement in tubulogenesis parameters. (C) Evaluation of intracellular ROS production following long-term photoexcitation protocol of ECFC cultures on polymer and glass substrates (glass dark, n = 629; glass light, n = 656; P3HT dark, n = 686; and P3HT light, n = 583). For each panel, the results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with unpaired Student’s t test (A and B) or one-way ANOVA coupled with Tukey post hoc test (C). P < 0.001. Fig. 5. Phototransduction mechanisms. (A) An electrically insulating, thermally conducting material (photoresist) is successfully used as an ECFC seeding substrate. (B) Photoresist long-term photoexcitation does not lead to sizable enhancement in tubulogenesis parameters. (C) Evaluation of intracellular ROS production following long-term photoexcitation protocol of ECFC cultures on polymer and glass substrates (glass dark, n = 629; glass light, n = 656; P3HT dark, n = 686; and P3HT light, n = 583). For each panel, the results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with unpaired Student’s t test (A and B) or one-way ANOVA coupled with Tukey post hoc test (C). *P < 0.001. To this goal, we realized ECFC cultures on top of polymer and glass control substrates, and we exposed them to the same optical stimulation protocol previously used in the tubulogenesis assay. ROS production was then evaluated by means of a fluorescence experiment based on the use of the well-known ROS probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Fig. 5C). Results show that light induces an in- crease in ROSproductionbothonglassandpolymersubstrates. Relative percentage variation amounts to +34 and +200%, respectively, thus pointing out that polymer surface photocatalytic activity plays a major role in the phototransduction phenomenon. angiogenesis in an NF-kB–dependent manner. We considered inter- cellular adhesion molecule 1 (ICAM1); vascular adhesion molecule 1 (VCAM1); selectin E (SELE), matrix metalloproteinases (MMPs) 1, 2, and 9; vascular endothelial growth factor A (VEGFA); cyclo- oxygenase 2 (COX2, PTGS2); and cyclin D1 (CCND1) (40). DISCUSSION h Therapeutic angiogenesis via autologous EPC transplantation repre- sents a promising strategy to preserve or, at least, partially restore cardiac function after myocardial infarction (24, 41). Nevertheless, the regenerative outcome of EPC-based therapies in preclinical studies was rather disappointing and did not lead to sufficient neovascularization of the ischemic heart (41). This led to the proposal to boost their angio- genic activity by using emerging technologies, including tissue engineer- ing of vascular niches, pharmacological preconditioning, or genetic and epigenetic reprogramming (42). ECFCs are regarded among the most suitable EPC subtypes to induce therapeutic angiogenesis and cardiac re- generation due to their high clonal proliferative potential and ability to assemble into capillary-like structures (23, 24). In addition, they can be easily isolated and expanded from the peripheral blood of patients and healthy donors. It has recently been suggested that their angiogenic activity could be boosted by targeting the intracellular Ca2+ toolkit (29). Here, we target ECFCs by adopting a fully different approach, i.e., by exploiting visible light as a modulation trigger and by the use of a thiophene-based conjugated polymer as the exogenous, light- responsive actuator. We demonstrate that photoexcitation of the organic material deterministically leads to robustly enhanced proliferation and tubulogenesis. Pharmacological assays, supported by electrophysiology experiments, allow the identification of TRPV1 selective excitation as a key player in the molecular pathway leading to macroscopic outcomes, as observed by quantitative analysis of the angiogenic response. Phototransduction mechanism Three different photostimulation mechanisms, active at the polymer/cell interface, have been proposed so far. These include (i) the creation of an interface capacitance, i.e., of a localized electric field, possibly affecting the cell membrane potential (11); (ii) photothermal processes, establishing a localized temperature in- crease upon polymer photoexcitation (13, 36); and (iii) photoelectro- chemical reactions, mainly oxygen reduction processes, leading to a local variation of extracellular and/or intracellular pH (33) and sizable production of reactive oxygen species (ROS), at a nontoxic concentra- tion, and intracellular calcium modulation (37). In electrophysiological experiments, carried out at a photoexcita- tion density higher than the one used in “chronic” stimulation by about two orders of magnitude, we clearly observe TRPV1 excitation, corre- sponding however to a small variation of the cell membrane potential, in the order of a few millivolts (Supplementary Materials). Thus, upon much lower light intensity, the effects of either direct photothermal channel activation and of photocapacitive charging are expected to be negligible. To further corroborate this hypothesis, we carry out control experiments aimed at disentangling photoelectrical from photothermal transduction processes. First, we use a different material as a cell-seeding substrate, char- acterized by optical absorption and heat conductivity similar to the ones typical of P3HT (13) but fully electrically inert (i.e., unable to sustain electronic charge generation upon photoexcitation). The Lodola et al., Sci. Adv. 2019;5:eaav4620 27 September 2019 5 of 12 5 of 12 Lodola et al., Sci. Adv. 2019;5:eaav4620 27 September 2019 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Of these, five are significantly up-regulated by light exposure in cells grown on P3HT substrates, namely, ICAM1 (+90% versus P3HT dark; P < 0.05), SELE (+1119%; P < 0.01), MMP1 (+242%; P < 0.01), MMP2 (+467%; P < 0.05), and MMP9 (+458%; P < 0.05) (Fig. 6C). Conversely, VCAM1, VEGFA, PTGS2, and CCND1 do not show relevant variation upon light stimulation (fig.S7A). Light excitation oncellsgrown on bare glass substrates does not show any significant effect as compared with control samples in dark conditions (fig. S7B). To this goal, we realized ECFC cultures on top of polymer and glass control substrates, and we exposed them to the same optical stimulation protocol previously used in the tubulogenesis assay. ROS production was then evaluated by means of a fluorescence experiment based on the use of the well-known ROS probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Fig. 5C). Results show that light induces an in- crease in ROSproductionbothonglassandpolymersubstrates. Relative percentage variation amounts to +34 and +200%, respectively, thus pointing out that polymer surface photocatalytic activity plays a major role in the phototransduction phenomenon. Altogether, data in Fig. 5 indicate that photoelectrochemical reac- tions induced by light at the interface between the organic semi- conducting polymer and the extracellular bath play a key role in triggering the observed enhancement in cellnetworkformation through indirect activation of the TRPV1 channel. The occurrence of faradaic phenomena at the polymer/bath interface may give rise to material deg- radation effects. The photostability of the polymer substrates was care- fully checked by optical absorption, photoluminescence, and Raman spectra measurements. By treating the samples with the same experi- mental protocol used for cell tubulogenesis assays (photoexcitation den- sity, pulses frequency, overall exposure duration, temperature, and humidity levels), no sign of irreversible polymer degradation was ob- served, as compared with nonilluminated samples (fig. S5). S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Results are expressed as means ± SEM of the relative percentage of p65 nuclei–positively stained cells to the total number of cells (glass dark, n = 151; glass light, n = 125; P3HT dark, n = 147; and P3HT light, n = 159). Ten fields per condition are analyzed. Data are obtained from two different experiments conducted on cells harvested from two different donors. (C) mRNA levels of intercellular adhesion molecule 1 (ICAM1), selectin E (SELE), and matrix metalloproteinases (MMP1, MMP2, and MMP9) are quantified by real-time polymerase chain reaction (PCR). Data are expressed as means ± SEM of percentage variation with respect to cells grown in the dark (n = 6). The significance of differences was evaluated with unpaired Student’s t test (C) or one-way ANOVA coupled with Tukey post hoc test (B). P < 0.05 and *P < 0.01. omotes p65 NF-kB nuclear translocation and induces the expression of proangiogenic genes in ECFCs. ECFCs seeded channel through direct photothermal transduction is not the pre- dominant process leading to enhanced tubulogenesis. been shown to be expressed and drive angiogenesis in human ECFCs (32). TRPV1 is a polymodal Ca2+-permeable channel that integrates multiple chemical and physical cues to sense major changes in the local microenvironment of most mammalian cells (43). TRPV1 is activated by either noxious heat (>42°C) and acidic solutions (pH < 6.5), whereas mild acidification (pH 6.3) of the extracellular milieus sensitizes TRPV1 to heat stimulation and results in channel activation at temperature thresholds (30° to 32°C) well below the normal one (43). ROS produc- tion is also expected to further contribute to TRPV1 activation, as pre- viously reported in mouse coronary endothelial cells (44), in which hydrogen peroxide elicits a depolarizing inward current at negative holding potentials. Likewise, ROS may stimulate TRPV1 to depolarize the membrane potential, thereby triggering trains of action potentials in airway C fibers (45, 46). We have previously demonstrated that polymer photoexcitation leads to generation of faradaic current, to electron transfer reactions at the polymer/electrolyte interface,andto sizable intracellular enhance- ment of ROS (37, 38). Briefly, optical excitation of P3HT polymer thin films leads to photoexcited species (Eq. 1), namely, singlets and charge states, which react with the oxygen dissolved in the cell medium, thus reducing oxygen (Eq. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Fig. 6. Light-induced photostimulation promotes p65 NF-kB nuclear translocation and induces the expression of proangiogenic genes in ECFCs. ECFCs seeded on P3HT samples and glass controls are subjected to long-term photostimulation protocol. Corresponding control samples are kept in dark conditions. After photo- stimulation, p65 NF-kB nuclear translocation (A and B) and mRNA levels of tubulogenic/angiogenic genes that have been shown to be activated downstream of NF-kB (C) are evaluated. (A) Representative images of immunofluorescence staining showing p65 NF-kB (green) nuclear translocation. Cell nuclei are detected by 4′,6-diamidino-2- phenylindole (DAPI) (blue). Scale bars, 50 mm. (B) Quantitative evaluation of p65 NF-kB nuclear translocation, as evidenced by colocalization experiments. Results are expressed as means ± SEM of the relative percentage of p65 nuclei–positively stained cells to the total number of cells (glass dark, n = 151; glass light, n = 125; P3HT dark, n = 147; and P3HT light, n = 159). Ten fields per condition are analyzed. Data are obtained from two different experiments conducted on cells harvested from two different donors. (C) mRNA levels of intercellular adhesion molecule 1 (ICAM1), selectin E (SELE), and matrix metalloproteinases (MMP1, MMP2, and MMP9) are quantified by real-time polymerase chain reaction (PCR). Data are expressed as means ± SEM of percentage variation with respect to cells grown in the dark (n = 6). The significance of differences was evaluated with unpaired Student’s t test (C) or one-way ANOVA coupled with Tukey post hoc test (B). P < 0.05 and **P < 0.01. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Fig. 6. Light-induced photostimulation promotes p65 NF-kB nuclear translocation and induces the expression of proangiogenic genes in ECFCs. ECFCs seeded on P3HT samples and glass controls are subjected to long-term photostimulation protocol. Corresponding control samples are kept in dark conditions. After photo- stimulation, p65 NF-kB nuclear translocation (A and B) and mRNA levels of tubulogenic/angiogenic genes that have been shown to be activated downstream of NF-kB (C) are evaluated. (A) Representative images of immunofluorescence staining showing p65 NF-kB (green) nuclear translocation. Cell nuclei are detected by 4′,6-diamidino-2- phenylindole (DAPI) (blue). Scale bars, 50 mm. (B) Quantitative evaluation of p65 NF-kB nuclear translocation, as evidenced by colocalization experiments. Long-term optical excitation recruits NF-kB and induces the expression of proangiogenic genes 2 The Ca2+-sensitive transcription factor NF-kB might provide the missing link between the influx of Ca2+ through TRPV1 and the in- crease in proliferation and tubulogenesis observed in ECFCs upon photostimulation (26). We therefore monitored the nuclear trans- location of the cytoplasmic p65 NF-kB subunit via immunofluorescence staining and mRNA levels of a number of genes induced during tubu- logenesis in an NF-kB–dependent manner (26, 40) (Fig. 6). Our data indicate that ECFCs seeded on polymer and subjected to light stimula- tion have a significantly enhanced p65 NF-kB nuclear translocation compared with the control conditions consisting of cells also seeded on P3HT but kept in dark conditions (+35% versus P3HT dark; P < 0.05; Fig. 6, A and B), and seeded on bare glass (+28% versus glass dark; P < 0.05; Fig. 6B). No differences were observed between samples seeded on glass, whetherthey were subjectedto opticalexcitation ornot (fig. S6). dd h h k d h f h y q y g g p All data unambiguously show that polymer photoexcitation leads to selective activation of the TRPV1 channel, which has recently In addition, we have checked the expression of nine genes whose ex- pression is known to be induced in endothelial cells during tubulogenesis/ 6 of 12 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Lodola et al., Sci. Adv. 2019;5:eaav4620 27 September 2019 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E As a consequence, the p65 NF-kB subunit is released from IkB inhibition and translocates into the nucleus (58) where it induces the expression of multiple proan- giogenic genes (40). Consistently, we found that optical excitation significantly boosted the nuclear translocation of p65 in ECFCs cultured on the conjugated polymer compared with those not exposed to light. Robust up-regulation of several angiogenic genes, such as ICAM, SELE, MMP1, MMP2, and MMP9, which are under NF-kB–dependent transcriptional control, was also consequently observed. Intriguingly, NF-kB also mediates VEGFA-induced gene expression and angiogen- esis in vascular endothelial cells (59, 60) through an increase in [Ca2+]i (61). These observations strongly hint at NF-kB as the Ca2+-sensitive decoder that translates optical excitation into an angiogenic response in human ECFCs interfaced with the light-sensitive conjugated polymer. through aquaporin AQP3, thereby triggering intracellular ROS signaling (47, 48). In line with our previous results, we have demon- strated here that intracellular ROS enhancement does occur in ECFCs upon photoexcitation of polymer thin films, thus contributing to TRPV1 activation. through aquaporin AQP3, thereby triggering intracellular ROS signaling (47, 48). In line with our previous results, we have demon- strated here that intracellular ROS enhancement does occur in ECFCs upon photoexcitation of polymer thin films, thus contributing to TRPV1 activation. Altogether, the evidence supports the hypothesis of a transduction mechanism mainly governed by photoelectrochemical reactions. More- over, these same observations could explain why TRPV4, which is also expressed in ECFCs (34), is not sensitive to optical modulation. Al- though TRPV4 is activated by moderate heat (24° to 38°C), it is sup- posed to be inhibited by local pH variation, although this is still a matter of debate (49, 50). ( ) On the one hand, the role attributed in the phototransduction mechanism to the capability of the polymer to generate and transport electronic charges, as well as to its photocatalytic activity in an aqueous environment, clearly implies the need for a biocompatible, visible light–responsive, semiconducting material. This excludes any possi- ble implementation of the reported technique by using a thermally conducting, electrically insulating plastic substrate. Suitable cell- seeding materials have to be selected and developed within the wide arena of organic semiconducting polymers. On the other hand, the key role played by ROS raises additional issues about material photo- stability, cell viability, and overall safety and reliability of the technique. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E The combined use of optical excitation and organic polymer tech- nology can open interesting perspectives for several different reasons. First, the use of light modulation allows unprecedented spatial and temporal resolution to be achieved in a fully reversible way. Light tem- poral and spatial patterns can be specifically designed and adapted to different in vitro cell models, allowing ideally endless combinations of possibilities, to finely tune overall output in cell proliferation and network formation. The demonstrated technology is minimally invasive, allows for massive parallelization of experiments, and can be virtually implemented in any cell therapy model in a straightforward way. In ad- dition, the use of different polymers, with lower energy gap and in the form of nanobeads, may pave the way to the optical enhancement of therapeutic angiogenesis in vivo. Further work is needed to understand whether the pattern and/or intensity of the illumination protocol may be adjusted to further boost the angiogenic response. For instance, the optical excitation protocol consisted of 30-ms-long light pulses that were delivered at 1 Hz for 4 (tubulogenesis) up to 36 (proliferation) hours. This is likely to result in oscillations in [Ca2+]i, which are known to deliver the most instructive signal for ECFCs to undergo angiogenesis by inducing the nuclear translocation of the p65 NF-kB subunit (26). As the frequency of intracellular Ca2+ oscillations can be artificially ma- nipulated to regulate NF-kB–dependent gene expression in virtually any cell type (62), we envisage an additional layer of specificity and control that could be exploited to further improve the angiogenic response to optical excitation. Future work will also be devoted to assess the outcome of optical modulation on patient-derived ECFCs. One of the main hurdles associated to autologous cell-based therapy is the impairment of the angiogenic activity of EPCs, including ECFCs harvested from cardiovascular patients (29). The therapeutic translation of our findings will require the demonstration that light-induced TRPV1 activation has shown that NF-kB triggers the transcriptional program underlying the angiogenic response to extracellular Ca2+ entry in ECFCs (26). Moreover, NF-kB activation in response to extracellular stimulation and Ca2+ entry through TRPV1 has also been demonstrated (56, 57). Under resting conditions, NF-kB is retained in the cytoplasm by the complex with the inhibitory protein IkB. An increase in [Ca2+]i results in IkB degradation by ubiquitination, which is triggered upon the Ca2+-dependent phosphorylation of IkB. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E We extensively verified that the main polymer optoelectronic properties are not substantially altered by the exposure to light and to incubating conditions. From the biological point of view, it is very well known that high ROS levels can induce highly toxic effects and, finally, lead to cell death. We notice, however, that the established photoactivation protocol (illuminator geometry and air flow, light photoexcitation den- sity, duty cycle, and repetition rate) has been implemented to avoid any detrimental effect. Accordingly, no toxicity effects were detected for the overall duration of the experiments, as proven by the robust increase in ECFC proliferation and tubulogenesis exposed to light. This ob- servation is consistent with the emerging notion that appropriate ROS levels can exert a signaling role and control angiogenesis in en- dothelial cells (51). Overall, our findings represent the proof of principle that optical modulation may be successfully exploited to directly control the fate of a progenitor cell population, i.e., ECFCs, which has been shown to support revascularization of ischemic tissues. The in vitro activation of ECFC angiogenic activity is made possible by the use of a bio- compatible, light-sensitive polymer as the phototransduction element. The combined use of optical excitation and organic polymer tech- nology can open interesting perspectives for several different reasons. First, the use of light modulation allows unprecedented spatial and temporal resolution to be achieved in a fully reversible way. Light tem- poral and spatial patterns can be specifically designed and adapted to different in vitro cell models, allowing ideally endless combinations of possibilities, to finely tune overall output in cell proliferation and network formation. The demonstrated technology is minimally invasive, allows for massive parallelization of experiments, and can be virtually implemented in any cell therapy model in a straightforward way. In ad- dition, the use of different polymers, with lower energy gap and in the form of nanobeads, may pave the way to the optical enhancement of therapeutic angiogenesis in vivo. Further work is needed to understand whether the pattern and/or intensity of the illumination protocol may be adjusted to further boost the angiogenic response. For instance, the optical excitation protocol consisted of 30-ms-long light pulses that were delivered at 1 Hz for 4 (tubulogenesis) up to 36 (proliferation) hours. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E 2) P3HT þ hn →P3HT ð1Þ P3HT þ O2 →P3HTþ þ O 2 ð2Þ ð1Þ ð2Þ On the basis of measurements carried out in cells seeded on the photoresist substrate, as well as on direct evaluation of a limited, local temperature increase upon light stimuli during the long-term photoexcitation protocol, we infer that the excitation of the TRPV1 The superoxide further evolves, leading to the generation of different ROS and, lastly, ending up with hydrogen peroxide production. It has been reported that extracellular H2O2 can cross the plasma membrane Lodola et al., Sci. Adv. 2019;5:eaav4620 27 September 2019 7 of 12 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E has shown that NF-kB triggers the transcriptional program underlying the angiogenic response to extracellular Ca2+ entry in ECFCs (26). Moreover, NF-kB activation in response to extracellular stimulation and Ca2+ entry through TRPV1 has also been demonstrated (56, 57). Under resting conditions, NF-kB is retained in the cytoplasm by the complex with the inhibitory protein IkB. An increase in [Ca2+]i results in IkB degradation by ubiquitination, which is triggered upon the Ca2+-dependent phosphorylation of IkB. As a consequence, the p65 NF-kB subunit is released from IkB inhibition and translocates into the nucleus (58) where it induces the expression of multiple proan- giogenic genes (40). Consistently, we found that optical excitation significantly boosted the nuclear translocation of p65 in ECFCs cultured on the conjugated polymer compared with those not exposed to light. Robust up-regulation of several angiogenic genes, such as ICAM, SELE, MMP1, MMP2, and MMP9, which are under NF-kB–dependent transcriptional control, was also consequently observed. Intriguingly, NF-kB also mediates VEGFA-induced gene expression and angiogen- esis in vascular endothelial cells (59, 60) through an increase in [Ca2+]i (61). These observations strongly hint at NF-kB as the Ca2+-sensitive decoder that translates optical excitation into an angiogenic response in human ECFCs interfaced with the light-sensitive conjugated polymer. Overall, our findings represent the proof of principle that optical modulation may be successfully exploited to directly control the fate of a progenitor cell population, i.e., ECFCs, which has been shown to support revascularization of ischemic tissues. The in vitro activation of ECFC angiogenic activity is made possible by the use of a bio- compatible, light-sensitive polymer as the phototransduction element. Isolation and cultivation of ECFCs ECFCs were isolated from peripheral blood and expanded as shown elsewhere (26). Blood samples (40 ml) collected in EDTA-containing tubes were obtained from healthy male human volunteers aged from 28 to 38 years. The Institutional Review Board at Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Foundation in Pavia approved all protocols and specifically approved this study. Informed written consent was obtained according to the Declaration of Helsinki of 1975 as revised in 2008. We focused on the so-called ECFCs, a subgroup of EPCs that are found in the CD34+ CD45−frac- tion of circulating mononuclear cells (MNCs), exhibit robust prolif- erative potential, and form capillary-like structures in vitro (23). To isolate ECFCs, MNCs were obtained from peripheral blood by den- sity gradient centrifugation on lymphocyte separation medium for 30 min at 400g and washed twice in EBM-2 with 2% fetal calf serum. A median of 36 × 106 MNCs (range, 18 to 66) was plated on fibronectin- coated culture dishes (BD Biosciences) in the presence of the endothe- lial cell growth medium EGM-2 MV (Lonza) containing endothelial basal medium (EBM-2), 5% fetal bovine serum (FBS), recombi- nant human (rh) EGF, rhVEGF, recombinant human Fibroblast Growth Factor-Basic (rhFGF-B), recombinant human Insulin-like Growth Factor-1 (rhIGF-1), ascorbic acid, and heparin and main- tained at 37°C in 5% CO2 and humidified atmosphere. Nonadherent cells were discarded after 2 days, and thereafter, medium was changed three times a week. The outgrowth of ECFCs from adherent MNCs was characterized by the formation of a cluster of cobblestone-shaped cells. That ECFC-derived colonies belonged to the endothelial line- age was confirmed by staining with anti-CD31, anti-CD105, anti- y For optical excitation of the polymer, a homemade petri cell culture illuminator, compatible with the use within the cell incubator, was designed and implemented. Its design included a black spacer made by fused filament fabrication, both to minimize overheating effects in the extracellular bath and to avoid unwanted light scattering/diffusion effects and cross-talk between different specimens. Optical excitation was provided by a green LED system, whose duty cycle, repetition rate, and intensity were set through a custom-made control circuit, compris- ing a microcontroller, a digital-to-analog converter, and an analog LED driver. The driver was connected to five green LEDs (SMB1N-525V-02; Roithner LaserTechnik GmbH, Vienna, Austria), with maximum emis- sion wavelength at 525 nm, each carrying a collimator lens reducing the emission angle to 22°. Semiconducting polymer and electrically insulating photoresist preparation Semiconducting polymer and electrically insulating photoresist preparation Regioregular P3HT (99.995% purity; Mn 54,000 to 75,000 molecular weight) was purchased from Sigma-Aldrich and used without any further purification. The samples for cell cultures were prepared by spin coating on a square 18 mm by 18 mm glass (VWR International) sub- strates carefully rinsed in subsequent ultrasonic baths of ultrapure water, acetone, and isopropanol. P3HT solution was prepared in chloro- benzene at a final P3HT concentration of 20 g/liter and spin coated on the cleaned substrates with a two-step recipe: (i) 3 s at 800 rpm and (ii) 60 s at 1600 rpm. Polymer film thickness is about 150 nm. Isolation and cultivation of ECFCs This way, up to five 3.5-cm petri dishes can be simultaneously treated with a homogeneous photoexcitation density of 40 mW/cm2. The long-term optical excitation protocol adopted for cell fate modulation consists of 30-ms-long pulses, followed by 70-ms-long darkconditions,continuouslyrepeatedforaminimumof4upto36hours in the case of tubulogenesis and proliferation assays, respectively. Electrophysiology Electrophysiological recordings were performed using a patch-clamp setup (Axopatch 200B; Axon Instruments) coupled to an inverted microscope (Nikon Eclipse Ti). ECFCs were measured in whole-cell configuration with freshly pulled glass pipettes (3 to 6 MW), filled with the following intracellular solution: 12 mM KCl, 125 mM K-gluconate, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM EGTA, 10 mM Hepes, and 10 mM ATP (adenosine 5′-triphosphate)–Na2. The extracellular solution contained the following: 135 mM NaCl, 5.4 mM KCl, 5 mM Hepes, 10 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2. Only single cells were selected for recordings. Acquisition was performed with the pCLAMP 10 software (Axon Instruments). Membrane currents were low pass filtered at 2 kHz and digitized with a sampling rate of 10 kHz (Digidata 1440 A; Molecular Devices). Data were analyzed with Clampfit (Axon Instruments) and Origin 8.0 (OriginLab Corporation). Microposit S1813 photoresist was purchased from Shipley and used without any further purification. Photoresist thin films were prepared by spin coating on cleaned substrates with a two-step recipe: (i) 3 min at 300 rpm and (ii) 30 s at 2600 rpm. Parameters were adjusted to ob- tain homogeneous films and similar optical absorbance to the one of the polymer thin films, at the same excitation wavelength used in the long-term stimulation protocol (see below). All films were thermally treated in an oven at 120°C for 2 hours for annealing and sterilization. To promote adhesion, samples were coated with fibronectin (from bovine plasma; Sigma-Aldrich) at a concentration of 2 mg/ml in phosphate-buffered saline (PBS) for at least 30 min at 37°C and then rinsed with PBS. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E This is likely to result in oscillations in [Ca2+]i, which are known to deliver the most instructive signal for ECFCs to undergo angiogenesis by inducing the nuclear translocation of the p65 NF-kB subunit (26). As the frequency of intracellular Ca2+ oscillations can be artificially ma- nipulated to regulate NF-kB–dependent gene expression in virtually any cell type (62), we envisage an additional layer of specificity and control that could be exploited to further improve the angiogenic response to optical excitation. Future work will also be devoted to assess the outcome of optical modulation on patient-derived ECFCs. One of the main hurdles associated to autologous cell-based therapy is the impairment of the angiogenic activity of EPCs, including ECFCs harvested from cardiovascular patients (29). The therapeutic translation of our findings will require the demonstration that light-induced TRPV1 activation boosts angiogenesis also in ECFCs derived from individuals affected by severe cardiovascular disorders, such as hypertension, atherosclerosis, ( ) The biophysical mechanisms whereby the photoactivation of TRPV1 stimulates in vitro angiogenesis in ECFCs deserve a more detailed discussion as well. Earlier work showed that TRPV1 stimu- lates proliferation and tube formation in vascular endothelial cells by mediating extracellular Ca2+ entry. The following increase in in- tracellular Ca2+ concentration ([Ca2+]i) leads to the recruitment of several downstream Ca2+-dependent decoders, such as endothelial ni- tric oxide synthase and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (52). Recently, TRPV1 was found to induce also proliferation and tube formation in ECFCs by mediating the uptake of the endocan- nabinoid anandamide (32). This study, however, did not investigate whether TRPV1 activation was per se able to stimulate ECFCs by engaging Ca2+-dependent pathways. Intracellular Ca2+ signaling is a crucial determinant of ECFC fate and behavior (26–28). Accordingly, light-induced ECFC proliferation and tube formation were markedly reduced by the pharmacological blockade of TRPV1-mediated Ca2+ entry with CPZ and RR and by preventing the subsequent increase in [Ca2+]i with BAPTA-AM. This finding endorses the view that optical excitation stimulates ECFCs through TRPV1-mediated ex- tracellular Ca2+ entry, and we suggest here that this occurs via downstream activation of transcriptional factor NF-kB. NF-kB has previously been shown to stimulate cell proliferation and tubulogen- esis in endothelial cells (53, 54) and in hepatocytes (55). Our group Lodola et al., Sci. Adv. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E 2019;5:eaav4620 27 September 2019 8 of 12 8 of 12 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E CD144, anti-CD146, anti–von Willebrand factor, anti-CD45, and anti-CD14 monoclonal antibodies and by assessment of capillary- like network formation in the in vitro tube formation assay. and heart failure. In this view, the combination of organic semiconduc- tors and genetic manipulation to increase endogenous TRPV1 expres- sion could be sufficient to restore the reparative phenotype of autologous ECFCs from cardiovascular patients. For our experiments, we have mainly used endothelial cells obtained from early-passage ECFCs (P2-4, which roughly encompasses a 15- to 18-day period) with the purpose to avoid, or maximally reduce, any potential bias due to cell differentiation. However, to make sure that the phenotype of the cells did not change throughout the experiments, in the preliminary experiments, we tested the immunophenotype of ECFCs at different passages, and we found no differences. We also tested whether functional differences occurred when early (P2)– and late (P6)–passage ECFCs were used by testing the in vitro capacity of capillary network formation in a Cultrex assay and found no differences between early- and late-passage ECFC-derived cells (data not shown). ROS detection H2DCF-DA (Sigma-Aldrich) was used for the intracellular detection of ROS. ECFCs were seeded onto polymer and control substrates and subjected to the same photoexcitation protocol used for the in vitro tube formation assay. Immediately after the end of the protocol, cell cultures were incubated with the ROS probe for 30 min. After careful washout of the excess probe from the extracellular medium, the flu- orescence of the probe was recorded (excitation/emission wave- lengths, 490/520 nm; integration time, 70 ms for H2DCF-DA) with an inverted microscope (Nikon Eclipse Ti) equipped with an Analog-WDM Camera (CoolSNAP MYO, Teledyne Photometrics). To minimize the effects of the spectral overlap between the polymer absorption and emission spectra, and the probe emission, samples were turned upside down by using a homemade chamber with a 500-mm-thick channel filled with extracellular medium. Variation of fluorescence intensity was evaluated over regions of interest covering single-cellareas, andreported values represent the average overmultiple cells. See figure captions for additional details about statistical analysis. Image processing was carried out with ImageJ and subsequently ana- lyzed with Origin 8.0. In vitro tube formation assay ECFC-derived cells from early-passage (P2 to P4) cultures were ob- tained by trypsinization and resuspended in EBM-2 supplemented with 2% FBS. EPC-derived cells (10 × 103) per well were plated in Cultrex basement membrane extract (Trevigen Inc., Gaithersburg, MD, USA) 10-mm fibronectin-treated cloning cylinders. Plates were then incu- bated at 37°C, 5% CO2, and capillary network formation was assessed starting from 4 to 24 hours later. At least three different sets of cultures were performed every experimental point. Quantification of tubular structures was performed after 8 hours of incubation by measuring the total length of structures per field with the aid of the ImageJ software (National Institutes of Health, USA; http://rsbweb.nih.gov/ij/). To eval- uate the role of TRPV1, the same protocol was repeated in the presence of the following drugs: BAPTA (30 mM), CPZ (10 mM), RN-1734 (20 mM), or RR (10 mM). Total RNA extraction and real-time polymerase chain reaction Cells were lysed in 0.5 ml of TRI Reagent (Sigma, catalog no. T9424), and total RNA was extracted according to the manufacturer’s protocol. One microgram of total RNA was retrotranscribed using SensiFAST cDNA Synthesis Kit (Bioline, London, UK, catalog no. BIO-65054). Real-time polymerase chain reaction (PCR) was performed using iTaq qPCRmastermix according to the manufacturer’sinstructions(Bio-Rad, Segrate, Italy, catalog no. 1725124) on a SFX96 Real-Time System (Bio- Rad). As a control, S18 ribosomal subunit was used, whose expression did not change across the conditions. For each gene, DCt was calculated by using the formula DCt = 2^(DCt(gene) – DCt(S18)). The data are expressed as a percentage variation between P3HT light and glass light conditions and P3HT dark and glass dark samples, respectively. Se- quences of oligonucleotide primers are listed in table S1. Immunofluorescence assay To examine NF-kB p65 subunit translocation into the nucleus in the individual ECFCs, the coverslips were fixed with 4% formaldehyde in PBS (20 min at RT) and permeabilized with 0.1% Triton X-100 in PBS (7 min at RT). Primary rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology, catalog no. Sc-372) was applied at a final dilution of 1:100 for 1 hour at 37°C in a humidified chamber. After three washes with PBS, secondary chicken anti-rabbit Alexa(488)- conjugated antibody (1:200; Invitrogen, catalog no. A-21441) was ap- plied for 1 hour at RT. After washing (three times in PBS), nuclei were counterstained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; 1:5000 dilution in PBS; 20 min at RT; Invitrogen, catalog no. D1306). Last, the coverslips with cells were mounted on microscope glass slides using Fluoroshield mount medium (Sigma, catalog no. F6182). Fluorescence images were taken with the same fluorescence mi- croscope used for the electrophysiology experiments, using standard DAPI and fluorescein isothiocyanate filters set for the acquisition of DAPI and Alexa(488) fluorescence emission, respectively. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E nant Raman spectra were recorded by using visible light excitation at 532 nm (HORIBA Jobin-Yvon HR800 micro-Raman spectrometer system). Laser power intensity on the sample was kept at values low- er than 0.03 mW to avoid laser-induced sample degradation. Spectra were typically recorded in the region 600 to 2000 cm−1 and were ca- librated against the 520.5 cm−1 line of an internal silicon wafer. The signal-to-noise ratio was enhanced by repeated acquisitions (100). The measurements were conducted at room temperature (RT), and the resulting spectral resolution was 0.4 cm−1. at 37°C (in 5% CO2 and humidified atmosphere), and cell growth was assessed after 36 hours since the beginning of the long-term il- lumination protocol. At this point, cells were recovered by trypsiniza- tion from all the dishes, and the cell number was assessed by counting in a hemocytometer. Preliminary experiments showed no unspecific or toxic effect for each agent when used at these concentrations. Each assay was repeated in triplicate. at 37°C (in 5% CO2 and humidified atmosphere), and cell growth was assessed after 36 hours since the beginning of the long-term il- lumination protocol. At this point, cells were recovered by trypsiniza- tion from all the dishes, and the cell number was assessed by counting in a hemocytometer. Preliminary experiments showed no unspecific or toxic effect for each agent when used at these concentrations. Each assay was repeated in triplicate. Polymer photostability assay Two sets of P3HT thin films (n = 12) were prepared as described above. The optical absorbance, the emission, and the Raman spectrum were measured immediately after fabrication. Then, all samples were exposed to ECFC growth medium (EBM-2 supplemented with 2% FBS) and in- cubated at 37°C, 5% CO2 for 24 hours. The first set was taken in dark conditions (n = 6), and the second one was treated with the same optical excitation protocol used in the tubulogenesis assays (n = 6). After incu- bation, absorption, emission, and Raman spectrum were measured again in the same conditions as before. Absorption spectra were recorded by using a spectrophotometer (PerkinElmer Lambda 1040) in transmission mode. Photoluminescence spectra were acquired by using a Jobin-Yvon spectrofluorometer; the excitation wavelength was set at the polymer absorption peak wavelength (530 nm). Reso- The significance of differences was evaluated with unpaired Student’s t test or one-way analysis of variance (ANOVA) coupled with Tukey or Dunnett’s post hoc test, as appropriate. Data are represented as means ± standard error of the mean (SEM). P < 0.05 was considered statistically significant. Statistical analysis was performed using the GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA). Proliferation assays h d Growth dynamics were evaluated by plating a total of 5 × 103 ECFC- derived cells into 10-mm fibronectin-treated cloning cylinders (5 × 104/cm2) in the presence of EGM-2 MV medium to facilitate the ad- hesion. After 12 hours, the medium was switched to EBM-2 supple- mented with 2% fetal calf serum. For the pharmacological treatment, one of compounds was added to the medium: BAPTA (30 mM), CPZ (10 mM), RN-1734 (20 mM), or RR (10 mM). Cultures were incubated Lodola et al., Sci. 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Author contributions: F.L., F.M., and M.R.A. planned the experiments. F.L. carried out the experimental measurements (electrophysiology, short- and long-term photoexcitation, evaluation of effects on proliferation, tubulogenesis, and ROS production). V.R. provided the ECFC models, took care of the cell cultures, and contributed to the tubulogenesis and proliferation experiments. G.T. prepared the polymer samples. A.D. designed, realized, and optimized the experimental setup for the long-term photoexcitation. L.T. and D.L. carried out the immunofluorescence and real-time PCR assays. P.C. contributed to the methodological discussion about gene expression. F.L. and M.R.A. wrote the main manuscript, with help from F.M. All authors contributed to the data interpretation and approved the final manuscript. Competing interests: The authors declare that they have no competing interests. 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Adv. 5, eaav4620 (2019). 12 of 12 Conjugated polymers optically regulate the fate of endothelial colony-forming cells F. Lodola, V. Rosti, G. Tullii, A. Desii, L. Tapella, P. Catarsi, D. Lim, F. Moccia and M. R. Antognazza Conjugated polymers optically regulate the fate of endothelial colony-forming F. Lodola, V. Rosti, G. Tullii, A. Desii, L. Tapella, P. Catarsi, D. Lim, F. Moccia and M. R. Antognazza DOI: 10.1126/sciadv.aav4620 (9), eaav4620. 5 Sci Adv DOI: 10.1126/sciadv.aav4620 (9), eaav4620. 5 Sci Adv DOI: 10.1126/sciadv.aav4620 (9), eaav4620. 5 Sci Adv ARTICLE TOOLS http://advances.sciencemag.org/content/5/9/eaav4620 MATERIALS SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2019/09/23/5.9.eaav4620.DC1 REFERENCES http://advances.sciencemag.org/content/5/9/eaav4620#BIBL This article cites 62 articles, 9 of which you can access for free PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions http://advances.sciencemag.org/content/5/9/eaav4620 Terms of Service Use of this article is subject to the registered trademark of AAAS. is a Science Advances Association for the Advancement of Science. No claim to original U.S. Government Works. 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https://openalex.org/W2141795335	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_S6_from_Maturation_Stage_of_T-cell_Acute_Lymphoblastic_Leukemia_Determines_BCL-2_versus_BCL-XL_Dependence_and_Sensitivity_to_ABT-199/22531948/1/files/39995158.pdf	Aragonese	null	Maturation Stage of T-cell Acute Lymphoblastic Leukemia Determines BCL-2 versus BCL-XL Dependence and Sensitivity to ABT-199	Cancer discovery	2,014	cc-by	2,080	V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 S p l e e n W e i g h t ( m g ) ETP-ALL ETP-ALL Spleen V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 100 200 300 400 500 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Vehicle ABT-199 ABT-263 ETP-ALL Bone Images V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 1000 S p l e e n W e i g h t ( m g ) * * Typical T-ALL V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Typical T-ALL Spleen Typical T-ALL Blood ETP-ALL Blood Vehicle ABT-199 ABT-263 Typical T-ALL Bone Images Supplemental Figure 6 Ni Chonghaile et. al. A B C D E F G H 0 1 2 0 20 40 60 80 100 Weeks H u m a n C D 4 5 % Vehicle ABT-199 ABT-263 * * 0 1 2 50 60 70 80 90 100 Weeks H u m a n C D 4 5 % * Vehicle ABT-199 ABT-263 V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 S p l e e n W e i g h t ( m g ) ETP-ALL ETP-ALL Spleen V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 100 200 300 400 500 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Vehicle ABT-199 ABT-263 ETP-ALL Bone Images V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 1000 S p l e e n W e i g h t ( m g ) * * Typical T-ALL V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Typical T-ALL Spleen Typical T-ALL Blood ETP-ALL Blood Vehicle ABT-199 ABT-263 Typical T-ALL Bone Images Supplemental Figure 6 Ni Chonghaile et. al. A B C D E F G H 0 1 2 0 20 40 60 80 100 Weeks H u m a n C D 4 5 % Vehicle ABT-199 ABT-263 * *** 0 1 2 50 60 70 80 90 100 Weeks H u m a n C D 4 5 % * Vehicle ABT-199 ABT-263 Ni Chonghaile et. al. V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 S p l e e n W e i g h t ( m g ) ETP-ALL ETP-ALL Spleen V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 100 200 300 400 500 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Vehicle ABT-199 ABT-263 ETP-ALL Bone Images V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 1000 S p l e e n W e i g h t ( m g ) * * Typical T-ALL V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Typical T-ALL Spleen Typical T-ALL Blood ETP-ALL Blood Vehicle ABT-199 ABT-263 Typical T-ALL Bone Images Supplemental Figure 6 Ni Chonghaile et. al. A B C D E F G H 0 1 2 0 20 40 60 80 100 Weeks H u m a n C D 4 5 % Vehicle ABT-199 ABT-263 * *** 0 1 2 50 60 70 80 90 100 Weeks H u m a n C D 4 5 % * Vehicle ABT-199 ABT-263 V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 S p l e e n W e i g h t ( m g ) ETP-ALL ETP-ALL Spleen V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 100 200 300 400 500 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Supplemental Figure 6 A B ETP-ALL Blood Ni Chonghaile et. al. C 0 1 2 0 20 40 60 80 100 Weeks H u m a n C D 4 5 % Vehicle ABT-199 ABT-263 * * V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 00 00 00 00 ETP-ALL ETP-ALL Spleen V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 100 200 300 400 500 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) ETP-ALL Blood pplemental Figure 6 Ni Chonghaile et. al. V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 S p l e e n W e i g h t ( m g ) ETP-ALL ETP-ALL Spleen V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 100 200 300 400 500 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Vehicle ABT-199 ABT-263 ETP-ALL Bone Images V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 1000 S p l e e n W e i g h t ( m g ) * * Typical T-ALL V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Typical T-ALL Spleen Typical T-ALL Blood ETP-ALL Blood Vehicle ABT-199 ABT-263 Typical T-ALL Bone Images Supplemental Figure 6 Ni Chonghaile et. al. A B C D E F G H 0 1 2 0 20 40 60 80 100 Weeks H u m a n C D 4 5 % Vehicle ABT-199 ABT-263 * *** 0 1 2 50 60 70 80 90 100 Weeks H u m a n C D 4 5 % * Vehicle ABT-199 ABT-263 B C 0 1 2 0 20 40 60 80 100 Weeks H u m a n C D 4 5 % Vehicle ABT-199 ABT-263 * * ETP-ALL Spleen V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 100 200 300 400 500 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) B ETP-ALL Spleen V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 100 200 300 400 500 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) ETP-ALL Blood g B C 0 1 0 20 40 60 80 100 Weeks H u m a n C D 4 5 % Ve AB AB * V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 S p l e e n W e i g h t ( m g ) ETP-ALL A C C B A D Vehicle ABT-199 ABT-263 ETP-ALL Bone Images D V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 1000 S p l e e n W e i g h t ( m g ) * * Typical T-ALL V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Typical T-ALL Spleen E F Human CD45% V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Typical T-ALL Spleen F V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 A b s o l u t e N u m b e r o f H C D 4 5 + c e l l s ( 1 0 6 ) Typical T-ALL Spleen Typical T-ALL Blood F G 0 1 2 50 60 70 80 90 100 Weeks H u m a n C D 4 5 % * ** Vehic ABT- ABT-2 Typical T-ALL Blood G 0 1 2 50 60 70 80 90 100 Weeks H u m a n C D 4 5 % * Vehicle ABT-199 ABT-263 G V e h i c l e A B T - 1 9 9 A B T - 2 6 3 0 200 400 600 800 1000 S p l e e n W e i g h t ( m g ) * *** Typical T-ALL E F E Typical T-ALL Blood Typical T-ALL S p l e e n W e i g h t ( m g ) Vehicle ABT-199 ABT-263 Typical T-ALL Bone Images H Typical T-ALL Bone Images H H
https://openalex.org/W2113719562	https://sajp.co.za/index.php/sajp/article/download/7/6	English	null	HIV/AIDS in Physiotherapy Education – Mainstreaming Using UN Principles	South African journal of physiotherapy	2,012	cc-by	6,738	HIV/AIDS in Physiotherapy Education – Mainstreaming Using UN Principles Abstract: HIV /AIDS remains at epidemic levels in South Africa with one in ten people affected by the HI virus. The United Nations program for AIDS (UNAIDS) has defined five principles that can be used to mainstream HIV. The five key principles are: the development of a clearly defined entry point; use of existing policies, structures and frameworks; advocacy sensitization and capacity building; distinguishing between the external (institutional interventions) and internal (staff risks and vulnerability) domain; and developing strategic partnerships. A proposed HIV curriculum for physiotherapists was been developed from a series of studies undertaken to understand the issues r mainstreaming. Myezwa H, PhD,1 Stewart A, PhD.1 1 University Of Witwatersrand. The purpose of this paper is to explore how each principle of mainstreaming can be ap education and curricula. Each mainstreaming principle was considered and ways in which it could be mainstreamed into physiotherapy training and practice, using data from the patients’, academics’ and clinicians’ experience were proposed.i The five principles can be used in South African settings. In principle one the aetiology, approaches to treatment, management of conditions and symptoms are used to identify entry points for introducing HIV teaching. Relevant examples of conditions associated with HIV include pain, stroke, specific muscle weakness patterns, fatigue and cardiopulmonary system problems (e.g. breathlessness). The role of physiotherapy in HIV management is also a good entry point. For principle two, policies were considered. Pertinent areas such as antiretroviral use, frameworks such as the International Classification of Function (ICF), Worthington’s rehabilitation framework and CBR can also be inte- grated with HIV physiotherapy curriculum and practice. Sensitised and aware staff as promoted in principle three, can advocate for affected patients and role model appropriate practice and attitudes in HIV management thus contributing to reducing stigma and burnout. Integrating policies with the need to avoid risks and correct inappro priate attitudes of staff is promoted and promoting strategic partnerships and collaborative work will help provide appropriate services. Using the mainstreaming principles allows for the inclusion of HIV content in physiotherapy education and prac- tice. Both physiotherapy curriculum and continuous professional development may use this information to respond appropriately. Research Research Article Introduction The manage ment of each of these systems is already taught and commonly seen in the clinical setting. The specific effects of HIV on multiple systems are important in teach ing and clinical reasoning. For example, HIV directly affects either progenitor cells or indirectly through opportunis tic infections, it can produce symptoms that are from multiple aetiologies, thus impacting on clinical reasoning. Similarly is: dyspnoea may be as a result of the direct effects on the haematologi cal system or the pathological effects of opportunistic and recurrent infections, like pneumocystic jiroveci pneumonia on oxygen exchange. Thus by implica tion, these differences in causes and their effects on treatment should be addressed during training by the respective area of speciality. Similarly, impairments such as pain, fatigue and loss of body mass commonly occur in HIV. Physiotherapy curricula include the basic structure, function and relevant pathogenesis of systems related to these impairments (CSP, 2008b) and therefore entry points for teaching on how HIV impacts on these systems can be identified. The emergence of HIV, however has affected the aetiology and morbidity of all the conditions traditionally treated and this is further complicated by problems of stigma, fear and the need for a very strong health and inter-sectoral support system. A decision thus would need to be made, as to how much content on HIV/AIDS should be included in under graduate or post graduate education. Recent graduates and academics (Myezwa 2008) felt that the way HIV 2. Mainstreaming should take place within existing policies and frame works and should be located within existing institutional structures. 3. Advocacy, sensitisation and capacity building to place health workers in a better position to undertake main streaming should take place. 4. A distinction between the external and internal domains must be made. Internal domains includes risks and vulnerability and the external domains include issues such as an institution undertaking interventions based on its mandate and capacity in support of national and local strate gic efforts Principle 1: A clearly defined entry point for mainstreaming HIV/AIDS The entry point is seen as a position in the core business, at which one can start to include the matter to be mainstreamed. In this case, the physiotherapy curricu lum and practice is the core business. The generic curriculum, that teaches basic aetiology, pathophysiology and a largely biopsychosocial approach to patient management presents a ready opportunity for mainstreaming manage ment of HIV/AIDS. 5. Developing strategic partnerships based on comparative advantage, cost effectiveness and collaboration Using the results of the four studies, out lined below as well as an extensive lite rature review mainstreaming principles were applied. An extensive literature review on HIV/AIDS was conducted as part of the process of collating information important for the development of a curriculum on HIV and placing it into a rehabilitation framework such as the International Classification of Function (ICF) (Myezwa 2008). The review includes explanations of the genesis of the disease, principal modes of transmis sion, evaluation of drug trials and other forms of treatment potentially used to slow down the degenerative process associated with HIV disease (Azzam 2006, Volberding et al 2003, Evans and Scadden 2000). Other topics were HIV virology and subsequent staging and antiretroviral therapy. These topics were found to be fundamental in the training of all health workers, includ ing the physiotherapist (Myezwa et al in press; Flaskrud & Ungvarski, 1999). Thus the current basic sciences are an already used entry point at which HIV can be included and should be reviewed to ensure they correspond with what is required by physiotherapists. The first study established referral patterns of patients with HIV to physio therapy. This study contributed to under standing the different conditions present in patients with HIV and which ones are suitable for physiotherapy (Myezwa et al., 2005). Furthermore, the results showed how many patients affected were referred for physiotherapy. The second study involved 80 in- patients and 51 out-patients living with HIV and outlined their impairments, activity limitations and participation restrictions (Myezwa et al., 2009). The third study explored a focus group discussion (FGD) on clinicians’ and academics’ perceptions and expe riences with HIV to enrich the picture of what topics should concern physio therapists (Myezwa 2008). A fourth study used a Delphi technique to test consensus on curriculum content among academic staff at all eight univer sities that offer physiotherapy training in South Africa (Myezwa et al., in press). Introduction outline of mainstreaming HIV. One of the main advantages of mainstream ing HIV is that it brings the issue to the fore and into the core business agenda, so that it becomes part and parcel of a programme. Similarly, in education, Hunt et al. (1998) refer to mainstream ing as a method of taking a key goal of a learning area from the periphery and placing it into the centre and within the entire curriculum. Hunt (1998) acknowledges mainstreaming within the curriculum at subject level as an effec tive means of incorporating an impor tant topic into the curriculum. The focus of this paper is thus on mainstream ing HIV/AIDS into the physiotherapy curriculum and the discussion is struc tured around the key principles of this process which according to the UNAIDS (2002) are: Human immune deficiency virus (HIV) is a vast and complex subject with many implications for physiotherapy educa tion and practice. This paper attempts to assess the inclusion of HIV/AIDS into a physiotherapy curriculum and practice according to the United Nations defined mainstreaming criteria. The results of four studies Myezwa et al., 2009, Myezwa 2008, Van As 2009, Myezwa et al., 2005 are utilised and discussed to illustrate how they affect the South African physiotherapy curriculum. Corresponding Author: Hellen Myezwa University Of Witwatersrand 7 York Road Park Town 2093 Johannesburg E-mail: Hellen.Myezwa@wits.ac.za Corresponding Author: Hellen Myezwa University Of Witwatersrand 7 York Road Park Town 2093 Johannesburg E-mail: Hellen.Myezwa@wits.ac.za The rationale for using this approach can be found in Verbrugenn’s (2004)’s SA Journal of Physiotherapy 2012 Vol 68 No 1 35 1. The development of a clearly defined entry point for mainstreaming HIV/ AIDS principles can be integrated using find ings from the four studies by Myezwa et al., (2005, 2008, 2009 and in press). The same results from each study may apply to more than one principle, although the application may be differ ent. An audit of all eight physiotherapy curricula to determine the extent of HIV inclusion revealed that the majority of physiotherapy curricula did not compre hensively include HIV (Myezwa 2008) Most of the relevant content was loosely woven into the curriculum and arbitrarily dependent on the interest of the lecturer. impairment and subsequent impact on the patients’ activity levels and quality of life was also ascertained (Myezwa et al., 2009, Rusch et al., 2004). Introduction Common systems, that are considered pertinent to physiotherapy practice, and are affected by HIV are cardiopulmonary (Diaz 1999), haematological (Evans & Scadden, 2000; Mars, 2004), metabolic (Mutimura et al., 2008a,c), neurological (Ricardo-Dukelow et al., 2007, Cherner et al., 2002) and musculoskeletal systems (Sattler et al., 1999; Sattler et al., 2002). Experiences of clinicians and academics emphasise the need to include HIV effects into the teaching of the different body systems. The manage ment of each of these systems is already taught and commonly seen in the clinical setting. The specific effects of HIV on multiple systems are important in teach ing and clinical reasoning. For example, HIV directly affects either progenitor cells or indirectly through opportunis tic infections, it can produce symptoms that are from multiple aetiologies, thus impacting on clinical reasoning. Similarly is: dyspnoea may be as a result of the direct effects on the haematologi cal system or the pathological effects of opportunistic and recurrent infections, like pneumocystic jiroveci pneumonia on oxygen exchange. Thus by implica tion, these differences in causes and their effects on treatment should be addressed during training by the respective area of speciality. Similarly, impairments such as pain, fatigue and loss of body mass commonly occur in HIV. Physiotherapy curricula include the basic structure, function and relevant pathogenesis of systems related to these impairments (CSP, 2008b) and therefore entry points for teaching on how HIV impacts on these systems can be identified. The emergence of HIV, however has affected the aetiology and morbidity of all the conditions traditionally treated and this is further complicated by problems of stigma, fear and the need for a very strong health and inter-sectoral support system. A decision thus would need to be made, as to how much content on HIV/AIDS should be included in under impairment and subsequent impact on the patients’ activity levels and quality of life was also ascertained (Myezwa et al., 2009, Rusch et al., 2004). Common systems, that are considered pertinent to physiotherapy practice, and are affected by HIV are cardiopulmonary (Diaz 1999), haematological (Evans & Scadden, 2000; Mars, 2004), metabolic (Mutimura et al., 2008a,c), neurological (Ricardo-Dukelow et al., 2007, Cherner et al., 2002) and musculoskeletal systems (Sattler et al., 1999; Sattler et al., 2002). Experiences of clinicians and academics emphasise the need to include HIV effects into the teaching of the different body systems. Principle 1: A clearly defined entry point for mainstreaming HIV/AIDS must be exposed to basic knowledge of HIV and its mode of transmission. Thereafter it would follow for all depart ments to identify specific areas within their courses where inclusion of HIV is pertinent. p The study exploring the perceptions of clinicians and academics categorised curricula concerns at macro, meso and micro levels (Myezwa 2008). Under macro level issues, participants sup ported the need to include HIV because of its high prevalence and thus a national priority. An understanding of the current policies and frameworks is important for physiotherapists and would help them in defining their role and making informed decisions. Only one of the seven universities, whose curricula were reviewed, reflected policies and frame works (Myezwa 2008). Verbruggens (2004) proposes the use of frameworks to locate and contextualise the specific place for HIV in any area of work. For physiotherapists, Worthington’s (2005) rehabilitation approach which under scores a multisectoral approach, and the ICF approach, (Anandan et al., 2006, Hwang & Nochajski, 2003) can be used as they account for disability in different life domains. Using ICF would include individual needs in physical, functional, psychological, spiritual, social and vocational aspects. At the meso level participants expressed a fragmented and uncoordinated approach to HIV curricula input. This may be indicative of the lack of teaching around a frame work that “pulls” the subject together. The impacts of HIV on the socio-eco nomic and socio-cultural environment of the patient should also be considered. The impact on the body systems results in a wide range of impairments, activity limitation and disability. Myezwa et al (2009),Van As et al., (2009); Zonta et al., (2003) ; Zonta et al., (2005) revealed clear patient profiles. Understanding the particular patient profiles allows the practitioner and the academic to give better guidance on the assessment of people with HIV or AIDS and to inte grate relevant HIV knowledge attitudes and skills. One line of thought is that people with HIV/AIDS already expe rience similar problems to other patient groups seen by physiotherapists and therefore skills and knowledge should be transferable and tapped. It is a valid argument that physiotherapists have the fundamental knowledge and skills to assess and treat patients, however further research is necessary to establish if this assumption is true. In some cases there maybe information that is particular to the HIV infected patient that may need to have attention. Principle 1: A clearly defined entry point for mainstreaming HIV/AIDS APPLYING MAINSTREAMING PRINCIPLES The paper provides how, within the teaching of physiotherapy, the five Recent graduates and academics (Myezwa 2008) felt that the way HIV Reports in the literature have explored the effect of HIV on body systems. The SA Journal of Physiotherapy 2012 Vol 68 No 1 36 manifestations (Nixon et al., 2010, 2005; Rusch et al., 2004). Aerobic exercise primarily changes the mitochondrial and cytosolic proteins while resisted exer cise increases muscle protein (Zinna & Yarasheski, 2003). The effects of exer cise on specific HIV conditions such as lipodystrophy (Mutimura et al., 2007) fatigue and body mass changes for example, were areas that experienced and newly qualified academic and clinical staff recommended input. Other interventions such as electrotherapy, use of aids and appliances massage are still being investigated for their effects on HIV/AIDS. These investigations have shown positive clinical effects, how ever, most studies are still challenged by small sample sizes, difficulties with blinding, standardisation and poor iso lation of treatment, due to the nature of treating HIV. (Fulk et al., 2004; Galantino et al., 1999; Posner et al., 2004; Shor- Posner et al., 2004; Worthington et al., 2005). Incorporation into teaching should therefore take cognisance of the levels of evidence. manifestations (Nixon et al., 2010, 2005; Rusch et al., 2004). Aerobic exercise primarily changes the mitochondrial and cytosolic proteins while resisted exer cise increases muscle protein (Zinna & Yarasheski, 2003). The effects of exer cise on specific HIV conditions such as lipodystrophy (Mutimura et al., 2007) fatigue and body mass changes for example, were areas that experienced and newly qualified academic and clinical staff recommended input. Other interventions such as electrotherapy, use of aids and appliances massage are still being investigated for their effects on HIV/AIDS. These investigations have shown positive clinical effects, how ever, most studies are still challenged by small sample sizes, difficulties with blinding, standardisation and poor iso lation of treatment, due to the nature of treating HIV. (Fulk et al., 2004; Galantino et al., 1999; Posner et al., 2004; Shor- Posner et al., 2004; Worthington et al., 2005). Incorporation into teaching should therefore take cognisance of the levels of evidence.i was included in the existing curricula was problematic and further analy sis indicated that institutions had not looked comprehensively at the issues of HIV content, mode of delivery and integration of HIV-related aspects in the general curriculum. Principle 1: A clearly defined entry point for mainstreaming HIV/AIDS An example of this is the reduced proximal muscle power seen in different patient groups (Myezwa et al., 2009) therefore specific assessment focus should be included in the cur riculum. Another example is why on an activity and participation level patients reported no community integration. They attributed this restriction to fear of not being able to find a toilet quickly enough thus had self-imposed restriction in movement and participation in com munity activity (Myezwa et al., 2009). In defining entry points, the challenge for the lecturer is to keep a balance in equipping the graduate with the neces sary knowledge while avoiding con tinuous additions to the curriculum and balancing undergraduate time. In spite of this, much of the identified HIV con tent can be included in existing content and need not lead to additional lectures or more time, but adding to how HIV changes the clinical picture and in doing this, mainstream the content. It is relatively easy to apply the ICF as most studies show how HIV causes numerous impairments that impact on function or quality of life (Myezwa et al 2009, 2011, Van As et al 2009., Anandan et al., 2006; Vidrine et al., 2003; Zonta et al., 2005). Specific aspects of func tion and quality of life such as mobility, instrumental activities and self-care activities were illuminated in these stu dies. These are areas with which physio therapy is concerned and therefore the ICF framework and Worthington’s framework are appropriate to be applied to HIV. The ICF which is in the physio therapy curriculum is considered as a generic approach that facilitates clini cal reasoning through the continuum Principle 2: Mainstreaming should take place within existing policies, philoso- phies and frameworks Policies and frameworks are assessed at two levels namely the macro and micro levels. In response to the call for national responses and incorporation of HIV into all programmes by UNAIDS, the government called for all sectors to respond by incorporating HIV into their core business (Department of Health, 2007b). All eight universities have HIV policies that guide the implementation of response to infection and inclusion in subject matter. For example, the University of the Witwatersrand policy states that all university undergraduates Another entry point for mainstream ing education about HIV could be during teaching on rehabilitation and physiotherapy interventions, including maintaining or increasing the level of physical activity and exercise. Exercise, in particular, as part of behaviour modi fication and change in life style, has been shown to provide a positive contribu tion to the prevention and care of HIV SA Journal of Physiotherapy 2012 Vol 68 No 1 37 mented inclusion of the subject, which was not dependent on current policies, prevalence clinicians’ experiences, the number of patients or their clinical pro file. Clinicians supported this interpre tation, by stating that their own input on HIV, as students, had been somewhat fragmented and resulted in knowledge gaps. Students and qualified therapists should be equipped with the neces sary skill to apply known principles of physiotherapy and rehabilitation to their treatment of HIV and they need an appro priate level of knowledge (Puckree et al., 2004; Puckree et al., 2002; Ukwakwe, 2000). Use of evaluation and self- directed learning to extend their capa city and skills in managing HIV should be actively encouraged to augment their ability to manage HIV, with its ever changing profile (Hunt et al., 1998). of problems i.e. impairment to envi ronment. Related to the ICF and the bio-psychosocial approach are service delivery models such as CBR. CBR is an already well understood concept that is taught to physiotherapists. CBR prin ciples allow the rehabilitation of a client within a community focussed paradigm (Helander,1994). The role of the physiotherapist will be fluid and will change depending on whether they are in a hospital, out- patient or community setting, or if the patient is in a period of wellness or ill ness. The hospital setting may call pre dominantly for their clinical skills and working within a multi-disciplinary team. Principle 3: Advocacy sensitisation and capacity Building Advocacy is a process of active sup port that involves acting as a liaison or canvassing for a particular cause to be considered (Jacobs & Jacobs, 2004). Once HIV is recognised as requiring advocacy it is necessary to get support from the people who are instrumental in the implementation of intervention programmes. What sets HIV apart from other conditions is its pervasive nature, a high prevalence and the complexities it presents for the clinical situation. One clinician sums it up in this statement: For example, the role of physiother apy in HIV falls within the educational outcome of understanding of physio therapeutic principles, philosophies and problem-solving with due consideration for disease processes. Working from what is known to the unknown is a known teaching and learning strategy (CSP, 2008b) and can it be used for sensitisa tion and capacity building ( Myezwa et al in press). For effective capacity build ing, gaps and omissions in the curriculum are a good starting and must be addressed for both the undergraduate and the quali fied staff. To effect real change, advocacy is needed to ensure the required change to the curriculum. Clinicians reported feeling over whelmed by the number of patients, their inability to define what their role is, and their limited background knowl edge of specific HIV related conditions. The presence of co-morbities in the patients seen and clinicians’ inability to deal with them was seen as problematic. In addition to building capacity that concerns clinical practice and teaching, HIV is a condition fraught with major complications that influence the indi vidual. Issues of stigma, disclosure and fear have been documented as almost paralysing health workers into inaction (Vance & Denham, 2008). This may underscore the need to build capacity among physiotherapists and deal with HIV in an inter-professional manner as the presenting problems are multifac torial. “I think it’s so vast and that it actually connects with so many other conditions that we could integrate it. HIV is part of everything because HIV affects your pathology, your recovery, your rehab, whatever.” (Clinician) For physiotherapy, mainstreaming HIV into education and practice one needs the facts to use as advocacy tools. The proposed curriculum content frame work developed from the results of all the studies by Myezwa et al (2005, 2008, and 2009, in press) can be used as an advocacy tool. Clinicians stated that their role in HIV management was unclear. Principle 3: Advocacy sensitisation and capacity Building No evidence of an outcome to increase the under standing of physiotherapeutic principles, philosophies and problem-solving with due consideration for the impact of HIV/ AIDS input was found (Myezwa 2008). Approaches to gaining a better under standing of the complexities of HIV management that are currently proposed include the use of problem-based learn ing, and inter-professional education. Other proposals include using people living with HIV/AIDS as facilitators of HIV education sessions and developing trans-disciplinary management mod els (Canadian Working Group on HIV and Rehabilitation, 2002b; Solomon et al., 2003b). Evidence suggests that problematic attitudes among health workers, as well as a lack of accurate information about HIV AIDS can make a significant negative impact on the health worker’s approach to HIV. Capacity building and training can improve knowledge, skills and attitude increasing the level of com fort in dealing with those who are HIV infected (Kent et al., 2005). However, many clinicians expressed feeling ill- equipped and some academics and cli nicians’ showed a lack of knowledge as to how to manage HIV/AIDS (Myezwa 2008). The same study showed that most topics were viewed as inadequately taught or not taught at all. Reports on the curricula presented an ad-hoc and frag Principle 2: Mainstreaming should take place within existing policies, philoso- phies and frameworks For example, therapists may need to respond to acute problems such as neurological, respiratory or neuromus cular problems with co-morbidities. Palliative approaches may also be required (Uwimana & Louw, 2007). In an out-patient setting, the therapist may be required, to use a wellness approach that needs the patient with HIV to focus on managing their risks and maintaining their health (O’Brien et al., 2007). Many of the skills required to fulfil these roles are already known to physiotherapists and they need to access them in an HIV context. Principle 5: Importance of developing strategic partnerships With regard to the internal domain, many staff anxieties emerged from the focus group discussions that included their knowledge gaps and inappropriate attitudes and perceptions (Myezwa 2008). Meeting knowledge gaps and addressing attitude problems in both undergraduate and qualified staff may impact on clinical decision-making and educational input. The impact on such fundamental educational activi ties may present a risk that HIV/AIDS will not be included appropriately into both education and practice. Another aspect that clinicians reported as hav ing a direct impact on their ability to assimilate and apply knowledge on HIV, was the lecturers’ attitudes and beliefs. Clinicians reported how the demeanour of the lecturer affected their impressions of HIV. Clinicians emphasised the need for a positive and informed outlook in managing HIV/AIDS, in particular, avoiding behaviours such as, treating the condition and not the person, being judgemental and viewing HIV, as a death sentence. This perception, on the part of students, supports Eisner’s (1992) notion that the impact of the implicit and null curriculum does influence what students learn. They recommended that important changes that help engender a positive outlook to the management of HIV/AIDS were that the prognosis has changed with the advent of ARVs and that opportunistic infections can be managed. Loss of hope, loss of morale, and patient overload affected therapists on a personal level. These responses are in no way peculiar to qualified therapists and it would be expected that students are affected in the same way. To mini mise these effects improved knowledge of HIV nature, impact on both body and health systems and counselling should be provided. The range and complexity of problems found when patients were assessed using the ICF checklist show that, in order to be effective in managing HIV patients therapists need to work with other sec tors such as social services, to enhance their rehabilitation outcomes and qua lity of life (Myezwa 2009). In addition disparities evident in the physiotherapy HIV curricula content, demonstrate the need for promoting partnerships between universities in order to maximize on expertise and experience. Strategic part nerships that ensure that mainstreaming and integration of HIV is done effi ciently and with some level of quality assurance will also require coorporation at the level of the regulatory and sup portive bodies such as other universities, the Health Professions Council of South Africa (HPCSA) and the South African Society of Physiotherapy (SASP). Principle 4: The distinction between the internal and external domains In Myezwa’s study (2008), academics and clinicians, in the ory embraced the policies and theories guiding input in relation to HIV, but did not appear to put these into practice. Principle 5: Importance of developing strategic partnerships The internal and external domain problems point to the need to also strengthen the collaboration between hospitals and other health care delivery institutions. Doing so will help to define the problem of HIV better and to respond through evidence based and expertise informed knowledge, practice and experience (Canadian Working Group on HIV and rehabilitation, 2002a). The Canadians recognised that this type of strategic partnership is required and they have formed partnerships to bring expertise together across sectors and within reha bilitation programmes. Principle 4: The distinction between the internal and external domains The internal domains refer to staff risks and vulnerability. The external domains refer to the institutional efforts in support of national, local and strategic efforts. Some results point to issues that threaten management and mainstreaming of HIV. In terms of the external domain, aca demic and clinical staff are aware of the need to include HIV as there are national calls to do so (Department of Education, SA Journal of Physiotherapy 2012 Vol 68 No 1 38 with current evidence and to create an environment where inclusion of HIV/ AIDS becomes a real possibility. A number of areas, such as the different body systems, pain, function, activity limitation, the effect of the environment and social determinants where physio therapists already work, have been pointed out as suitable entry points for introducing HIV/AIDS information. Ensuring that HIV content is incorpo rated in these areas would successfully fulfil the application of the first main streaming principle. The level of detail and the process of learning should be determined by the educators themselves. Unless educators embrace the new con tent required and successfully integrate it into curricula, the mainstreaming of HIV will remain ineffective and fragmented. Any clinician or academic using these mainstreaming principles can examine their input and identify where HIV has an impact. The ICF is a tool to use in framing the content in each area of teach ing. For inclusion in the curriculum, academics and practitioners, perhaps through the use of continuous profes sional development, need to be sensi tised to what is involved and need to be trained in areas where gaps in knowl edge, practice and inappropriate attitudes exist. To ensure HIV is mainstreamed into curricula and practice, partnerships between universities, practice areas such as hospitals and communities, patient organisations and regulatory bodies, should be developed. one (Myezwa 2009), and principle three all contribute to both internal and exter nal domain impacts. Structural issues such as problems experienced around disclosure of HIV status and increased work load have a negative impact on staff, both personally and professionally, for example, (Ukwakwe, 2000). 1999; Department of Health, 2007b;). In addition, the inclusion and integra tion of HIV is required in response to the increased prevalence and chang ing clinical picture. References Anandan N, Braveman B, Kielhofner G, Forsyth K. 2006. Impairments and perceived competence in persons living with HIV/AIDS. Work: A Journal of Prevention, Assessment and Rehabilitation 27:255-66 Azzam R, Kedzierska K, Leeansyah E, Chan H, Doischer D, Gorry PR, Cunningham AL, Crowe SM, Jaworowski A 2006 Impaired complement mediated phagocytosis by HIV type 1 infected human monocyte derived macrophages involves camp dependent mechanism. AIDS Research and Human Retroviruses 22:619-29 Azzam R, Kedzierska K, Leeansyah E, Chan H, Doischer D, Gorry PR, Cunningham AL, Crowe SM, Jaworowski A 2006 Impaired complement mediated phagocytosis by HIV type 1 infected human monocyte derived macrophages involves camp dependent mechanism. AIDS Research and Human Retroviruses 22:619-29 Conclusion Identifying entry points and applying mainstreaming principles one reveals challenging points of entry in both edu cation and practice. The range of options indicates the opportunity for introducing a topic so varied and changing as HIV/ AIDS. It raises the need to be abreast Canadian Working Group on HIV and rehabilita tion. 2002a Canadian Working Group on HIV and Rehabilitation. http://www.hivandrehab.ca/ EN/index.php Actual gaps perceived in HIV curricu lum content, discussed under principle 39 SA Journal of Physiotherapy 2012 Vol 68 No 1 Jacobs K, Jacobs L. 2004. Quick Reference Dictionary for OT. Massechusettes USA: Slack incorporated O’Brien K, Davis A, Strike C, Young N, Bayoumi A. 2007. Putting disability in to context. Factors that influence the experiences of “disabi lity” for adults living with HIV/AIDS. In WCPT Vancouver Canada Canadian Working Group on HIV and Rehabilitation 2002b Policy Issues on Rehabilitation in the Con text of HIV Disease. A background and position paper. http://www.hivandrehab.ca/EN/index.php Kent A, Myer L, Flisher AJ, Mathews C, Lombard C 2005 Introducing HIV/AIDS to South African health sciences students. Medical Teacher 27:647-9 Cherner M, Masliah E, Ellis RJ, Marcotte TD, Moore DJ 2002 Neurocognitive dysfunction pre dicts postmortem findings of HIV encephalitis. Neurology 59:1563-7 Posner GS, Miguez MJ, Hernandez-Reif M, Perez-Then E 2004 Massage treatment in HIV-1 infected Dominican children: A preliminary report on the efficacy of massage therapy to preserve the immune system in children without antiretro viral medication. The Journal of Alternative and Complementary Medicine 10:1093-5 Mutimura E, Stewart A, Rheeder P, Crowther NJ. 2007. Metabolic function and the prevalence of lipodystrophy in a population of HIV-infected African subjects receiving highly active Antiretroviral therapy. Journal of Acquired Immune Deficiency Syndrome. 46:451-5 CSP. 2008b. CSP Physiotherapy Curriculum Framework Website for Curriculum Framework http://www.csp.org.uk/director/libraryandpublica tions.cfm Puckree T, Chetty BJ, Govender V, Ramparsad S, Lin J 2004 Are physiotherapy graduates adequately prepared to manage HIV/AIDS patients. South African Journal of Physiotherapy 60:7 - 10 Department of Education. 1999. Department of Education Response to HIV/AIDS. http://wced.wcape.gov.za/planning&devel/sup port/special_ed/hiv_aids/National_policy_on_ HIV-AIDS.pd accessed October 2009 Myezwa H, M’kumbuzi V. 2003. Participation in Community Based Rehabilitation programmes in Zimbabwe: Where are we. Asia Pacific Disability Rehabilitation Journal 14:18-29 Puckree T, Kasiram R, Moodley M, Singh RM, Lin J 2002 Physiotherapists and Human Immuno deficiency Virus / Acquired Immune Deficiency Syndrome: Knowledge and prevention: A study in Durban, South Africa. International Journal of Rehabilitation Research 25:231-4 Department of Health. 2007b. Conclusion HIV/AIDS and STI national strategic plan for South Africa 2007-2011: HIV, AIDS and disability in South Africa South African National AIDS council 2008, Johannesburg Mars M 2004 HIV - implications for exercise in treatment and rehabilitation. South African Journal of Physiotherapy 60:9-17 Mutimura E, Crowther NJ, Cade TW, Yara sheski KE, Stewart A 2008a Exercise training reduces central adiposity and improves metabolic indices in HAART-treated HIV-positive subjects in Rwanda: A randomized controlled trial. AIDS Research Human Retroviruses 24:15-23 Ricardo- Dukelow M, Kadiu I, Rozek W, Schlautman J, Persidsky Y Ciborowski P, Kanmogne D Gendelman H 2007 HIV-1 infected monocyte-derived macrophages affect the human brain micro-vascular endothelial cell proteome: New insights into blood-brain barrier dysfunction for HIV-1-associated dementia Journal of Neuro- immulnology 185:37-46 Diaz PT, King MA, Pacht ER, Wewers MD, Gadek JE 1999 The pathophysiology of pulmonary diffusion impairment in Human Immunodeficiency Virus infection. American Journal of Respiratory Critical Care Medicine 160:272-7 exercise training on quality of life in HAART- treated HIV-positive Rwandan subjects with body fat redistribution. Quality of Life Research 17:377-85 Eisner, E. W. (1992). Curriculum ideologies. In The educational imagination (3rd ed., pp. 47-107): Macmillan Rusch M, Nixon S, Schilder A, Braitstein P, Chan K, Hogg R 2004 Impairments, activity limi tations and participation restrictions: Prevalence and associations among persons living with HIV/ AIDS in British Columbia. Health and Quality of Life Outcomes 2:46 Evans RH, Scadden DT 2000 Haematological aspects of HIV infection. Best Practice and Research Clinical Haematology 13:215-30 Myezwa H, Stewart A, Solomon P, in press Topics On HIV/AIDS for inclusion into a physical therapy curriculum; consensus through a modified Delphi technique. Journal of physical Therapy Education Flaskrud JH, Ungvarski P 1999 Overview and Update of HIV Disease, HIV/AIDS a Guide to Primary Care Management: J.H. 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University of the Witwatersrand Myezwa H, Stewart A, Musenge E Nesara P 2009 Assessment of HIV-positive in-patients using the International Classification of Functioning, Disability and Health (ICF), at Chris Hani Baragwanath Hospital, Johannesburg. African Journal of AIDS research Myezwa H, 2008 Mainstreaming HIV into physio therapy curriculum and practice. PhD thesis. University of the Witwatersrand Myezwa H, Stewart A, Musenge E Nesara P 2009 Assessment of HIV-positive in-patients using the International Classification of Functioning, Disability and Health (ICF), at Chris Hani Baragwanath Hospital, Johannesburg. African Journal of AIDS research Sattler FR, Schroeder ET, Dube MP, Jaque SV, Martinez C, Blanche PJ, Azen S, Krauss RM 2002 Metabolic effects of Nandrolone Decanoate and resistance training in men with HIV. American Journal of Physiology Endocrinology Metabolism 283:1214-22 Galantino MLA, Eke-Okoro ST, Findley TW, Condoluci D 1999 Use of noninvasive elec troacupuncture for the treatment of HIV-related peripheral neuropathy: A pilot study. Journal of Alternative and Complementary Medicine 5:135-42 Helander E. 1994. Prejudice and dignity. An introduction to Community Based Rehabilitation UNDP series. Myezwa H, Stewart A, ⁪Mbambo N, Nesara P 2005 Status of referral to physiotherapy among HIV positive patients at Chris Hani Baragwanath Hospital Johannesburg South Africa The South African Journal of Physiotherapy: 63 (2) 27-31 Solomon P, Salvatori P, Guenter D. 2003b. Inter- professional professional problem-based learning course on rehabilitation issues in HIV. Medical Teacher 25:408-13 Hunt A, Adamson B, Higgs J, Harris L 1998 University education and the physiotherapy pro fessional. Physiotherapy 84:264-73 Shor-Posner G, Miguez M-J, Hernandez-Reif M, Perez-Then E, Fletcher M 2004 Massage treatment in HIV-1 infected Dominican children: A pre liminary report on the efficacy of massage therapy to preserve the immune system in children without Antiretroviral medication: 1093-5 Shor-Posner G, Miguez M-J, Hernandez-Reif M, Perez-Then E, Fletcher M 2004 Massage treatment in HIV-1 infected Dominican children: A pre liminary report on the efficacy of massage therapy to preserve the immune system in children without Antiretroviral medication: 1093-5 Zonta MB, de Almeida SM, de Carvalho TM, Werneck LC 2005 Evaluation of AIDS-related disability in a general hospital in Southern Brazil. The Brazilian Journal of Infectious Diseases 9:479-88 Nixon 2010 Nixon S, O’Brien K, Glazier R, Tynan A 2005 Aerobic exercise interventions for adults living with HIV/AIDS. Cochrane Database Systematic Review 2 Hwang J, Nochajski S 2003 The International Classification of Function, Disability and Health (ICF) and its application with AIDS. Journal of Rehabilitation 69:4-14 SA Journal of Physiotherapy 2012 Vol 68 No 1 40 SA Journal of Physiotherapy 2012 Vol 68 No 1 Ukwakwe CBU 2000 Systemised HIV/AIDS education for student nurses at the University of Ibadan, Nigeria: Impact on knowledge, attitudes and compliance with universal precautions. Journal of Advanced Nursing 32:416-24 patients with AIDS disease. The Brazilian Journal of Infectious Diseases 7:301 - 6 patients with AIDS disease. The Brazilian Journal of Infectious Diseases 7:301 - 6 Vance R, Denham S. 2008. HIV/AIDS related stigma: Delivering appropriate nursing care. Teaching and Learning in Nursing 3:59-66 Vidrine DJ, Amick BC, Gritz ER, Arduino RC. 2003. Functional status and overall Quality of Life in a multiethnic HIV-positive population. AIDS Patient Care and STDs 17:187-97 Uwimana J, Louw Q. 2007. Effectiveness of palliative care including physiotherapy in HIV patients a literature review South African Journal of Physiotherapy 63:41 - 6 Volberding PA, Baker KR, Levine AM. 2003. Human Immunodeficiency Virus. Hematology 2003:294-313 UNAIDS. 2002. UNAIDS mainstreaming HIV/AIDS: A Conceptual Framework and ImplementingPrinciples.Accra http://www.unaid srstesa.org/Documents/thematic_areas/main streaming.html accessed April 2009 Worthington C, Myers T, O’Brien K, Nixon S, Cockerill R 2005 Rehabilitation in HIV/AIDS: Development of an expanded conceptual frame work. AIDS Patient Care & STDs 19:258-71 Verbrugenn B 2004 Mainstreaming HIV AIDS. Organisation Paper Frankfurt: GTZ/UNAID Zinna E, Yarasheski KE 2003 Exercise treat ment to counter act protein wasting of chronic diseases Current Opinion in Clinical Nutrition and Metabolic Care 6:87-93 Zinna E, Yarasheski KE 2003 Exercise treat ment to counter act protein wasting of chronic diseases Current Opinion in Clinical Nutrition and Metabolic Care 6:87-93 Zonta MB, de Almeida SM, de Carvalho TM, Werneck LC 2003 Functional assessment of Van As M, Myezwa H, Stewart A, Maleka D, Musenge E 2009 The international classification of function (ICF) in adults visiting the HIV outpatient clinic at a regional hospital in Johannesburg, South Africa. Aids Care 21:1 50-58 Zonta MB, de Almeida SM, de Carvalho TM, Werneck LC 2003 Functional assessment of If you’re looking for extra special service where the practitioner as well as the patient’s needs are handled with the utmost care ... AccountsdotCom is the solution! Nixon 2010 Phone now 012 653-7200 or 012 653-7122 or fax 012 653-7850 or e-mail accountsdotcom@gmail.com If you’re looking for extra special service where the practitioner as well as the patient’s needs are handled with the utmost care ... AccountsdotCom is the solution! Phone now 012 653-7200 or 012 653-7122 or fax 012 653-7850 or e-mail accountsdotcom@gmail.com SA Journal of Physiotherapy 2012 Vol 68 No 1 41
https://openalex.org/W4307810463	https://kth.diva-portal.org/smash/get/diva2:1712957/FULLTEXT01	English	null	Evaluation of the structural response of two in-service thin flexible pavements under heavy vehicle loading during different seasons by built-in sensors	International journal of pavement engineering/The International journal of pavement engineering	2,022	cc-by	10,546	International Journal of Pavement Engineering ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gpav20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=gpav20 Introduction These roads play an important role in the Swedish economy in particular for the mining and timber industries. In recent years, Longer Heavier Vehicles (LHVs) have become increasingly more popular. LHVs have a higher load-carrying capacity compared to conventional heavy- duty vehicles which can be achieved by increasing the num- ber of axles, length of the vehicles, axle load, and total vehicle weight. Multiple studies have reported that the use of LHVs leads to improvements in transportation cost eﬃciency (Ortega 2014; Sanchez Rodrigues 2015) and improved environmental performance (Knight 2008; Christi- dis and Leduc 2009). Most of the research on the impact of LHVs within the transport sector focuses on the vehicles themselves and not on the potential increase in pavement deterioration (Pålsson 2017). The inclusion of the impact of LHVs on pavement construction and maintenance-related costs could improve the accuracy of economic analyses related to LHVs. p g The environmental variables such as temperature and moisture, that surround pavement structures aﬀect consider- ably their structural behaviour (Doré and Zubeck 2009). It is therefore important to account for the seasonal variation of the environmental variables when analysing the performance of pavements, in particular in areas with large seasonal vari- ations. The temperature and moisture distribution in the cross-section is largely inﬂuenced by the boundary conditions of the system. Thus, larger variations occur in the upper part of the structure due to the proximity to the surface boundary. The temperature variations aﬀect the stiﬀness of the asphalt layers. Frost penetrating into the structure makes the unbound layers stiﬀand moisture lubricates the granular layers and soft- ens them, particularly during spring thaw (Simonsen and Isacsson 1999; Salour and Erlingsson 2013a; Salour 2015; Erlingsson et al. 2017). The maximum allowable gross vehicle weight (GVW) in Sweden has increased from 60 tons in 1996, to 64 tons in 2015 by the Swedish Transport Administration (Erlingsson et al. 2018). On a limited number of roads, a higher loading of 74 tons is allowed since 2018 as well, an increase which is planned to be implemented on the entire road network in the future (Natanaelsson and Eriksson 2020). A large number of roads in the national network are low volume roads which typically consist of a thin asphalt layer or layers placed over one or more granular layers on top of a compacted subgrade. KEYWORDS Thin ﬂexible pavement; built-in sensors; in-situ response measurement; long heavy vehicle; numerical modelling; seasonal variation © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 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, dis- tribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Denis Saliko denis.saliko@vti.se Denis Saliko & Sigurdur Erlingsson To cite this article: Denis Saliko & Sigurdur Erlingsson (2022): Evaluation of the structural response of two in-service thin flexible pavements under heavy vehicle loading during different seasons by built-in sensors, International Journal of Pavement Engineering, DOI: 10.1080/10298436.2022.2138875 To link to this article: https://doi.org/10.1080/10298436.2022.2138875 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 29 Oct 2022. Submit your article to this journal Article views: 122 View related articles View Crossmark data © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 29 Oct 2022. Submit your article to this journal Article views: 122 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=gpav20 INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING https://doi.org/10.1080/10298436.2022.2138875 Evaluation of the structural response of two in-service thin ﬂexible pavements under heavy vehicle loading during diﬀerent seasons by built-in sensors Denis Saliko a,b and Sigurdur Erlingsson a,c aPavement Technology, Swedish National Road and Transport Research Institute VTI, Linköping, Sweden; bDepartment of Building Materials, KTH Royal Institute of Technology, Stockholm, Sweden; cFaculty of Civil and Environmental Engineering, University of Iceland, Reykjavik, Iceland ARTICLE HISTORY Received 20 November 2021 Accepted 17 October 2022 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 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, dis- tribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT ABSTRACT Long Heavy Vehicles (LHV) are considered more eﬃcient and environmentally friendly transportation of goods compared to conventional trucks. Thus, the maximum allowable gross vehicle weight (GVW) in Sweden was increased on part of the road network from 64 to 74 tons in 2018 by increasing the vehicles’ length and the number of axle groups per vehicle but not the axle load limits. This change in loading conditions is expected to lead to changes in the structural response and degradation rate of thin pavements on the low-volume road network. To improve our understanding of thin pavements behaviour exposed to multiple axle loadings two thin pavement structures located in the north of Sweden were instrumented with road response and climate sensors. Four measurement campaigns were carried out within one year by in-situ stress and strain measurements from the built-in sensors as LHV passes over at normal speed. The recorded response was compared with numerical calculations based on multilayer elastic theory (MLET). Values of stresses and strains showed a generally good agreement with high values of coeﬃcient of determination R2 during diﬀerent seasons when the asphalt stiﬀness values were adjusted based on temperature and granular layer stiﬀness values based on moisture. Introduction A new Mechanistic-Empirical (M-E) pavement design and performance prediction tool is currently under development in Sweden. It is essential that the response analysis part of the tool can predict the actual induced response in the pavement struc- ture due to the heavy loadings under the prevailing ambient climate conditions. It is therefore important that the M-E scheme considers the shift in vehicle types and the various axle conﬁgurations of the vehicles with higher GVW. Several methods are available for modelling the induced response of D. SALIKO AND S. ERLINGSSON 2 2 pavement structures, among them the multilayer elastic theory (MLET) and the ﬁnite element method (FEM). FEM is able to model more complex loading conditions, geometries and can incorporate more advanced material models. MLET requires several background assumptions such as isotropic and hom- ogenous layers extending inﬁnitely in the horizontal direction and the applied loading being circular with constant pressure (Burmister 1945; Huang 2004; Erlingsson and Ahmed 2013). However, due to faster computation times, MLET has become the common option of choice in pavement design (ARA Inc. 2004; Loulizi et al. 2006). It has further been shown that the diﬀerences in calculated pavement response values between MLET and FEM are negligible in many cases (Saevarsdottir and Erlingsson 2015; Fladvad and Erlingsson 2022). The vali- dation of the pavement response models requires ﬁeld testing (Ahmed and Erlingsson 2013; Pereira and Pais 2017; Gkyrtis et al. 2021) through instrumented road sections in which sen- sors are embedded within the pavement structure to measure the actual response (Gusfeldt and Dempwolﬀ1967; Hicks and Finn 1970; Terrell and Krukar 1970; Al-Qadi et al. 2004; Blanc et al. 2019; Duong et al. 2019). surface. The εMU coils and SPCs were installed in the granular layers of the structure to measure the induced vertical strain and stress respectively. More details on the sensors used in this study can be found in Saevarsdottir et al. (2016). y The installation procedure began with the removal of the top AC layer along 20 m in one of the two lanes. Afterwards, two trenches each of them 1.2 m wide were excavated with a distance of 4 m away from each other. The storage of the exca- vated material was done separately for each layer to avoid mix- ing of material from diﬀerent layers. All existing layer thicknesses were determined during the excavation process. Introduction After the excavation was completed, the sensors were installed inside the pavement structure. The excavated material was placed back and compacted layer-by-layer. Finally, a new layer of AC (ABT16) was repaved. The procedure of the sensor installation is illustrated in Figure 1. Pictures taken during the installation procedure are shown in Figure 2. The layer types and thicknesses of the cross-sections of the two pavement structures are illustrated in Figure 3. Both struc- tures are old pavement structures and have had a maintenance history over the years. In the case of Lv373, the original cross- section consisting of cutback AC placed on top of a subbase and a sandy clay layer has been overlaid two times. The ﬁrst time the structure was overlaid by an unbound granular material (UGM) base course, and a thin surface treatment. Later a new unbound subbase and base course, and a 10 cm AC layer were added. Lv515 is a simpler structure consisting of a thin 4 cm AC layer and a 15 cm unbound base course placed above a natural sandy subgrade. g In this paper, the response signals obtained from two instrumented thin ﬂexible pavements subjected to loading by three diﬀerent LHVs have been evaluated at four diﬀerent times of measurements over 12 months. The primary aim is to investigate the eﬀects of seasonal variations of the environ- mental variables on the response of pavement structures due to LHV. Furthermore, the response signals have been compared against MLET-calculated values. The layer stiﬀness values were obtained by falling weight deﬂectometer (FWD) backcal- culation (Irwin 2002) and simultaneously adjusted for temp- erature and moisture variations to reﬂect the actual ﬁeld conditions. The measurement-calculation comparison of the instrumented test sections was used to validate the numerically predicted structural response to be used in the new M-E calcu- lation scheme. The installation of the sensors was performed in a bottom- to-top manner. First, the εMU coils were installed in one of the walls of a trench while SPCs were installed in the adjacent wall. After the εMU coils and SPCs were placed at a certain depth, the stored material from that layer was placed back and com- pacted. This was done for all the granular layers. Once all the UGM layers were placed back and compacted, ASGs were installed. Introduction The ASGs were not installed on top of the material located in the trench but on top of the base course outside of the trench. This was done to obtain a response that resembles more closely the existing conditions as the material in the trench was disturbed by the excavation. A new layer of AC (ABT16) was thereafter repaved on top of the ASG sensors. Description of the test sites and instrumentation procedure For this research presented here, two road sections were selected to be instrumented during autumn 2017 (Saliko and Erlingsson 2021). The sections are located in the northern part of Sweden along the roads Lv373 (section 58,980 m) and Lv515 (section 1360 m) notations by which they are referred to in this paper. The sections are located approxi- mately 10 km apart from each other. The coordinates for the location of Lv373 are N65.33741, E20.4675 and for Lv515 N65.31813, E20.29687. Monitoring the climate seasonal variation The seasonal variation of the environmental variables was monitored using thermocouples, time-domain reﬂectometer (TDR) probes, and frost rods. The thermocouples were used to monitor the temperature variation within the AC layer. For Lv373, the thermocouples were installed at 2.5, 5, and 7.5 cm depths. In the case of Lv515, the thermocouples were installed only at 2 cm depth due to the structure’s AC layer thickness limitation. The recording interval for the thermo- couples was set to one hour. To monitor the cross-sectional moisture variation, TDR probes were installed in the UGM of the structures. The TDR probes were placed on a 200 cm rod at 5 diﬀerent depths ranging from 36 cm to 196 cm with 40 cm spacing intervals. The volumetric moisture content values were logged every 20 min. The recorded temperature To monitor the mechanical response of the structures, asphalt strain gauges (ASGs), soil pressure cells (SPCs), and strain measuring units (εMU coils) were used. Two trenches were made in each of the structures in order to obtain two set-ups of instrumentation which reduces the risk of loss of data due to possible malfunctions. The ASGs were installed underneath the asphalt concrete (AC) layer in two directions, along the wheel path and perpendicular to it. This was done to measure the longitudinal and transversal strains in the bottom of the asphalt concrete layer when loading was applied on the INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 3 3 Figure 1. Illustration of the pavement instrumentation procedure (a) excavation procedure in plan-view, (b) excavation procedure in cross-section-view, (c) installation positions for the sensors, and (d) completed installation. Figure 1. Illustration of the pavement instrumentation procedure (a) excavation procedure in plan-view, (b) excavation procedure in cross-section-view, (c) installation positions for the sensors, and (d) completed installation. instrumentation procedure (a) excavation procedure in plan-view, (b) excavation procedure in cross-section-view, (c) installation mpleted installation. and volumetric moisture content values for both test sites are shown in Figures 4 and 5. interpolation between two consecutive sensors where one shows positive and the other negative temperature values to obtain the 0°C isotherm. The recorded temperature distri- bution and interpolated frost depth for the period 1 October 2018 until 22 June 2019 are presented in Figures 6 and 7. Monitoring the climate seasonal variation For both structures, the temperature of the asphalt layer and the moisture content in the granular layers have been monitored for a whole year from 1 August 2018 to 31 August 2019. The TDR probes recorded a low moisture content during the winter period followed by a sudden increase during the spring thaw and a gradual decrease during the following recov- ery period. Peaks in the moisture content levels are recorded during rainy events in the summer and autumn seasons. Since these structures are located in the north of Sweden, the spring thaw period starts in late April and is over in the middle of June. A limitation that applies to the data is the maximum depth of 200 cm due to the length of the frost rod. Nevertheless, it was possible to obtain the beginning of the freezing period, the approximate rate of frost penetration, the beginning of the thawing period, and the approximate rate of thawing. Backcalculation of the structural response from FWD Cross-sectional temperature variation and frost depth corresponding to the 0°C isotherm for Lv373 from 01 October 2018 to 30 June 2019. The dotted line represents the two dates when response measurements were made. INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 5 Figure 6. Cross-sectional temperature variation and frost depth corresponding to the 0°C isotherm for Lv373 from 01 October 2018 to 30 June 2019. The dotted line represents the two dates when response measurements were made. Figure 7. Cross-sectional temperature variation and frost depth corresponding to the 0°C isotherm for Lv515 from 01 October 2018 to 30 June 2019. The dotted line represents the two dates when response measurements were made. Figure 7. Cross-sectional temperature variation and frost depth corresponding to the 0°C isotherm for Lv515 from 01 October 2018 to 30 June 2019. The dotted line represents the two dates when response measurements were made. the main focus on the spring thaw period. Each testing cam- paign included falling weight deﬂectometer (FWD) measure- ments and sensor response registrations under controlled application of passing LHV vehicles. thoroughly layer by layer it was not possible to achieve the same compaction level as in the undisturbed material. This is reﬂected also in the FWD deﬂection bowls from 50 kN load- ing as shown in Figure 9. The FWD measurements were performed to later backcal- culate the stiﬀness values of the layers. Three diﬀerent load levels of 30kN, 50kN, and 65kN were applied. The loading was applied directly on top of the sensors. Afterwards, the 50kN load level was used as a reference for the comparison between the measured FWD deﬂection bowls and the calcu- lated bowls using the MLET-based software ERAPave (Erlings- son and Ahmed 2013). The algorithm used for the backcalculation procedure is illustrated in Figure 8. As illustrated in Figure 9, the graphical comparisons between the measured and MLET-calculated deﬂection bowls show a generally good agreement. An exception to this is the backcalculation for road 515 during the August 2019 campaign illustrated in Figure 9(d) in which a lower qual- ity of ﬁt can be noticed. Backcalculated and measured deﬂec- tion bowls from FWD loading with 30 and 65 kN exhibited a similar quality of ﬁt and therefore it was decided to initially perform a linear MLET simulation to determine whether it would be possible to capture the general response behaviour of the two pavement structures. Backcalculation of the structural response from FWD To monitor the temperature distribution and frost depth in the cross-section of the structures, frost rods were used. The frost rods were made of 41 temperature sensors placed every 5 cm on a 200 cm long bar. The logging was performed at 30-minute intervals. The frost penetration was obtained either directly from a temperature sensor recording 0°C or by linear Four diﬀerent testing campaigns over one year have been per- formed. The dates when the tests were carried out were 29–30 August 2018, 8–9 May 2019, 12–13 June 2019, and 21–22 August 2019. The dates were selected to capture the seasonal variation of the structural behaviour of the test sections with Figure 2. Pictures taken during the installation of the sensors used for the instrumentation procedure (a) ASGs – Asphalt Strain Gauges, (b) SPC – Soil Pressure Cell, and (c) εMU coil – Strain Measuring Unit coil. stallation of the sensors used for the instrumentation procedure (a) ASGs – Asphalt Strain Gauges, (b) SPC – Soil Pressure Cell, and il Figure 2. Pictures taken during the installation of the sensors used for the instrumentation procedure (a) ASGs – Asphalt Strain Gauges, (b) SPC – Soil Pressure Cell, and (c) εMU coil – Strain Measuring Unit coil. 4 D. SALIKO AND S. ERLINGSSON Figure 3. The layer and sensor conﬁgurations for the (a) Lv373 cross-section, and (b) Lv515 cross-section. Figure 3. The layer and sensor conﬁgurations for the (a) Lv373 cross-section, and (b) Lv515 cross-section. Figure 4. Recorded values at Lv373 for (a) the daily average AC temperature and (b) the moisture content variation of the unbound granular layers. Figure 4. Recorded values at Lv373 for (a) the daily average AC temperature and (b) the moisture content variation of the unbound granular layers. Figure 5. Recorded values at Lv515 for (a) the daily average AC temperature and (b) the moisture content variation of the unbound granular layers. Figure 5. Recorded values at Lv515 for (a) the daily average AC temperature and (b) the moisture content variation of the unbound granular layers. Figure 5. Recorded values at Lv515 for (a) the daily average AC temperature and (b) the moisture content variation of the un es at Lv515 for (a) the daily average AC temperature and (b) the moisture content variation of the unbound granular layers. INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 5 Figure 6. Backcalculation of the structural response from FWD For the backcalculation procedure, both the surface deﬂec- tion bowl obtained through the deﬂection sensors of the FWD equipment and the response of the instrumented sensors at the time when the loading was applied were used. The sensors’ response was checked by comparing the longitudinal and transversal strain recorded by ASGs to the calculated values at the bottom of the AC layer, recorded vertical stresses by SPCs, and vertical strains by εMU coils to the MLET-calcu- lated values at their corresponding depths. The measurements were divided into two groups. Measurements performed out- side the trench corresponding to the ASG sensors and measurements performed inside the trench corresponding to the εMU coils and SPCs. The reason for this choice was to take into consideration the disturbance in the excavated and recompacted material inside the trenches. Even though the material inside the trenches was inserted back and compacted In the process of backcalculating the layer stiﬀness values, the inﬂuence of temperature on the AC layers and the inﬂu- ence of moisture in the UGM was considered. In this study, the stiﬀness of the AC layer was adjusted according to (Erlingsson 2012) ET = ET,ref · e−b(T−Tref ) (1) (1) where ET is the predicted asphalt stiﬀness, Tref is the reference temperature (10°C), ET,ref is the asphalt stiﬀness 6500 MPa at the reference temperature, and b is a regression constant of 0.065. For Lv373, the AC surface and AC base were combined into a single layer as their individual thicknesses were only a small Figure 8. Flowchart illustrating the backcalculation procedure used in the study 6 D. SALIKO AND S. ERLINGSSON 6 D. SALIKO AND S. ERLINGSSON 6 Figure 8. Flowchart illustrating the backcalculation procedure used in the study. part of the total thickness of the cross-section. The tempera- ture value recorded at the time of FWD measurements from the thermocouple at 5.5 cm depth in the middle of the asphalt layer was used. For Lv515, the value at a depth of 2.0 cm was used. variation in moisture was recorded by the TDR probes. The volumetric moisture content values recorded by the TDR probes were converted into the degree of saturation and then implemented in the model. The default model par- ameters for coarse-grained material were used, a = −0.3123, b = 0.3, and ks = 6.8157 (ARA Inc. 2004). Backcalculation of the structural response from FWD Two diﬀerent opti- mal resilient moduli (Mr,opt) as 90 and 85 MPa and the opti- mal degree of saturation (Sopt) values were used as 20% and 15%, respectively. The higher values for the undisturbed material outside the trench are due to the higher degree of compaction. The variation of the stiﬀness values of UGM layers has been adjusted using the MEPDG MR-moisture model (Leong and Rahardjo 1997; ARA Inc. 2004; Andrei et al. 2009) expressed as a function of the degree of saturation log MR MR,opt = a + b −a 1 + exp (b + ks(S −Sopt)) (2) (2) (2) The backcalculation was performed iteratively. An initial set of layer stiﬀness values were assumed. To quantify and minimise the diﬀerence between the measured and the calcu- lated values, the root mean square error RMSE was used where MR is the predicted resilient modulus, MR,opt is the opti- mal reference modulus, S is the degree of saturation, Sopt is the optimal degree of saturation, a, b, and ks regression parameters and β = ln(–b/a). RMSE = n i=1 (yi −ˆy)2 n (3) The adjusted stiﬀness values according to temperature and degree of saturation are illustrated in Figure 10. (3) For the backcalculation procedure, the cross-sections of the structures were divided into sublayers. This was done to take into account the variation in moisture levels and whether the part of the structure during the measurements was frozen or thawed. For both structures, the frozen part of the cross- section was estimated at the time of measurement which is marked by a dotted line in Figures 6 and 7. In the case of Lv515, the subgrade was considerably thick and a large where yi represents a measured datapoint, ŷ a calculated data- point, and n is the total number of datapoints. Three RMSE values were deﬁned for the error minimisation procedure. The ﬁrst RMSE value was used to compare the sur- face deﬂections measured by the FWD to the calculated surface deﬂections by MLET at those locations. The typical RMSE values for the FWD backcalculation were in the range of 3–5 INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 7 Figure 9. Deﬂection bowls under 50kN FWD loading compared to MLET calculations for the campaign (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 9. Backcalculation of the structural response from FWD Deﬂection bowls under 50kN FWD loading compared to MLET calculations for the campaign (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 9. Deﬂection bowls under 50kN FWD loading compared to MLET calculations for the campaign (a) August 2018, (b 2019. N FWD loading compared to MLET calculations for the campaign (a) August 2018, (b) May 2019, (c) June 2019, and (d) August with the exception of the backcalculation for road 515 during the August 2019 campaign which had an RMSE value of 11. The second RMSE value was used to compare the stress and strain values recorded by the ASGs, εMU coils, and SPCs to the MLET-calculated stress and strain values at the known location of each respective type of sensor. Typical RMSE values for individual built-in sensor response under FWD loading range between 6 and 12. The third RMSE value was obtained through the diﬀerence between the assumed Mr – recorded degree of saturation values and the Mr-moisture model. The values of the RMSE used to adjust for the temperature and moisture variations ranged between 1 and 4. The stiﬀness values at the dates of measurements obtained through the iterative procedure are given in Tables 1 and 2. Both structures were partially frozen during the May and June measurement campaigns. This has been considered in the backcalculation procedure according to the depth interval where the frozen part of the cross-section was located (dfrost). For Lv373 it was possible to capture the upper boundary of the frozen part of the cross-section but not the full extension of the frozen part. For Lv515 it was possible to capture the upper boundary of the frozen part only during the May cam- paign. For the June campaign at Lv515, it was not possible to capture neither the upper nor the lower depth of the frozen Figure 10. Adjusted values of AC stiﬀness according to temperature and stiﬀness of granular layers according to the degree of saturation. Figure 10. Adjusted values of AC stiﬀness according to temperature and stiﬀness of granular layers according to the degree of saturation. D. SALIKO AND S. ERLINGSSON 8 8 8 Table 1. Backcalculated stiﬀness values at each measurement date for Lv373. Out. and In. represent the value sets outside and inside the trench. Layer Thickness Stiﬀness (MPa) 29 August 2018 8 May 2019 12 June 2019 21 August 2019 (cm) Out. In. Backcalculation of the structural response from FWD The values of the frost depth were obtained through a statistical-based empirical correlation developed for the con- ditions in Sweden, in which the frost penetration depth is obtained as a function of the freezing index determined by air temperature registrations at a speciﬁc location (Erlingsson and Saliko 2020). The empirical values were used as an initial estimate which was further reﬁned by interpolating the slope of the seasonally frozen zone from Figures 6 and 7. The stiﬀ- ness of the frozen layer was set to 2800 MPa which is in agree- ment with previous studies (Salour and Erlingsson 2013b). part. This was due to the 200 cm limited length of the frost rods. The values of the frost depth were obtained through a statistical-based empirical correlation developed for the con- ditions in Sweden, in which the frost penetration depth is obtained as a function of the freezing index determined by air temperature registrations at a speciﬁc location (Erlingsson and Saliko 2020). The empirical values were used as an initial estimate which was further reﬁned by interpolating the slope of the seasonally frozen zone from Figures 6 and 7. The stiﬀ- ness of the frozen layer was set to 2800 MPa which is in agree- ment with previous studies (Salour and Erlingsson 2013b). Backcalculation of the structural response from FWD Out. In. Out. In. Out. In. 1. Asphalt Concrete 10 6107 6107 10,756 10,756 4464 4464 5764 5764 2. Base Course 12 383 341 376 336 331 296 380 329 3. Subbase 26 266 197 259 191 256 187 248 177 4. Base Course 12 169 118 166 116 147 102 154 103 5. Tarmac 13 149 138 232 201 158 152 188 182 6. Subbase 30 148 88 143 84 169 101 161 97 7. Sandy Clay 15 129 76 117 71 133 79 131 77 8. Subgrade (dfrost) 42 64 51 2800 2800 49 39 47 37 9. Subgrade (dfrost) 45 74 74 2800 2800 2800 2800 68 68 10. Subgrade Unfrozen 1 75 75 105 105 72 72 76 76 Table 1. Backcalculated stiﬀness values at each measurement date for Lv373. Out. and In. represent the value sets outside and inside the trench. Stiﬀness (MPa) Table 2. Backcalculated stiﬀness values at each measurement date for Lv515. Out. and In. represent the value sets outside and inside the trench. Layer Thickness Stiﬀness (MPa) 30 August 2018 9 May 2019 13 June 2019 22 August 2019 (cm) Out. In. Out. In. Out. In. Out. In. 1. Asphalt Concrete 4 5160 5160 10,421 10,421 4605 4605 6138 6138 2. UGM Base 15 180 145 150 135 195 160 185 155 3. SG Sub.1 (TDR1) 37 93 76 90 74 100 82 97 78 4. SG Sub.2 (TDR2) 40 94 78 91 76 106 86 98 82 5. SG Sub.3 (TDR3) 40 85 73 78 69 95 80 89 75 6. SG Sub.4(TDR4,dfrost) 14 77 67 2800 2800 83 73 77 67 7. SG Sub.5 (dfrost,June) 10 85 85 2800 2800 2800 2800 87 87 8. SG Sub.6 16 95 95 2800 2800 2800 2800 97 97 9. SG Sub.7 (TDR5,dfrost) 84 107 107 2800 2800 2800 2800 108 108 10. SG Sub.8 (Natural SG) 1 107 107 95 95 96 96 108 108 Table 2. Backcalculated stiﬀness values at each measurement date for Lv515. Out. and In. represent the value sets outside an Stiﬀness (MPa) ted stiﬀness values at each measurement date for Lv515. Out. and In. represent the value sets outside and inside the trench. Stiﬀness (MPa) part. This was due to the 200 cm limited length of the frost rods. INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 9 INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 9 Figure 12. Half-axle and tyre conﬁguration, spacing, and IDs for the LHVs used in this study. All distances in cm. INTERNATIONAL JOURNAL OF PAVEMENT ENG Figure 12. Half-axle and tyre conﬁguration, spacing, and IDs for the LHVs used in this study. All distances in cm. The conﬁguration of vehicle loading (single vs. dual tyres, tyre pressures, number of axles, load per axle, total load, and total length) was diﬀerent for each of the three LHVs used. LHV1 had 9 axles, two of them having single tyres and seven with dual tyres, with a total length centre-to-centre of the ﬁrst to the last axle of 20.5 m and a total weight of approxi- mately 74 tons. LHV2 had seven axles, the driving axle with single tyres while the rest of the axles had dual tyre conﬁgur- ation. The centre-to-centre length between the ﬁrst and last axle for LHV2 was 19.74 m and the total weight was approxi- mately 64 tons. LHV3 had eight axles, seven of them with single tyres and one with dual tyres, an axle centre-to-centre total length of 20.8 m, and a total weight of approximately 68 tons. The axle weights and tyre pressures for each axle are shown in Table 3. The LHV1 had the lowest variation in axle loads, the lowest calculated average value of axle load and the lowest tyre pressure, despite having the highest total weight. LHV2 had the highest average axle load since the weight was distributed over a lower number of axles compared to the two other vehicles. For LHV3 multiple single tyres were used in the axel conﬁguration. The largest load for a single axle was measured in the case of LHV3. Vehicle response modelling The response of the structure under loading by LHVs was measured at each testing campaign afterwards performing the FWD testing. Three diﬀerent types of LHVs were used, one of which is shown in Figure 11. The loading was applied by driving the vehicles at a constant speed of 60 km/h (50 km/h at Lv515 in May 2019) over the sensors in a way that the outer wheels would be aligned along the wheel path directly on top of the built-in sensors. The location of the sen- sors had previously been marked on the surface. Figure 11. The LHV2 used for the response measurements. The axles are shown according to the used naming convention. Figure 11. The LHV2 used for the response measurements. The axles are shown according to the used naming convention. Results of the vehicle induced response For the calculations of the structural response of the test sec- tions under moving load by the LHVs, the MLET-based soft- ware ERAPave (Erlingsson and Ahmed 2013; Saevarsdottir et al. 2016; Ahmed and Erlingsson 2017) was used. The stiﬀ- ness values of the AC layers were adjusted for the tempera- ture at the time of testing and the stiﬀness values of the UGM layers were adjusted according to the moisture levels (Tables 1 and 2). The loading was modelled according to Table 3 and Figure 12 except to simplify the modelling pro- cess and to lower the computational time, the tyre pressure was considered constant at 850 kPa for all tyres which is A naming convention was established to identify each type of LHVs as well as individual axles and axle groups. Axles located close to each other are considered to belong to the same axle group. The ID assigned to an axle represents ﬁrst the axle group and then the number of the axle within that group. The axle conﬁgurations, identiﬁcation names, and axle spacing in centimetres for all three LHVs are shown in Figure 12. Before each testing campaign, the axle weights were measured by static weighing each LHV and the tyre pressures were measured using a regular tyre pressure gauge. Table 3. Measured axle loads F and tyre pressures p for the three LHVs used in the study. Type S and D represent single vs. dual tyres respectively. Axle LHV1 LHV2 LHV3 ID Type p F Type p F Type p F (–) (–) (kPa) (kN) (–) (kPa) (kN) (–) (kPa) (kN) A1-1 S 840 83.36 S 850 74.53 S 890 93.16 A2-1 D 750 83.85 D 700 93.65 D 700 114.25 A2-2 D 750 82.38 D 700 91.20 S 920 67.67 A2-3 S 840 65.70 – – – – – – A3-1 D 850 87.77 D 900 91.20 S 870 94.14 A3-2 D 850 90.22 D 900 89.73 S 860 95.61 A4-1 D 850 78.45 D 900 101.50 S 870 69.14 A4-2 D 850 79.43 D 900 100.03 S 890 72.08 A4-3 D 850 80.41 – – – S 890 74.53 ΣF 731.58 641.85 680.58 D. SALIKO AND S. ERLINGSSON 10 placement is longitudinal or transversal, the sensors can suc- cessfully capture the structural behaviour of the pavement in case the loading is applied at the correct position. Results of the vehicle induced response close to the average of all the tyre pressures. A sensitivity analysis on the inﬂuence of the variation of tyre pressure was performed which showed that the impact of diﬀerent tyre pressures is negligible for the range of the tested vehicles. The details of the sensitivity analysis have been omitted due to space and scope limitations. Any minor diﬀerences in driv- ing speed and positioning in transversal direction due to human error in driving were neglected. The vertical strain signals recorded by the εMU coils were noisier compared to the other types of sensors, in par- ticular in the case of Lv373 which consisted of several layers. These sensors were also observed to be the least durable. For Lv373, the two bottom εMU coils from one of the trenches were malfunctioning during three out of the four testing campaigns. For Lv515, the uppermost εMU coil from one of the trenches malfunctioned during the May 2019 campaign which increased to three εMU coils malfunctioning during the measurements of August 2019. Comparisons between the measured and calculated responses from the structures are shown in Figures 13–19. As shown, the strain registrations from the longitudinal ASGs exhibit the lowest diﬀerence between the measured response signals and the MLET calculations. The accuracy was lower in the case of transversal ASGs as can be inferred from Figures 14 and 17. It was observed that the ASGs placed in the transversal direction were sensitive to the lat- eral position of the axle applying the load. Small lateral devi- ations in the load application led to measured values that were lower than the expected values. Depending on the pos- ition of the axle applying the load, diﬀerent parts of the registration curves matched the calculations. In cases when the ﬁrst wheel was positioned correctly, the ﬁrst peak of the registered curve corresponding to the driving axle matched the calculations as shown in Figure 14(a). In cases when the driver adjusted the positioning near the end of the time when the truck was passing on top of the sensors, the last peaks of the registration corresponding to the trailer axles matched the calculations as shown in Figure 14(b). In the case of ASGs, not considering whether their The stresses measured by the SPCs showed reasonable prediction accuracy. Results of the vehicle induced response The vertical stresses recorded by the SPCs located in the upper part of the structure were found to depend more on the temperature of the AC layer than the moisture of the UGM layers in which they were located. The environmental-related variables such as the temperature of the AC and the moisture of the UGM layers did not have a large eﬀect on the vertical stresses recorded by the SPCs located at deeper layers. Instead, the properties of the load- ing conﬁgurations such as the axle load, and tyre conﬁgur- ation (single vs. dual tyres) were found to have the main inﬂuence. The vertical strains recorded by the εMU coils showed noi- sier signals compared to the other types of sensors. However, in most cases, the measured values ﬁtted adequately with the Figure 13. Longitudinal horizontal strain εxx response registrations from ASG compared to MLET calculations at Lv373 under LHV1 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 13. Longitudinal horizontal strain εxx response registrations from ASG compared to MLET calculations at Lv373 under LHV1 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 11 Figure 14. Transversal horizontal strain εyy response registrations from ASG compared to MLET calculations at Lv373 under LHV2 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 14. Transversal horizontal strain εyy response registrations from ASG compared to MLET calculations at Lv373 under LHV2 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 15. Vertical stress σzz response registrations from SPC at 35 cm depth compared to MLET calculations at Lv373 under LHV3 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 15. Vertical stress σzz response registrations from SPC at 35 cm depth compared to MLET calculations at Lv373 under LHV3 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. D. SALIKO AND S. ERLINGSSON 12 Figure 16. Longitudinal horizontal strain εxx response registrations from ASG compared to MLET calculations at Lv515 under LHV1 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 16. Comparisons between multilayer linear and nonlinear modelling The comparison was made for the August 2018 measure- ment campaign at Lv515 subjected to loading by LHV1 between two selected measurements and the corresponding calculations. The comparisons were made for the longitudinal strain under the AC layer recorded by the ASG at 4 cm depth as shown in Figure 20(a) and for the vertical stress recorded by the SPCs at 38 cm depth as shown in Figure 20(b). The bottom of the AC layer and the top of the subgrade were selected as they are known to be locations where critical strains in the structure occur. The comparison between the measured response and numerical calculations using linear MLET-based software showed a good ﬁt, indicating that the linear elastic theory is a suitable method for the response modelling. However, the viscoelastic behaviour of the asphalt layers and the stress-dependent nonlinear behaviour of the UGM layers is therefore neglected. To further investigate the signiﬁcance of the abovementioned factors, two additional modelling approaches were set up for comparisons, that is assuming, the AC layers exhibit a viscoelastic behaviour, but all other layers are linear elastic materials, and thereafter that the UGM (base course and subbase) were modelled as stress- dependent linear elastic materials but the asphalt layers and the subgrade were modelled as linear elastic materials. As before the ERAPave software was used. A detailed description of the approach can be found in Ahmed and Erlingsson (2017). The approach is based on the elastic- viscoelastic correspondence principle in which the Laplace transform of the constitutive equations for linear viscoelastic problems is formulated similarly to homogenous and isotro- pic linear elastic problems (Huang 2004; Kim 2011). The creep compliance of the asphalt materials is therefore required as an input for the viscoelastic modelling. A power function derived from complex modulus (Baumgaertel and Winter 1989; Tschoegl 2012) was used for the Based on the comparison shown in Figure 20, minor diﬀer- ences were found between the calculated responses by the lin- ear elastic, viscoelastic, and the k-θ model. The viscoelastic and the k-θ model were found to oﬀer an improvement in the range of 1–4% over the linear elastic model in the prediction of the response peaks shown in Figure 20. The temperatures during the times when the response testing was performed were relatively low, which has led to a less pronounced visco- elastic behaviour of the AC. Results of the vehicle induced response . (4) where D´(ω) is the storage creep compliance, D′′(ω) is the loss creep compliance, ω is the frequency, Γ is the gamma function, and D0, D1, and m are the parameters for the power function creep compliance curve. For the comparison between the measurement campaigns, the peak strain values from the longitudinal ASGs were taken into consideration. These sensors were selected as they oﬀered the highest reliability. The lowest strains under the AC layer for both structures were recorded during the May 2019 measurement campaign. During the May 2019 campaign, the temperature of the AC layer was the lowest compared to the other measurement campaigns. The highest temperature and the highest longitudinal strains under the AC layer were recorded during the June 2019 campaign. Similar temperature values were recorded for the August 2018 and August 2019 measurement campaigns, however, some diﬀerences in strain values were observed. For the August 2019 campaign, the recorded AC temperature was slightly lower, but the moisture recorded by TDR1 at 36 cm depth was lower than in the August 2018 measure- ment campaign. This is an indicator that the strains at the bottom of the AC layer depend not only on the stiﬀness of the AC layer but also on the support from the granular base course under the asphalt. For the power function of the creep compliance curve, the parameters were taken from a previous study D0 = 1.47E-07, D1= 1.27E-03, and m = 0.06 (Ahmed and Erlingsson 2016). g To model the stress-dependent nonlinear behaviour in the granular layers, the k-θ model (Uzan 1985; Lekarp et al. 2000; Leischner 2016) expressed in a normalised form was used. Mr = k1pa 3p pa k2 (5) (5) where k1 and k2 are model constants derived experimentally, p is the mean normal stress equal to 1/3 of the sum of the prin- cipal stresses, and pa is the reference pressure at 100 kPa. The model has been shown to capture the response of UGMs when subjected to rolling wheel loading (Erlingsson 2010; Saevarsdottir and Erlingsson 2015). The constants used in the modelling process were taken from previous studies as k1= 900, and k2 = 0.4 (Tseng and Lytton 1989; Rahman and Erlingsson 2015). Results of the vehicle induced response Longitudinal horizontal strain εxx response registrations from ASG compared to MLET calculations at Lv515 under LHV1 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 17. Transversal horizontal strain εyy response registrations from ASG compared to MLET calculations at Lv515 under LHV2 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 17. Transversal horizontal strain εyy response registrations from ASG compared to MLET calculations at Lv515 under LHV2 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 13 Figure 18. Vertical stress σzz response registrations from SPC at 38 cm depth compared to MLET calculations at Lv515 under LHV3 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 18. Vertical stress σzz response registrations from SPC at 38 cm depth compared to MLET calculations at Lv515 under LHV3 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 19. Vertical strain εzz response registrations from εMU coil at 84 cm depth compared to MLET calculations at Lv515 under LHV1 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. Figure 19. Vertical strain εzz response registrations from εMU coil at 84 cm depth compared to MLET calculations at Lv515 under LHV1 for (a) August 2018, (b) May 2019, (c) June 2019, and (d) August 2019. 14 D. SALIKO AND S. ERLINGSSON 14 viscoelastic modelling of the asphalt layer. viscoelastic modelling of the asphalt layer. calculated values. Deviations were noticed mainly for the three last peaks that correspond to the strains caused by the last tri- dem trailer axle of the LHV1 as shown in Figure 19(c,d) for the εMU at 76 cm depth. The increase in strains under loading by the last tridem axle was observed mainly in combination with increased moisture content in the structure. This was observed only for LHV1 and not for the other two vehicles. The exact reason for this is unknown. A possible hypothesis is that the increase in measured strains comes due to a local temporary pore water pressure build-up that dissipates afterwards. D(t) = D0 + D1tm D′(v) = D0 + D1G(m + 1)v−m cos (mp(2k + 1/2)) D′′(v) = D1G(m + 1)v−m sin (mp(2k + 1/2)) k = 0, + 1, + 2 . . Evaluation of the modelling accuracy wider range as the sensors were located at multiple depths compared to the longitudinal ASGs which were located at a single depth. For the longitudinal strains, two separate clusters corresponding to each of the sections are observed. This is due to the diﬀerence in AC layer thickness between the two sec- tions, higher strains occur in the case of Lv515 due to the lower thickness of the AC layer. To evaluate the accuracy of the modelling by MLET, the peaks of the measured curves for each of the sensor types were com- pared against the calculated peak values. The comparison is illustrated in Figure 21. Each data point shown in the plot cor- responds to one peak of the response curves obtained by the loading of one axle of a speciﬁc LHV. The plot includes the peaks from the response of all three types of LHVs. The results are grouped based on the section corresponding to where the measurements were made. To statistically quantify the accu- racy of the ﬁt, the coeﬃcient of determination R2 and the nor- malised root mean square error NRMSE were used. The NRMSE was deﬁned as The highest NRMSE value, lowest R2 value and lowest pre- diction accuracy were estimated in the case of the transversal horizontal strains εyy recorded by transversal ASGs. The measured values for this type of sensor were lower compared to the calculated values due to diﬃculties in maintaining the correct lateral position during the testing campaigns. This is an indicator that the response and usability of this type of sen- sor highly depend on the correct positioning of the vehicle in the transversal direction during the testing. This also indicates that in most cases there will be a lateral wander when the vehicles pass above the structure. This could lead to fatigue performance estimates to be on the conservative side, as typi- cally they assume that the loading follows the wheel path. NRMSE = RMSE ymax −ymin (4) (4) where ymax represents the largest measured datapoint, and ymin represents the smallest measured datapoint. The NRMSE was used to facilitate the comparison between datasets of diﬀerent scales. The number of data points n,the R2 value, and the NRMSE value are given in the plots. A high R2 value of 0.888 was calculated in the case of the εMU coils. The NRMSE value for the εMU coils was calculated to be 0.05. Comparisons between multilayer linear and nonlinear modelling Due to the abovementioned reasons, it was decided to continue with linear elastic model- ling due to its simplicity and faster computational time which would also be an advantage later for performance pre- diction calculations. In other scenarios at higher temperatures and larger loading magnitude variations, it would be relevant to look further into the eﬀect of the viscoelasticity of the asphalt and the nonlinear behaviour of the granular materials. INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 15 Figure 20. Comparison between measured responses, linear elastic modelling (LE), viscoelastic modelling (VE), and nonlinear k-theta modelling (k-theta) for (a) longi- tudinal asphalt strain gauge ASG, and (b) soil pressure cell SPC under LHV1 in measurements in August 2018. Figure 20. Comparison between measured responses, linear elastic modelling (LE), viscoelastic modelling (VE), and nonlinear k-theta modelling (k-theta) for (a) longi- tudinal asphalt strain gauge ASG, and (b) soil pressure cell SPC under LHV1 in measurements in August 2018. Evaluation of the modelling accuracy When looking at individual sensor responses for Lv373 some deviations between the measured and calculated values were observed for the εMU sensors that were located between two layers. This occurred in the cases when a layer was thin and it was not possible to place the εMU coil sensor only within that layer. This is shown in Figure 21(c) where the As shown in Figure 21, the best ﬁt between the measured and calculated values indicated by a higher R2 value and lower NRMSE was obtained in the case of the longitudinal horizontal strain εxx from the ASGs and the vertical stress σzz from the SPCs. A slightly higher R2 value was calculated for the SPCs due to the spread of the data points over a 16 D. SALIKO AND S. ERLINGSSON 16 Figure 21. Validation of calculations against measurements for (a) longitudinal ASGs, (b) transversal ASGs, (c) SPCs, and (d) EMUs for all four measurements campaigns. 16 D. SALIKO AND S. ERLINGSSON Figure 21. Validation of calculations against measurements for (a) longitudinal ASGs, (b) transversal ASGs, (c) SPCs, and (d) EMUs for all four measure Figure 21. Validation of calculations against measurements for (a) longitudinal ASGs, (b) transversal ASGs, (c) SPCs, and (d) EMUs for all four measurements campaigns. Figure 21. Validation of calculations against measurements for (a) longitudinal ASGs, (b) transversal ASGs, (c) SPCs, and (d) EMUs gainst measurements for (a) longitudinal ASGs, (b) transversal ASGs, (c) SPCs, and (d) EMUs for all four measurements campaigns. under loading from LHVs. The variation of the temperature, moisture content, and frost penetration in the cross-section of the pavement structures was monitored successfully by ther- mocouples (temperature in the AC layer), moisture rod (moisture variation in the granular layers), and frost rod (temperature granular layers). points representing Lv373 show a larger scatter compared to the points representing Lv515. However, the spread of the εMU coil sensors over a large range of depths led to a high cal- culated R2 value. INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING 17 17 the spring thaw period. This is essential in order to use the method in an M-E calculation scheme. Ahmed, A. and Erlingsson, S., 2016. Viscoelastic response modelling of a pavement under moving load. Transportation Research Procedia, 14, 748–757. doi:10.1016/j.trpro.2016.05.343. The longitudinal tensile strains at the bottom of the asphalt layer were primarily inﬂuenced by the temperature of the asphalt layer, with lower recorded strains at low temperatures. Increased moisture content in the unbound granular material leads to lower stiﬀness of unbound granular layers and increas- ing longitudinal tensile strains. Ahmed, A.W. and Erlingsson, S., 2017. Numerical validation of visco- elastic responses of a pavement structure in a full-scale accelerated pavement test. International Journal of Pavement Engineering, 18 (1), 47–59. doi:10.1080/10298436.2015.1039003. Al-Qadi, I.L., et al., 2004. The Virginia smart road: the impact of pave- ment instrumentation on understanding pavement performance. Journal of the Association of Asphalt Paving Technologists, 73 (3), 427–465. Comparisons of measured and calculated longitudinal hori- zontal strains and vertical stresses and strains gave high coeﬃ- cients of determination R2. The transversal horizontal strain calculations gave slightly higher values than the measured ones reducing the coeﬃcient of determination. The transversal horizontal strains are however highly dependent on the lateral position of the applied load. As it was not possible to keep a correct positioning, lower registered values were observed compared to the calculated values as expected. Andrei, D., Witczak, M.W., and Houston, W.N., 2009. Resilient modulus predictive model for unbound pavement materials. In: Contemporary topics in ground modiﬁcation, problem soils, and geo-support. 401– 408. doi:10.1061/41023(337)51. ARA Inc., 2004. Guide for mechanistic–empirical design of new and reha- bilitated pavement structures. Final Rep., NCHRP Project 1-37A. Available from: https://onlinepubs.trb.org/onlinepubs/archive/ mepdg/guide.htm. Baumgaertel, M. and Winter, H.H., 1989. Determination of discrete relax- ation and retardation time spectra from dynamic mechanical data. Rheologica Acta, 28 (6), 511–519. doi:10.1007/BF01332922. Minor reliability issues occurred with the εMU coils in some of the testing campaigns due to malfunctions. The regis- trations from the εMU coil sensors were noisier compared to the other types and in some cases stopped functioning. The reliability of the conﬁguration could be improved by increas- ing the number of εMU coils or by using more durable sensors. Further improvements could be made in the response captur- ing accuracy by adding more ASGs in the transversal direction to account for the lateral deviations in vehicle positioning. INTERNATIONAL JOURNAL OF PAVEMENT ENGINEERING Blanc, J., et al., 2019. Monitoring of an experimental motorway section. Road Materials and Pavement Design, 20 (1), 74–89. doi:10.1080/ 14680629.2017.1374997. Burmister, D.M., 1945. The general theory of stresses and displacements in layered systems. I. Journal of Applied Physics, 16 (2), 89–94. doi:10. 1063/1.1707558. Christidis, P. and Leduc, G., 2009. Longer and heavier vehicles for freight transport. JRC 52005. European Commission, Joint Research Centre, Institute for Prospective Technological Studies, 1–40. Available from: https://ec.europa.eu/transport/sites/transport/ﬁles/modes/road/ events/doc/2009_06_24/2009_jrc52005.pdf. p g Non-linear MLET including viscoelastic behaviour of the asphalt layers or stress dependency of the granular layers only marginally improved the calculations compared to the linear MLET solutions. It was therefore concluded to include the linear MLET-based approach in the new M-E design and prediction tool that is under development in Sweden using a climatic model to adjust the stiﬀness properties of the diﬀerent pavement layers. The response validation carried out in this study was performed only at two locations for a limited range of temperatures and loading magnitudes. The eﬀects of higher temperatures and larger loading magnitudes should be further studied in order to investigate the impact of the vis- coelasticity of the AC layers and the nonlinear behaviour of the granular materials. j Doré, G. and Zubeck, H.K., 2009. Cold regions pavement engineering. Reston, VA: New York: ASCE Press; McGraw-Hill. 978-0-07- 160088-0. Duong, N.S., et al., 2019. Continuous strain monitoring of an instrumen- ted pavement section. International Journal of Pavement Engineering, 20 (12), 1435–1450. doi:10.1080/10298436.2018.1432859. Erlingsson, S., 2010. Impact of water on the response and performance of a pavement structure in an accelerated test. Road Materials and Pavement Design, 11 (4), 863–880. doi:10.1080/14680629.2010. 9690310. Erlingsson, S., 2012. Rutting development in a ﬂexible pavement struc- ture. Road Materials and Pavement Design, 13 (2), 218–234. doi:10. 1080/14680629.2012.682383. Erlingsson, S. and Ahmed, A., 2013. Fast layered elastic response program for the analysis of ﬂexible pavement structures. Road Materials and Pavement Design, 14 (1), 196–210. doi:10.1080/14680629.2012.757558. Erlingsson, S., Rahman, M.S., and Ahmed, A., 2018. Impact of longer and heavier vehicles on the performance of asphalt pavements: A labora- tory study. In: A. Loizos, I.L. Al-Qadi, and A. (Tom) Scarpas, eds. Bearing capacity of roads, railways and airﬁelds. 1st ed. CRC Press, 483–490. doi:10.1201/9781315100333-63. Disclosure statement No potential conﬂict of interest was reported by the author(s). Erlingsson, S., Rahman, S., and Salour, F., 2017. Characteristic of unbound granular materials and subgrades based on multi stage RLT testing. Transportation Geotechnics, 13, 28–42. doi:10.1016/j.trgeo.2017.08.009. ORCID Denis Saliko http://orcid.org/0000-0002-0370-3866 Sigurdur Erlingsson http://orcid.org/0000-0002-4256-3034 Fladvad, M. and Erlingsson, S., 2022. Modelling the response of large-size subbase materials tested under varying moisture conditions in a heavy vehicle simulator. Road Materials and Pavement Design, 23 (5), 1107– 1128. doi:10.1080/14680629.2021.1883462. Funding This work was supported by Traﬁkverket. Erlingsson, S. and Saliko, D., 2020. Correlating air freezing index and frost penetration depth – a case study for Sweden. In: C. Raab, ed. Proceedings of the 9th international conference on maintenance and rehabilitation of pavements – Mairepav9. Vol. 76. Springer International Publishing, 847–857. doi:10.1007/978-3-030-48679-2_79 Conclusions The data obtained from environmental sensors was used together with the FWD backcalculation to obtain layer stiﬀness values. The backcalculated stiﬀness values were thereafter used as input for the linear MLET-based response modelling. The MLET numerical simulation due to the three LHV´s loadings where the material properties of the pavement layers were updated according to the environmental readings gave good results. The modelling was relatively accurate, this was true for both the late summer measurements as well as during Two thin pavement structures located in the northern part of Sweden have been instrumented with road response and cli- matic sensors. Registrations from LHV passes at normal speed have been gathered during diﬀerent seasons. The read- ings have further been compared with the results from numerical calculations using MLET-based software. ASGs, SPCs and εMU coils were successfully used to pro- vide induced response data throughout diﬀerent seasons References Ahmed, A. and Erlingsson, S., 2013. Evaluation of permanent defor- mation models for unbound granular materials using accelerated pave- ment tests. Road Materials and Pavement Design, 14 (1), 178–195. doi:10.1080/14680629.2012.755936. Gkyrtis, K., Armeni, A., and Loizos, A., 2021. A mechanistic perspective for airﬁeld pavements evaluation focusing on the asphalt layers’ behav- iour. International Journal of Pavement Engineering, 1–14. doi:10.1080/ 10298436.2021.1995733. D. SALIKO AND S. ERLINGSSON 18 Journal of Pavement Engineering, 16 (7), 587–601. doi:10.1080/ 10298436.2014.943209. Gusfeldt, K.H. and Dempwolﬀ, K.R., 1967. Stress and strain measure- ments in experimental road sections under controlled loading con- ditions. 2nd international conference on the structural design of asphalt pavements. Ann Arbor, Michigan. 663–669. Saevarsdottir, T. and Erlingsson, S., 2015. Modelling of responses and rut- ting proﬁle of a ﬂexible pavement structure in a heavy vehicle simu- lator test. Road Materials and Pavement Design, 16 (1), 1–18. doi:10. 1080/14680629.2014.939698. g Hicks, R.G. and Finn, F.N., 1970. Analysis of results from the dynamic measurements program on the San Diego test road. Association of Asphalt Paving Technologists, 39, 153–185. Available from: https:// trid.trb.org/view/101320. Saevarsdottir, T., Erlingsson, S., and Carlsson, H., 2016. Instrumentation and performance modelling of heavy vehicle simulator tests. International Journal of Pavement Engineering, 17 (2), 148–165. doi:10.1080/10298436.2014.972957. g Huang, Y.H., 2004. Pavement analysis and design. 2nd ed Upper Saddle River, NJ: Pearson/Prentice Hall. 978-0-13-142473-9. Irwin, L.H., 2002. Backcalculation: an overview and perspective. In: Pavement evaluation conference, 2002, Roanoke, VI. Saliko, D. and Erlingsson, S., 2021. Damage investigation of thin ﬂexible pavements to longer heavier vehicle loading through instrumented road sections and numerical calculations. Road Materials and Pavement Design, 22 (sup1), S575–S591. doi:10.1080/14680629.2021. 1899964. Kim, J., 2011. General viscoelastic solutions for multilayered systems sub- jected to static and moving loads. Journal of Materials in Civil Engineering, 23 (7), 1007–1016. doi:10.1061/(ASCE)MT.1943-5533. 0000270. Salour, F., 2015. Moisture inﬂuence on structural behaviour of pavements: ﬁeld and laboratory investigations. PhD thesis. Architecture and the Built Environment, KTH Royal Institute of Technology. Available from: https://www.diva-portal.org/smash/get/diva2:796833/ SUMMARY01.pdf. Knight, I., et al., 2008. Longer and/or longer and heavier goods vehicles (LHVs) – a study of the likely eﬀects if permitted in the UK: ﬁnal report. TRL Limited. Available from: http://data.parliament.uk/Deposited Papers/Files/DEP2008-1410/DEP2008-1410.pdf. Leischner, S., et al., 2016. Design of thin surfaced asphalt pavements. Procedia Engineering, 143, 844–853. doi:10.1016/j.proeng.2016.06.138. Salour, F. and Erlingsson, S., 2013a. Moisture-sensitive and stress-depen- dent behavior of unbound pavement materials from in situ falling weight deﬂectometer tests. 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https://openalex.org/W2749031264	https://europepmc.org/articles/pmc5563892?pdf=render	English	null	How empathic is your healthcare practitioner? A systematic review and meta-analysis of patient surveys	BMC medical education	2,017	cc-by	6,030	* Correspondence: jeremy.howick@phc.ox.ac.uk 1Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford OX2 6GG, UK Full list of author information is available at the end of the article Howick et al. BMC Medical Education (2017) 17:136 DOI 10.1186/s12909-017-0967-3 Howick et al. BMC Medical Education (2017) 17:136 DOI 10.1186/s12909-017-0967-3 © 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. How empathic is your healthcare practitioner? A systematic review and meta-analysis of patient surveys J. Howick1* , L. Steinkopf2, A. Ulyte3, N. Roberts4 and K. Meissner2,5 Eligibility criteria We included any study where patients rated their practi- tioners’ empathy using the CARE measure. We included ratings of any practitioner including nurses, doctors, alter- native practitioners, and medical students. We included studies in any language, provided that the translation of the CARE questionnaire was validated. Supporting the view that empathic care should be en- couraged, the extent to which healthcare practitioners ex- press empathy seems to be lacking in some cases, [13–16] and it may decline with time in practice [17]. The in- creased burden of paperwork, which takes up a quarter of practitioner time, [18] may be a barrier to empathic care. However we do not know the prevalence of inadequate empathy. If adequate empathy is rare, then patients and practitioners would both likely benefit if practitioners re- inforced how they display empathy. In this study, we aimed to address this gap by conducting a systematic re- view of patient ratings of practitioner empathy. We excluded studies that used other measures of em- pathy, because only CARE has been validated. An added benefit of this approach is that it reduced heterogeneity. We excluded studies where practitioners were reported to have been trained in empathy prior to being rated by pa- tients, since we were interested in pre-training empathy ratings. Where the publications included surveys of more than one group of practitioners the surveys were treated independently. CARE asks patients to answer 10 questions about the consultation with their practitioner such as whether the practitioner: made the patient feel at ease, really listened and understood, showed compassion, and explained things clearly (see Additional file 1). Each question can be answered by ticking one of five options: poor, fair, good, very good, excellent, does not apply, with the low- est being given a score of ‘1’, and the highest a score of ‘5’. Hence, the maximum CARE score is 50. The devel- opers of the CARE measure have produced normative values based on administration of their questionnaire [27]. They found that the mean CARE score was 45.75, and that 5% of CARE scores fell above 48.32, and 5% fell below 40.72. An obstacle to empathy research is that practitioner em- pathy is difficult to define theoretically [19, 20]. Objectives Our primary objective was to measure the extent to which patients (of any type) report their healthcare practitioners (of any type) to be empathic. Our secondary objective was to compare differences in empathy ratings between differ- ent practitioner groups (male versus female, consultation times, different types of practitioners, and practitioners in different countries). Abstract BMC Medical Education (2017) 17:136 Howick et al. BMC Medical Education (2017) 17:136 Page 2 of 9 Page 2 of 9 Information sources and search Information sources and search We searched the following databases: MEDLINE (OvidSP) [1946–09/03/2016], Embase (OvidSP) [1974 to 2016 March 08], PsycINFO (OvidSP) [1967–09/03/2016], Cinahl (EBSCOHost), Science & Social Science Indexes (Web of Science, Thomson Reuters) [1945–09/03/2016], Cochrane Central Register of Controlled Trials [Issue 2 of 12, February 2016], Cochrance Database of Systematic Reviews [Issue 3 of 12, March 2016] and Database of Abstracts of Reviews of Effects [issue 2 of 4, April 2015] (via Cochrane Library, Wiley) and Pubmed (see Additional file 2 for search strategy). We also searched the Web of Science Core Collection, Scopus and Google Scholar for studies that have cited the CARE measure, [25] and any record that includes the full name of the measure (consultation and relational empathy). Additionally, we Background CARE scores before and after training, however there were insufficient studies to complete this analysis. We also had insufficient data to perform the proposed analyses comparing practitioners with 10 years or more experience with those who had less than 10 years experience. Neither of these changes was related to our main study aim. A growing number of randomized trials show that when healthcare practitioners are encouraged to enhance how they express empathy, this can reduce patient pain, [1, 2] lower patient anxiety, [3] increase patient satisfaction, [4, 5] im- prove medication adherence, [6, 7] and ameliorate other pa- tient health outcomes. [8–11]. For example, Chassany’s [1] empathy training intervention for general practitioners (GPs) (n = 180) reduced pain in osteoarthritis patients (n = 842) by one point on a 10-point VAS (P < 0.0001). These modest benefits are comparable to many pharmaceutical interven- tions without the adverse events. Hence some authors have recently called for efforts to encourage empathic care [12]. Eligibility criteria At the same time there is an emerging consensus that empathy can be operationalized as a healthcare practitioner’s ability to understand a patient’s point of view, express this under- standing, and make a recommendation that reflects the shared understanding [21, 22]. More importantly for present purposes, while empathy is measured using differ- ent scales, [23, 24] only one patient-rating of practitioner empathy demonstrated evidence of reliability, [25] internal validity and consistency: CARE [25, 26]. From a patient health perspective, patient ratings of practitioner empathy are likely to be important. We therefore limited our review to studies that used the CARE measure. Abstract Background: A growing body of evidence suggests that healthcare practitioners who enhance how they express empathy can improve patient health, and reduce medico-legal risk. However we do not know how consistently healthcare practitioners express adequate empathy. In this study, we addressed this gap by investigating patient rankings of practitioner empathy. Methods: We conducted a systematic review and meta-analysis of studies that asked patients to rate their practitioners’ empathy using the Consultation and Relational Empathy (CARE) measure. CARE is emerging as the most common and best-validated patient rating of practitioner empathy. We searched: MEDLINE, Embase, PsycINFO, Cinahl, Science & Social Science Citation Indexes, the Cochrane Library and PubMed from database inception to March 2016. We excluded studies that did not use the CARE measure. Two reviewers independently screened titles and extracted data on average CARE scores, demographic data for patients and practitioners, and type of healthcare practitioners. Results: Sixty-four independent studies within 51 publications had sufficient data to pool. The average CARE score was 40.48 (95% CI, 39.24 to 41.72). This rank s in the bottom 5th percentile in comparison with scores collected by CARE developers. Longer consultations (n = 13) scored 15% higher (42.60, 95% CI 40.66 to 44.54) than shorter (n = 9) consultations (34.93, 95% CI 32.63 to 37.24). Studies with mostly (>50%) female practitioners (n = 6) showed 16% higher empathy scores (42.77, 95% CI 38.98 to 46.56) than those with mostly (>50%) male (n = 6) practitioners (34.84, 95% CI 30.98 to 38.71). There were statistically significant (P = 0.032) differences between types of providers (allied health professionals, medical students, physicians, and traditional Chinese doctors). Allied Health Professionals (n = 6) scored the highest (45.29, 95% CI 41.38 to 49.20), and physicians (n = 39) scored the lowest (39.68, 95% CI 38.29 to 41.08). Patients in Australia, the USA, and the UK reported highest empathy ratings (>43 average CARE), with lowest scores (<35 average CARE scores) in Hong Kong. Conclusions: Patient rankings of practitioner empathy are highly variable, with female practitioners expressing empathy to patients more effectively than male practitioners. The high variability of patient rating of practitioner empathy is likely to be associated with variable patient health outcomes. Limitations included frequent failure to report response rates introducing a risk of response bias. Future work is warranted to investigate ways to reduce the variability in practitioner empathy. Keywords: Empathy, Consultation, Communication, Practitioner, Expectations Howick et al. Protocol and registration The protocol for this review was published in PROSPERO (record no. CRD42016037456). We made two changes to the protocol. In the protocol we proposed to analyze Howick et al. Sensitivity and subgroup analyses Sensitivity and subgroup analyses We conducted four preplanned subgroup analyses. Protocol and registration BMC Medical Education (2017) 17:136 Page 4 of 9 contacted authors of studies to ask whether they are aware of any additional studies. and consultation time with respect to CARE scores we performed a multivariable regression analysis, with gen- Table 1 Study groups included in meta-analysis (n = 64 published in 51 articles) (Continued) Pollak (2015) US Physicians/Specialists 2 N/A 21 N/A 46.0 (4.2) Price (2006) UK TCM practitioners 15 N/A 52 N/A 42.4 (6.9) Price (2008) UK Physicians/Primary Care 35 N/A 2550 10.2 (5.5) 43.2 (7.7) Quaschning (2013) Germany Mixed N/A N/A 402 N/A 41.5 (7.3) Rees (2014) UK Allied health professionals N/A N/A 225 N/A 43.1 (7.8) Scales (2008) US Allied health professionals 1 N/A 411 N/A 47.6 (4.4) Scarpellini (2014) Brazil Physicians/Specialists 12 N/A 12 N/A 41.4 (6.0) Scheffer (2013a) Germany Medical Students N/A N/A 103 N/A 45.4 (5.5) Scheffer (2013b) Germany Medical Students N/A N/A 94 N/A 41.7 (9.0) Steinhausen (2014) Germany Physicians/Specialists N/A N/A 120 N/A 38.0 (9.8) Tran (2012) Australia Physicians/Primary Care 3 N/A 38 15.0 (4.0) 43.4 (4.2) Weiss (2015) UK Mixed 51 0.69 207 N/A 43.0 (7.4) Wong (2013) Hong Kong Physicians/Primary Care 9 N/A 1030 7.7 (4.7) 34.4 (7.8) Wu (2015a) China Physicians/Specialists N/A N/A 199 N/A 39.6 (8.3) Wu (2015b) China Physicians/CAM N/A N/A 146 N/A 41.2 (8.6) Wu (2015c) China Physicians/Specialists N/A N/A 139 N/A 38.4 (8.7) Yu (2015a) Hong Kong Physicians/not specified 6 0.33 179 4.5 (2.4) 29.2 (7.4) Yu (2015b) Hong Kong Physicians/Specialists 7 0.57 207 10.5 (8.6) 35.5 (8.9) Yu (2015c) Hong Kong Physicians/Primary Care 14 0.50 435 7.4 (4.8) 35.7 (8.3) Zilliacus (2011a) Australia Physicians/Specialists N/A N/A 178 N/A 41.3 (9.6) Zilliacus (2011b) Australia Genetic Counselor N/A N/A 152 N/A 44.6 (7.8) Table 1 Study groups included in meta-analysis (n = 64 published in 51 articles) (Continued) contacted authors of studies to ask whether they are aware of any additional studies. contacted authors of studies to ask whether they are aware of any additional studies. and consultation time with respect to CARE scores we performed a multivariable regression analysis, with gen- der and consultation time included as the independent variables, and CARE scores included as the dependent variable. Protocol and registration BMC Medical Education (2017) 17:136 Page 3 of 9 Table 1 Study groups included in meta-analysis (n = 64 published in 51 articles) Study Country Type of Providers N Providers % Female N Patients Mean (SD) consultation time (min) Mean CARE score (SD) Aomatsu (2014) Japan Physicians/Primary Care 9 N/A 272 17.2 (14.3) 38.4 (8.6) Attar (2012) India Physicians/Specialists N/A 1.00 53 N/A 29.4 (10.9) Bikker (2005) UK Physicians/CAM 9 N/A 187 50.1 (14.0) 45.0 (7.0) Bikker (2015) UK Nurses 17 N/A 774 13.0 (7.6) 45.9 (5.9) Birhanu (2012) Ethiopia Mixed N/A N/A 768 6.3 (2.6) 31.3 (8.3) Buecken (2012) Germany Physicians/Specialists N/A N/A 541 N/A 39.9 (9.1) Chen (2015) Hong Kong Medical Students 158 0.39 9 15.0 (N/A) 35.8 (7.3) Chung (2012) South Korea Physicians/CAM 1 0.00 143 5.0 (N/A) 38.0 (6.9) Chung, Yip (2016) Hong Kong TCM practitioners N/A N/A 514 N/A 34.2 (8.1) Fogarty (2013) Australia TCM practitioners 1 1.00 18 60.0 (N/A) 49.8 (0.6) Fritzsche (2011a) China Physicians/Specialists 2 N/A 28 N/A 45.0 (5.2) Fritzsche (2011b) China Physicians/Specialists 5 N/A 37 N/A 36.7 (7.7) Fritzsche (2011c) China Physicians/CAM 4 N/A 31 N/A 42.9 (7.3) Fung (2009) Hong Kong Physicians/Primary Care 13 N/A 228 5.7 (3.9) 31.8 (8.9) Griffin (2014a) UK Physicians/Primary Care N/A N/A 444 N/A 39.7 (9.9) Griffin (2014b) UK Nurses N/A N/A 444 N/A 30.4 (9.5) Gu (2015) Hong Kong N/A 332 N/A 332 N/A 31.0 (9.3) Hanzevacki (2015) Croatia Physicians/Primary Care 8 N/A 568 6.8 (N/A) 35.9 (4.2) Jani (2012) UK Physicians/Primary Care 47 N/A 163 9.5 (4.5) 43.8 (6.9) Johnson (2012) UK Mixed 21 N/A 1103 N/A 45.2 (6.2) Johnston (2015) UK Mixed 17 N/A 30 N/A 39.9 (8.7) Joice (2010) UK Psychotherapist N/A N/A 141 N/A 39.0 (8.0) Kersten (2012) UK TCM practitioners N/A N/A 213 N/A 42.2 (6.8) Lafreniere (2015) US Physicians/Specialists 44 0.57 244 41.2 (23.4) 44.6 (6.7) LaVela (2015) US Physicians/Specialists N/A N/A 389 N/A 40.1 (9.9) Lee (2012) South Korea Physicians/CAM 1 0.00 110 N/A 36.0 (8.4) Lelorain (2015) France Physicians/Specialists 28 N/A 201 26.0 (14.0) 38.4 (8.9) MacPherson (2003) UK TCM practitioners N/A N/A 135 N/A 45.5 (6.7) Menendez (2015) US Physicians/Specialists 4 N/A 112 11.0 (7.0) 46.0 (6.8) Mercer (2004) UK Physicians/Primary Care N/A N/A 10 N/A 39.2 (10.8) Mercer (2005) UK Physicians/Primary Care 26 N/A 3044 N/A 40.9 (8.8) Mercer (2008a) UK Physicians/Primary Care 5 0.60 323 10.0 (N/A) 42.4 (8.1) Mercer (2008b) UK Physicians/Specialists 31 N/A 1582 N/A 43.8 (6.6) Mercer (2008c) UK Physicians/Specialists 25 N/A 1015 N/A 43.5 (7.4) Mercer (2011) Hong Kong Physicians/Primary Care 20 0.30 984 5.5 (2.9) 34.6 (8.8) Murphy (2013a) UK Allied health professionals N/A N/A 13 N/A 43.4 (7.4) Murphy (2013b) UK Physicians/Primary Care N/A N/A 86 N/A 43.9 (7.6) Neumann (2007) Germany Physicians/Specialists N/A N/A 326 N/A 37.1 (11.1) Nezenega (2013) Ethiopia Mixed N/A N/A 531 7.1 (4.4) 35.9 (8.5) Ohm (2013) Germany Medical Students 30 0.73 5 N/A 41.3 (6.3) Parrish (2016) US Physicians/Specialists 5 N/A 112 10.0 (5.6) 43.0 (8.0) Place (2016a) UK Allied health professionals N/A 53 N/A 45.7 (5.1) Place (2016b) UK Allied health professionals N/A 217 N/A 46.3 (5.6) Howick et al. Data collection, extraction, and management After piloting the extraction sheet by two authors (JH, KM), two authors (LS, AU) independently screened all titles and abstracts and extracted data. Discrepancies were resolved with discussion by a third author (JH). We extracted data about: type of practitioner, percentage female practitioners, country, average CARE score, and individual CARE scores (where available). Sensitivity and subgroup analyses We conducted four preplanned subgroup analyses. 1. Longer (>10 min) consultations compared with shorter (≤10 min) consultations. This was based on average consultation times in UK general practice [29]. We assessed risk of bias within studies by measuring response rates. It was not feasible to assess risk of bias across studies, for example by conducting a funnel plot since there was no reason to suspect higher (or lower) CARE scores varying with sample size. There was insuf- ficient data to investigate risk of bias across studies. p 2. Gender: average empathy ratings of mostly (>50%) female compared with average ratings of mostly (>50%) male practitioners. Statistical analyses were performed using the program Comprehensive Meta Analysis [28]. We provided the mean and 95% confidence interval of the CARE score. We contacted study authors via email to obtain missing data with respect to participants, outcomes, or summary data. Participant data were analysed as reported. We conducted preplanned subgroup analyses to assess the extent to which proportion of female practitioners, con- sultation duration, type of practitioner, and country played a role. To evaluate the predictive value of gender 3. When there were at least three studies within the same country, we conducted a subgroup analysis with those three countries, and compared it with the complement. We chose three studies because fewer than three makes meta-analysis problematic and in- creases the likelihood of basing conclusions on anomalous results. 4. Types of practitioners (physicians, medical students, alternative practitioners, etc.). If there were at least three studies that measured patient ratings of Page 5 of 9 Page 5 of 9 Howick et al. BMC Medical Education (2017) 17:136 Howick et al. BMC Medical Education (2017) 17:136 Howick et al. BMC Medical Education (2017) 17:136 Twenty-two studies reported consultation times. Longer consultations (≥10 min; n = 13) scored higher (42.60, 95% CI 40.69 to 44.52) than shorter (<10 min; n = 9) consultations (34.93, 95% CI 32.66 to 37.21). This differ- ence of 7.67 points (15%) between longer and shorter consultations was highly significant (P < 0.001). Twelve studies provided data on the gender of practitioners (Table 2). Studies with predominantly female practi- tioners (n = 6) showed higher empathy scores (42.77, 95% CI 38.98 to 46.56) than those with predominantly male practitioners (n = 6, 34.85, 95% CI 30.98 to 38.71). This difference of 7.92 points (16%) was statistically sig- nificant (P = 0.004). Sensitivity and subgroup analyses We conducted four preplanned subgroup analyses. specific types of practitioners, we conducted a subgroup analysis of this group, and compared it with the complement. Fig. 1 PRISMA Flow diagram Main results Our search yielded 392 independent records, of which 69 studies met our inclusion criteria (see Supplemental Material). Of these, 64 independent study groups (within 51 publications) had sufficient data to be included in our meta-analysis (see Table 1, Fig. 1, Additional file 3). See Additional file 4 for excluded studies. The 64 study groups were from 15 different countries: UK (n = 23), USA (n = 6), Hong Kong (n = 9), Germany (n = 7), Australia (n = 4), China (n = 6), Ethiopia (n = 2), South Korea (n = 2), and one study from each of Brazil, Croatia, France, India, and Japan. The types of practi- tioners included primary care physicians, practitioners of Traditional Chinese Medicine (TCM), medical students, allied health professionals, and other specialists. Fifty-five study groups could be included in the pre- planned subgroup analysis by country (Table 2). Highest empathy scores were found in Australia (n = 4, 44.88, 95% CI 42.63 to 47.14), USA (n = 6, 44.56, 95% CI 42.71 to 46.40) and UK (n = 23, 43.07, 95% CI 42.11 to 44.04). Scores were lowest in Hong Kong (n = 9, 33.46, 95% CI 31.94 to 34.99). Scores in Germany (n = 7, 40.72, 95% CI 39.02 to 42.44) and China (n = 6, 40.61, 95% CI 38.68 to 42.55) were in-between. We added an exploratory ana- lysis by country including all 64 study groups and found The average CARE score for the 64 study groups was 40.48 (95% CI, 39.24 to 41.72) (see Table 2, Fig. 2). Fig. 1 PRISMA Flow diagram Fig. 1 PRISMA Flow diagram Howick et al. BMC Medical Education (2017) 17:136 Page 6 of 9 Table 2 Summary of results from subgroup analyses Analysis No. Main results studies Average CARE score (95% confidence interval) P-value for difference (if applicable) Overall 64 40.48 (39.24 to 41.72) n/a Longer versus shorter consultations 22 Longer consultations (<10 min) 13 42.60 (40.69 to 44.52) <0.001 Shorter consultations (≥10 min) 9 34.93 (32.66 to 37.21) Proportion of female practitioners 12 < 50% female practitioners 6 34.85 (30.98 to 38.71) 0.004 ≥50% female practitioners 6 42.77 (38.98 to 46.56) By Country 55 UK 23 43.08 (42.11 to 44.04) No significant difference between UK, USA, Australia, Germany and China lower than USA and Australia, Hong Kong lower than all other countries USA 6 44.56 (42.71 to 46.40) Australia 4 44.88 (42.63 to 47.14) Germany 7 40.73 (39.02 to 42.44) China 6 40.61 (38.68 to 42.55) Hong Kong 9 33.46 (31.94 to 34.99) By type of provider 53 Allied Health Professionals 5 45.29 (41.38 to 49.20) 0.032 Medical Students 4 41.35 (36.91 to 45.79) Physicians 39 39.68 (38.29 to 41.08) Traditional Chinese Doctors 5 42.98 (39.15 to 46.81) Fig. 2 Comparison of average CARE score within subgroups Howick et al. BMC Medical Education (2017) 17:136 Page 7 of 9 Table 4 Multivariable regression analysis, with proportion of female practitioners and consultation time as independent variables and CARE scores as dependent variable (n = 8) Variable Coefficient (ß) Standard error 95% CI Wald χ2 P-value Intercept 33.40 2.82 27.87 to 38.93 11.84 <0.0001 Proportion of female practitioners 1.04 8.05 −14.74 to 16.82 0.13 0.897 Consultation duration 0.26 0.11 0.04 to 0.48 2.27 0.023 Table 4 Multivariable regression analysis, with proportion of female practitioners and consultation time as independent variables and CARE scores as dependent variable (n = 8) that scores in India (n = 1, 29.49, 95% CI 24.18 to 34.80) were lower than those in Hong Kong. Scores in the UK, USA and Australia were highest (See Additional file 5). We found at least three studies each measured empathy in the following types of providers: physicians, medical students, allied health professionals, and practitioners of Traditional Chinese Medicine (Table 2). There was statis- tically significant heterogeneity between these (P = 0.032), with allied health professionals scoring the highest (n = 5, 45.29, 95% CI 41.38 to 49.20), and physicians scoring the lowest (n = 39, 39.68, 95% CI 38.29 to 41.08). We found no differences between primary care physicians, special- ists, and complementary and alternative medicine (CAM) providers, (P = 0.386) (see Table 3). Discussion We found that patient rating of practitioner empathy is highly variable, with some practitioners being reported to express empathy much less effectively to patients than others. Female practitioners, allied health professionals, those who spend more time with patients, and practi- tioners from Australia, the US, and the UK seem to dis- play empathy more effectively than other practitioners. In addition, the average care score we identified was low in comparison with normative values, falling in the low- est 5% of CARE scores measured by the developers of the questionnaire [27]. The highly variable scores we found are likely to be associated with variable patient outcomes [9–11, 30]. Risk of bias The response rate was reported in 20 of the 53 studies (38%), with the average rate being high (69%, ranging from 21% to 100%). The uncertainty about the remaining re- sponse rates entails a risk of response bias. Main results Another strength is that it used measures of the only validated patient-rated measure of practitioner empathy. As such, it provides a good indication of the differences between perceived empathy across gender, disciplines, and countries. A multivariable regression analysis was performed to analyze the predictive value of gender and consultation time with respect to CARE scores. Consultation duration was the only significant predictor for CARE scores (Table 4). There are also several potential limitations. First, our method for measuring the difference between female and male practitioners was likely to be an underestimate. If studies with majority female practitioners resulted in greater patient-rated empathy, it is reasonable to assume that if all the practitioners were female, the difference between male and female practitioners would have been greater. In the context of this observational research we do not know whether the additional time caused female practitioners to be more empathic, or whether female practitioners’ higher empathy caused them to spend more time with patients, or whether these two factors cannot be separated. Second, response bias [26, 31, 32] could have affected the results. Patients who know they are rating their practitioners may wish to please their practitioners, [33] for example by giving them higher scores than they otherwise would [31, 32]. The lack of response rate reporting in most of the studies makes the extent of this problem unclear. Furthermore, selection bias might have influenced the results: the CARE ques- tionnaire could be delivered in areas where the empathy of the practitioners is believed to be anomalous (either particularly high or particularly low). Next, the compari- son between countries could have been influenced by the number of studies per country. Specifically, some of the countries with low scores had very few studies (Croatia had 1, Ethiopia had 2, and India had 1). More- over in spite of validation of CARE translations, patients in different countries may have divergent prior expecta- tions and beliefs about what it means to be an empathic practitioner. Finally, the comparison with normative values (resulting in the average score we found being in the lowest 5%) is problematic. In spite of being relatively low, the average score is still above 40. Further work needs to be done to investigate the meaning of average CARE scores References 1. Chassany O, Boureau F, Liard F, Bertin P, Serrie A, Ferran P, Keddad K, Jolivet-Landreau I, Marchand S. Effects of training on general practitioners' management of pain in osteoarthritis: a randomized multicenter study. J Rheumatol. 2006;33(9):1827–34. Strengths and limitations This is the first systematic review to investigate the extent to which healthcare practitioners are empathic. Table 3 CARE scores by physician specialty Analysis No. studies Average CARE score (95% confidence interval) CAM 5 40.83 (37.78 to 43.87) Primary Care 14 38.96 (37.14 to 40.76) Specialists 19 40.49 (38.93 to 42.05) Page 8 of 9 Howick et al. BMC Medical Education (2017) 17:136 Page 8 of 9 Page 8 of 9 Page 8 of 9 Howick et al. BMC Medical Education (2017) 17:136 Publisher’s Note Implications for clinical practice and clinical research Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Implications for clinical practice and clinical research The way different healthcare practitioners express em- pathy to patients is low (on average) in comparison with normative scores, and highly variable. Given the likely association between practitioner empathy and patient outcomes, further research is now warranted to investi- gate how these findings can be used to improve patient care. Future reports of the CARE questionnaire should include all the potentially relevant factors we have iden- tified here, especially details about response rates, and also consultation duration, gender, experience of practi- tioners, and other demographic details of patient raters and practitioners. Additional files (DOCX 48 kb) brief intervention targeting predominantly non-verbal communication in general practice consultations. Brit J Gen Pract. 2015;65(635):e351–6. 6. Kim SS, Kaplowitz S, Johnston MV. The effects of physician empathy on patient satisfaction and compliance. Eval Health Prof. 2004;27(3):237–51. 7 Attar HS Chandramani S Impact of physician empathy on migraine g p ; ( ) 6. Kim SS, Kaplowitz S, Johnston MV. The effects of physician empathy on patient satisfaction and compliance. Eval Health Prof. 2004;27(3):237–51. Additional file 5: CARE scores by country (all 64 studies included). Additional subgroup analysis by country. (DOCX 16 kb) Additional file 5: CARE scores by country (all 64 studies included). Additional subgroup analysis by country. (DOCX 16 kb) 7. Attar HS, Chandramani S. Impact of physician empathy on migraine disability and migraineur compliance. Annals of Indian Academy of Neurology. 2012;15(Suppl 1):S89–94. Availability of data and materials All data available in manuscript, supplemental material, or by contacting authors. All data available in manuscript, supplemental material, or by contacting authors. 13. Parker SM, Clayton JM, Hancock K, Walder S, Butow PN, Carrick S, Currow D, Ghersi D, Glare P, Hagerty R, et al. A systematic review of prognostic/end-of- life communication with adults in the advanced stages of a life-limiting illness: patient/caregiver preferences for the content, style, and timing of information. J Pain Symptom Manag. 2007;34(1):81–93. Funding JH 11. Mistiaen P, van Osch M, van Vliet L, Howick J, Bishop FL, Di Blasi Z, Bensing J, van Dulmen S. The effect of patient-practitioner communication on pain: a systematic review. Eur J Pain. 2015;20(5):675–88. JH was supported by the Nuffield Department of Primary Care Health Sciences. KM received support from the Theophrastus Foundation and the Schweizer-Arau Foundation, Germany. 12. Howick J, Rees S. Overthrowing barriers to empathy in healthcare: empathy in the age of the internet. J R Soc Med. 2017:141076817714443. http://journals.sagepub.com/doi/full/10.1177/0141076817714443. Author details 1 1Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford OX2 6GG, UK. 2Institute of Medical Psychology, Faculty of Medicine, LMU Munich, 80336 Munich, Germany. 3Faculty of Medicine, Vilnius University, 01513 Vilnius, Lithuania. 4Bodleian Health Care Libraries, Knowledge Centre, University of Oxford, Old Road Campus Research Building, Headington, Oxford OX3 7DQ, UK. 5Division Health Promotion, University of Applied Sciences Coburg, 95450 Coburg, Germany. Received: 1 November 2016 Accepted: 25 July 2017 Received: 1 November 2016 Accepted: 25 July 2017 Acknowledgments 8. Di Blasi Z, Harkness E, Ernst E, Georgiou A, Kleijnen J. Influence of context effects on health outcomes: a systematic review. Lancet. 2001;357(9258): 757–62. Bridget Johnson, Stewart Mercer, Vincent Chung, and Michelle Dossett shared their data with us when it was missing from published reports. Sir Muir Gray came up with the title for the paper. Claire Madigan provided some useful suggestions to improve the manuscript. 9. Derksen F, Bensing J, Lagro-Janssen A. Effectiveness of empathy in general practice: a systematic review. Br J Gen Pract. 2013;63(606):e76–84. 10. Kelm Z, Womer J, Walter JK, Feudtner C. Interventions to cultivate physician empathy: a systematic review. BMC Med Educ. 2014;14:219. Authors’ contributions JH drafted the protocol; KM helped develop the protocol. JH piloted the data extraction form with help from KM. NR conducted the search; LS and AU completed the searches, conflicts were resolved by discussion with JH. KM conducted the statistical analyses. JH drafted the final manuscript with help from KM. LS, AU, and NR also helped to develop the final manuscript. All authors read and approved the final manuscript. 14. Abraham A. Care and compassion: report of the health service ombudsman on ten investigations into NHS care of older people, fourth report if the health service commissioner for England; session 2010–2011. In. Edited by service PaH. London: The Stationary Office; 2011. 15. Francis R. Report of the mid Staffordshire foundation NHS trust public inquiry volumes 1–3, HC-898-I-III. In. London: The Stationary Office; 2013. Ethics approval and consent to participate Not relevant (systematic review). 16. Davies HT, Mannion R. Will prescriptions for cultural change improve the NHS? BMJ. 2013;346:f1305. pp Not relevant (systematic review). 17. Neumann M, Edelhauser F, Tauschel D, Fischer MR, Wirtz M, Woopen C, Haramati A, Scheffer C. Empathy decline and its reasons: a systematic review of studies with medical students and residents. Acad Med. 2011;86(8):996–1009. 17. Neumann M, Edelhauser F, Tauschel D, Fischer MR, Wirtz M, Woopen C, Haramati A, Scheffer C. Empathy decline and its reasons: a systematic review of studies with medical students and residents. Acad Med. 2011;86(8):996–1009. Consent for publication Not relevant (systematic review). Competing interests The authors declare that they have no competing interests. Additional files 2. Vangronsveld KL, Linton SJ. The effect of validating and invalidating communication on satisfaction, pain and affect in nurses suffering from low back pain during a semi-structured interview. Eur J Pain. 2012;16(2):239–46. 2. Vangronsveld KL, Linton SJ. The effect of validating and invalidating communication on satisfaction, pain and affect in nurses suffering from low back pain during a semi-structured interview. Eur J Pain. 2012;16(2):239–46. Additional file 1: The CARE Measure Questionnaire © Stewart W Mercer 2004. Actual questionnaire used within studies to measure patient perception of practitioner empathy (permission obtained). (DOCX 108 kb) Additional file 1: The CARE Measure Questionnaire © Stewart W Mercer 2004. Actual questionnaire used within studies to measure patient perception of practitioner empathy (permission obtained). (DOCX 108 kb) 3. Fujimori M, Shirai Y, Asai M, Kubota K, Katsumata N, Uchitomi Y. Effect of communication skills training program for oncologists based on patient preferences for communication when receiving bad news: a randomized controlled trial. J Clin Oncol. 2014;32(20):2166–72. Additional file 2: Search Strategy. Search terms used to identify studies for electronic searches. 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https://openalex.org/W4366274310	https://www.researchsquare.com/article/rs-2772368/latest.pdf	English	null	Maternal and Live-birth Outcomes of Intrahepatic Cholestasis of Pregnancy after in vitro fertilization	Research Square (Research Square)	2,023	cc-by	4,397	Rajluxmee Beejadhursing Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Fei Li (  736802127@qq.com ) Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Le Xu Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Rajluxmee Beejadhursing Conclusion ICP patients who underwent IVF did not experience adverse pregnancy outcomes compared with those who conceived spontaneously. Given that there was no significant difference in prognosis and stillbirth incidence in the third trimester for IVF women with ICP, we suggest following the routine management. Results Numerical values of transaminases (ALT, AST) and TBA are significantly lower in the IVF group than that in the SC group (p < 0.05). The incidence of GDM was higher in the IVF group than in SC group (30.6% vs 16%, p < 0.05). The cesarean section rates are higher in the IVF group (97.2% vs 85.4%, p < 0.05). On the other hand, the prevalence of premature rupture of membranes was higher in the SC group (10.7%) while none was reported in the IVF-ICP group. Other maternal comorbidities and neonatal outcomes were similar between the two groups. DOI: https://doi.org/10.21203/rs.3.rs-2772368/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at BMC Pregnancy and Childbirth on January 8th, 2024. See the published version at https://doi.org/10.1186/s12884-024-06248-x. Page 1/11 Page 1/11 Page 1/11 Background Intrahepatic cholestasis of pregnancy (ICP) is an idiopathic disease of pregnancy while knowledge about ICP after IVF outcomes is scarce, especially the exact maternal characteristics and pregnancy outcomes. Our aim is to evaluate the impact of in vitro fertilization (IVF) on the perinatal outcomes of intrahepatic cholestasis of pregnancy. Methods A retrospective study of 242 patients with intrahepatic cholestasis of pregnancy enrolled between 2019– 2021 was carried out. Data were analyzed from the medical archives of the Huazhong university of science and technology, Tongji Hospital. The data analysis was conducted with SPSS 20.0 statistical software, results below 0.05 indicated significance. 3. Exclusion Criteria Incomplete case data, twin pregnancy, multiple pregnancy, extrahepatic biliary tract obstructions, viral hepatitis, autoimmune hepatitis (AIH), HELLP syndrome and fatty liver of pregnancy. 4. Statistical Processing Introduction Intrahepatic cholestasis of pregnancy (ICP) is an idiopathic disease of pregnancy. Pruritus and elevated serum total bile acid (TBA) concentrations are the main clinical manifestations, but patients can recover after delivery [1]. The incidence rates ranged from 0.3–5.6% of pregnancies [2]. ICP is a significant cause of adverse perinatal outcomes in the third trimester, such as fetal distress, neonatal pneumonia, neonatal asphyxia and even fetal death [3]. It can also be associated with premature rupture of membranes, postpartum hemorrhage and increase the risk of gestational diabetes and gestational hypertension [4, 5]. Although a few recent studies suggest that assisted reproduction treatments may promote the occurrence and development of ICP, knowledge about ICP after IVF outcomes in China is scarce, especially the exact maternal characteristics and pregnancy outcomes [6–8]. Therefore, we aim to explore the impact of IVF Page 2/11 on perinatal outcomes associated with ICP and to find the best management model for IVF-ICP pregnant women. 4. Statistical Processing All analyses were conducted with SPSS 20.0 statistical software, and results below 0.05 indicated significance. We analyzed the data at 95% confidence interval for mean with standard deviation. Nonparametric Pearson Chi-square test and independent samples t-test were used for comparison between means and percentages. Continuous data are presented as medians and interquartile ranges or as means with standard deviations, and categorical data as counts and percentages. 1. General Information This study was a retrospective analysis of data from 242 pregnant women complicated with ICP, including 36 conceived through IVF and 206 spontaneous conceived (SC). All of the patients were delivered from January 2019 to December 2021 in the Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. As shown in Table 1, there was no significant difference in age, pregnancy and delivery histories between the two groups (p > 0.05). 2. Diagnostic criteria ICP was defined as presence of pruritus in the absence of rash, together with raised level of serum bile acids (cut-off level 10 µmol/L) and/or raised level of serum ALT (> 40 U/L), and by normalization of biochemical parameters after delivery. Gestational diabetes mellitus (GDM) was established following 75 g oral glucose tolerance test. Gestational diabetes mellitus (GDM) was established following 75 g oral glucose tolerance test. Materials And Methods 1. General Information Results The mean gestational age of both groups at onset of ICP was 35.5 weeks. Woman in IVF pregnancy cohorts were more likely to have higher rates of GDM compared to SC group (30.6% vs 16%, p < 0.05). In addition, IVF group also had a striking cesarean section rates (97.2% vs 85.4%, p < 0.05). Conversely, the prevalence of premature rupture of membranes was more common in SC group (10.7%) but no such occurrence was reported in the IVF-ICP group. Meanwhile, numerical values of transaminases Page 3/11 (ALT, AST) and TBA are greatly reduced in the IVF group than that in the SC group (p < 0.05) (Table 1). The proportion of TBA exceeding 100 µmol/L, defined as the severe ICP, was 2.8% in the IVF group while 9.2% in the SC group. (ALT, AST) and TBA are greatly reduced in the IVF group than that in the SC group (p < 0.05) (Table 1). The proportion of TBA exceeding 100 µmol/L, defined as the severe ICP, was 2.8% in the IVF group while 9.2% in the SC group. (ALT, AST) and TBA are greatly reduced in the IVF group than that in the SC group (p < 0.05) (Table 1). Th i f TBA di 100 l/L d fi d h ICP 2 8% i h IVF hil (ALT, AST) and TBA are greatly reduced in the IVF group than that in the SC group (p < 0.05) (Table 1). (ALT, AST) and TBA are greatly reduced in the IVF group than that in the SC group (p < 0.05) (Table 1). The proportion of TBA exceeding 100 µmol/L, defined as the severe ICP, was 2.8% in the IVF group while 9.2% in the SC group. Other maternal complications and neonatal outcomes, such as postpartum hemorrhage, gestational hypertension, preterm labor, had no significant differences between two groups (Table 2). Table 1 Maternal characteristics among the SC and IVF groups Characteristics Overall (n = 242) SC group (n = 206) IVF group (n = 36) P-Value Age (years) 29.9 ± 4.3 29.6 ± 4.5 31. Results 9 ± 3.8 0.286 Onset time of symptoms (weeks) 35.5 ± 3.4 35.5 ± 3.1 35.5 ± 4.7 0.414 Gestational age at delivery (weeks) 36.3 ± 2.5 36.2 ± 2.5 36.7 ± 2.2 0.358 primipara 130 100 30 - multigravida 112 106 6 - Serum ALT(IU/L) 126.9 ± 183.8 139.3 ± 192.6 55.9 ± 95.8 0.01 Serum AST(IU/L) 104.1 ± 153.7 112.3 ± 161.3 57.4 ± 87.9 0.025 TBA (µmol/L) 42.4 ± 34.5 44.8 ± 35.7 29.1 ± 23.1 0.005 Table 1 Maternal characteristics among the SC and IVF groups Table 1 Maternal characteristics among the SC and IVF groups Page 4/11 Page 4/11 Table 2 Analysis of maternal complications and neonatal outcomes Items Overall (n = 242) SC group (n = 206) IVF group (n = 36) P-Value Gestational hypertension 34 26 8 0.127 GDM 44 33 (16%) 11 (30.6%) 0.037 PROM 22 22 0 0.040 Postpartum hemorrhage 14 12 2 0.949 Vaginal delivery 31 30 1 <0.05 Cesarean section 211 176 35 <0.05 Preterm labor 125 107 18 0.830 Meconium-stained amniotic fluid 53 47 6 0.531 Stillbirth 0 0 0 - Asphyxia neonatorum 52 48 4 0.816 Low birth weight 78 67 11 0.816 Table 2 Analysis of maternal complications and neonatal outcomes Discussion ICP is a polygenic disorder and its specific pathogenesis has not been fully elucidated. In recent years, many literatures have reported that cholestasis may also be related to the mutation of genes encoding bile acid metabolic transporters such as ABCB11, ABCB4, ABCCC2, ATP 8B1, TJP2, which result in the abnormal function of bile salt export pump in hepatocytes [14]. The presence of high levels of hormones such as estrogen and progesterone in assisted reproductive technology are more likely to induce ICP, and tend to cause more adverse pregnancy outcomes [15, 16]. Redundant estrogen accumulates in liver cells, leading to decrease activity of Na+/K+-ATPase, which can inhibit the synthesis of hepatic biliary proteins, and it leads to obstruction of bile salt uptake and excretion [17]. Supplementary progestin administration in IVF patients could be an important trigger for bile acid retention, thereby causing adverse events, such as premature birth, respiratory disorders, meconium-stained amniotic fluid and even stillbirth [18]. Serum bile acids stimulate the release of prostaglandins from the uterus and decidua, increase the expression of oxytocin receptors in uterine smooth muscle, and induce preterm labor [19, 20]. Furthermore, high concentrations of bile acids cause hypoxia which can affect the fetal vagus nerve, whose stimulation can increase intestinal peristalsis and result in meconium-stained of amniotic fluid. As the fetus inhales the feces which deposit in the respiratory circulatory system, excessive bile acids can result in atelectasis, pulmonary edema, neonatal lung injury or even asphyxia [21, 22]. The most unfortunate outcome of ICP is stillbirth whose exact mechanism has yet to be fully clarified. Compared with adults, fetal cardiomyocytes are more susceptible to bile acids. Once bile acids enter systemic circulation, they can directly act on the heart, reducing cardiac systolic function and inducing fetal arrhythmia [23, 24]. It has also been suggested that bile acids stimulate blood vessels on the surface of the placental chorion, causing vasospasm and decreasing placental perfusion, which leads to sudden death [25]. It has been reported that the incidence of stillbirth is 18.3% at 36 weeks, while it increases to 33.6% at 39 weeks [26]. However, there were no cases of stillbirth in our study and we were unable to explore this aspect. Prompt and effective management of severe ICP patients significantly reduces the risk of adverse perinatal outcomes [27]. Discussion Interestingly, this study demonstrated that even ICP incidence was higher, but TBA, AST and ALT mean values are significantly lower in ICP pregnancies resulting from IVF. Only 1/36 patients of the IVF cohort reported a TBA that surpassed 100 µmol/L. Certain studies have shown that concentration of serum bile acid is proportional to perinatal complications. Once TBA exceeds 40 µmol/L, for each 1 µmol/L increase, the incidence of perinatal complications will go up by 1–2%. If it reaches 100 µmol/L, the risk of stillbirth will increase by 30 times [9, 10]. Although assisted reproduction probably induces ICP, our findings suggest that it does not significantly increase adverse outcomes. One possible explanation may be the benefit of more focused prenatal care and intensive treatment of patients who had recourse to IVF. The study demonstrated that no obvious difference between the two groups in incidence of pre-preeclampsia, postpartum hemorrhage and coagulation disorders. Nevertheless, IVF patients are more likely to develop gestational diabetes. In our study, pregnancies conceived by IVF has a higher incidence rate of GDM, OR 1.91 (95% CI 1.06–3.42). One of the common causes of infertility is ovulatory disorders. Such patients would normally present with serious endocrinological disorders, such as insulin resistance, or carry high risk factors for obstetrical diseases, for instance, obesity [11]. On the other hand, excessive progesterone could alter the function of the major hepatic bile acid receptors, which may enhance the precarious situation in ICP patients [12]. Further experimental report has revealed that estrogen could induce disorders of blood sugar, which may result in liver injury and exacerbate cholestasis [13]. With regard to Interestingly, this study demonstrated that even ICP incidence was higher, but TBA, AST and ALT mean values are significantly lower in ICP pregnancies resulting from IVF. Only 1/36 patients of the IVF cohort reported a TBA that surpassed 100 µmol/L. Certain studies have shown that concentration of serum bile acid is proportional to perinatal complications. Once TBA exceeds 40 µmol/L, for each 1 µmol/L increase, the incidence of perinatal complications will go up by 1–2%. If it reaches 100 µmol/L, the risk of stillbirth will increase by 30 times [9, 10]. Although assisted reproduction probably induces ICP, our findings Page 5/11 Page 5/11 ICP patients resulting from IVF, more attention should be paid to gestational glucose status where earlier management will alleviate the burden of disease in later pregnancy. Discussion For IVF patients with ICP in our study, operative delivery was personally preferred to minimize emergence of adverse perinatal events, which reflected as a high rate (97.2%) of cesarean section. However, prenatal monitoring and the timing of delivery must be a comprehensive consideration. The time of delivery should not only depend on the concentrations of serum total bile acid, but on feedback from prenatal monitoring such as Non-Stress Test, fetal growth, gestational age and the effectiveness of medical treatment. Our study demonstrated that the time to termination of pregnancy is around 36 weeks and does not increase the incidence of stillbirth. If appropriately extending the gestational age could reduce the incidence of iatrogenic premature birth. However, investigations on the optimal termination of ICP pregnancies with varying concentrations of serum total bile acid and larger sample studies must be carried out. Moreover, we understand that the journey to a successful pregnancy may not be easy and expectant mothers, particularly women who underwent fertility treatment, will readily opt for scheduled Page 6/11 cesarean surgery at any slight discrepancy. Therefore, patient education is crucial in informing would-be parents of the risks of surgery, and reassuring them that with the progress of medicine, adverse are less likely to happen given the frequency of antenatal care, modalities of prenatal testing and ways to manage any possible undesirable situation. In addition, the inclusion of all members of the multidisciplinary team involved in the process, namely, gynecologists, midwives, neonatologists, pediatricians, and physicians will definitely comfort patients that there is a whole team ensuring the safety of everybody. This study has analyzed IVF as a modern effective way of achieving pregnancy that does not increase adverse complications in ICP. Therefore, it is advisable to continue routine prenatal check-up. Our findings may help in reducing the waste of medical resources and avoiding excessive emotional tension relating to any infertility etiologies. However, there are two notable limitations. First, the sample size was limited and a large sample and comparative studies should be encouraged to get a more comprehensive understanding of the impact of IVF on ICP. Second, there were no cases of stillbirth in our study and we were unable to explore this aspect. Still, the data in this study permit a general assessment of the effect of assisted reproduction on ICP patients. Conclusion Through this retrospective, hospital-based cohort study, we concluded that ICP patients who underwent IVF did not have increased risk of preterm labor, gestational hypertension, and postpartum hemorrhage when compared with cohorts who conceived spontaneously. This may be due to the fact that IVF patients are closely monitored and active interventions happen throughout pregnancy. Regrettably, ICP women in our study, especially after IVF, will receive excessive attention from themselves and their surrounding families throughout their pregnancy. They overeat because they unreasonably worry about the lack of nutrition of the fetus, which leads to the increase of obesity during pregnancy and the incidence rate of GDM. At the same time, they mistakenly believe that a natural birth is likely to damage the fetus, so they insist on a cesarean section. Therefore, we suggest that ICP pregnant women after IVF pregnancy should follow the doctor's advice to conduct scientific and appropriate pregnancy monitoring and treatments, so as to ensure necessary treatments and avoid GDM and unnecessary cesarean sections. GDM GDM Gestational Diabetes Mellitus Gestational Diabetes Mellitus Gestational Diabetes Mellitus Authors' contributions LX and RB were responsible for the collection, arrangement, analysis and conclusion of data. FL was a supervisor, revised the manuscript. YZ contributed to writing, editing, and revised the manuscript. All authors read and approved the final manuscript. Ethical approval and consent to participate Informed consent was obtained before the survey from each participant in the current study. We got permission for our study from the Tongji hospital ethics committee. All methods were carried out in accordance with relevant guidelines and regulations. All participants gave written informed consent to participate in the study. Availability of data and materials The datasets used and/or analyzed during the current study will be available from the corresponding author on reasonable request. Conflict of interest The authors report no conflicts of interest. The authors report no conflicts of interest. ICP ICP Intrahepatic Cholestasis of Pregnancy IVF In Vitro Fertilization TBA Serum Total Bile Acid SC Spontaneous Conceived Page 7/11 References 1. V. Geenes, and C. Williamson, “Liver disease in pregnancy,” Best Pract Res Clin Obstet Gynaecol, vol. 29, no. 5, pp. 612-24, Jul, 2015, doi:10.1016/j.bpobgyn.2015.04.003. 1. V. Geenes, and C. Williamson, “Liver disease in pregnancy,” Best Pract Res Clin Obstet Gynaecol, vol. 29, no. 5, pp. 612-24, Jul, 2015, doi:10.1016/j.bpobgyn.2015.04.003. Page 8/11 2. S. Basaranoglu, E. Agacayak, F. Ucmak, S. Y. Tunc, A. Deregozu, Z. M. Akkurt, N. Peker, M. Acet, H. Yuksel, and T. 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https://openalex.org/W3004135256	https://zenodo.org/record/5654776/files/MRS_Advances_Version%20Zenodo.pdf	English	null	All-Inkjet-Printed Humidity Sensors for the Detection of Relative Humidity in Air and Soil—Towards the Direct Fabrication on Plant Leaves	MRS advances	2,020	cc-by	7,365	Figure 1 A) Scheme describing the capacitive RH sensor geometry. B) Top picture: silver IDE before CAB deposition. Bottom picture: IDE after CAB deposition. All-Inkjet-Printed Humidity Sensors for the Detection of Relative Humidity in Air and Soil—Towards the Direct Fabrication on Plant Leaves All-Inkjet-Printed Humidity Sensors for the Detection of Relative Humidity in Air and Soil—Towards the Direct Fabrication on Plant Leaves Walid Ait-Mammar1, Samia Zrig1, Nathalie Bridonneau1, Vincent Noël1, Eleni Stavrinidou2, Benoît Piro1, Giorgio Mattana1,* 1 Sorbonne Paris Cité, ITODYS, UMR 7086 CNRS, Univ. Paris Diderot, 15 rue J-A de Baïf, Cedex 13 75205 Paris, France 2Laboratory of Organic Electronics, Department of Science and Technology, Linköping University, SE-601 74, Norrköping, Swede *corresponding author: giorgio.mattana@univ-paris-diderot.fr Figure 2 Optical image of an inkjet-printed RH sensor on a leaf (left). Inset: zoom on the sensor, the IDE is visible as well as two small drops of conductive silver paste and the probes used for measurements. Figure 3 Example of capacitance vs frequency graph. Inset: equivalent circuit used to extract capacitance from impedance measurements. Example of capacitance vs frequency graph. Inset: equivalent circuit used to extract capacitance from impedance measurements. e measurements. Frequency [Hz] Capacitance [pF] Frequency [Hz] Capacitance [pF] 20 16.78 1.01E6 16.766 20200 16.761 1.03E6 16.774 40400 16.729 1.05E6 16.782 60600 16.708 1.07E6 16.79 80800 16.692 1.09E6 16.798 101000 16.68 1.11E6 16.805 121000 16.67 1.13E6 16.813 141000 16.662 1.15E6 16.821 162000 16.655 1.17E6 16.829 182000 16.649 1.19E6 16.838 202000 16.645 1.21E6 16.846 222000 16.641 1.23E6 16.855 242000 16.638 1.25E6 16.865 263000 16.636 1.27E6 16.875 283000 16.633 1.29E6 16.884 303000 16.632 1.31E6 16.894 323000 16.627 1.33E6 16.903 343000 16.627 1.35E6 16.913 364000 16.626 1.37E6 16.922 384000 16.626 1.39E6 16.932 404000 16.627 1.41E6 16.942 424000 16.629 1.43E6 16.952 444000 16.631 1.45E6 16.964 465000 16.632 1.47E6 16.974 485000 16.633 1.49E6 16.986 505000 16.635 1.52E6 16.996 525000 16.636 1.54E6 17.008 545000 16.639 1.56E6 17.019 566000 16.642 1.58E6 17.031 586000 16.645 1.6E6 17.042 606000 16.648 1.62E6 17.054 626000 16.651 1.64E6 17.066 646000 16.655 1.66E6 17.079 667000 16.659 1.68E6 17.092 687000 16.663 1.7E6 17.104 707000 16.668 1.72E6 17.117 727000 16.673 1.74E6 17.129 747000 16.677 1.76E6 17.142 768000 16.682 1.78E6 17.155 788000 16.687 1.8E6 17.169 808000 16.692 1.82E6 17.183 828000 16.697 1.84E6 17.197 848000 16.703 1.86E6 17.212 869000 16.709 1.88E6 17.226 889000 16.715 1.9E6 17.24 909000 16.722 1.92E6 17.254 929000 16.729 1.94E6 17.269 950000 16.745 1.96E6 17.284 970000 16.752 1.98E6 17.299 990000 16.758 2E6 17.314 Figure 4 Static characterization (expressed in terms of normalized capacitance) on three different devices: ■ upsweep curve, ● downsweep curve. The solid lines represent Boltzmann fits (in both cases: R2 > 0.99). Relative Humidity [%] Normalised Capacitance (Upsweep) [-] Standard Deviation (Upsweep) [-] Normalised Capacitance (Downsweep) [-] Standard Deviation (Downsweep) [-] 20 1 0 1 0 30 1.017 1E-3 1.00409 0.00173 40 1.03 1E-3 1.0076 0.00341 50 1.07 0.03 1.01265 0.00595 60 1.19 0.06 1.02716 0.0231 70 1.37 0.08 1.08499 0.06145 80 1.45 0.12 1.16554 0.09481 90 1.51 0.2 1.505 0.19515 Figure 5 Example of a sensor dynamic response (○ capacitance percentage variation, ■ RH). Example of a sensor dynamic response (○ capacitance percentage variation, ■ RH). p y p ( p p g , ) Time [s] R.H. [%] ΔC [%] Time [s] R.H. Figure 5 [%] ΔC [%] 3.126 20 0 1000.563 20 1.44737 4.596 20 0 1002.039 20 1.43889 6.067 20 -0.00848 1003.51 20 1.43748 7.537 20 -0.00565 1004.98 20 1.43182 9.008 20 -0.01131 1006.451 20 1.42193 10.479 20 -0.01979 1007.922 20 1.39507 11.949 20 -0.02262 1009.391 20 1.38659 13.425 20 -0.02544 1010.863 20 1.37528 14.895 20 -0.02968 1012.339 20 1.36398 16.366 20 -0.02968 1013.81 20 1.3555 17.837 20 -0.03392 1015.279 20 1.34136 19.306 20 -0.03816 1016.751 20 1.33853 20.779 20 -0.04382 1018.222 20 1.32864 22.25 20 -0.04947 1019.692 20 1.31309 23.724 20 -0.05936 1021.161 20 1.30744 25.196 20 -0.06219 1022.639 20 1.2933 26.667 20 -0.07209 1024.11 20 3.04315 28.138 20 -0.0735 1025.58 20 4.03822 29.607 20 -0.07915 1027.051 20 4.02267 31.079 20 -0.08622 1028.522 20 2.82124 32.549 20 -0.08905 1029.993 20 1.24383 34.025 20 -0.09187 1031.463 20 1.23959 35.497 20 -0.10318 1032.939 20 1.22546 36.967 20 -0.10601 1034.41 20 1.21274 38.438 20 -0.1159 1035.88 20 1.30179 39.908 20 -0.11166 1037.351 20 3.93221 41.379 20 -0.12297 1038.822 20 3.95624 42.85 20 -0.11873 1040.293 20 3.94069 44.326 20 -0.12862 1041.763 20 3.92938 45.797 20 -0.13286 1043.239 20 2.67566 47.267 20 -0.13993 1044.71 20 1.48836 48.738 20 -0.14983 1046.18 20 3.90112 50.207 20 -0.14983 1047.651 20 3.89263 51.677 20 -0.15407 1049.122 20 2.39155 53.15 20 -0.16113 1050.592 20 2.4283 54.626 20 -0.16679 1052.064 20 3.84882 56.097 20 -0.17244 1053.54 20 3.23962 57.568 20 -0.17668 1055.01 20 2.83396 59.038 20 -0.18658 1056.48 20 3.83751 60.509 20 -0.19082 1057.951 20 3.82903 61.979 20 -0.20071 1059.422 20 3.82196 63.45 20 -0.20919 1060.893 20 3.82055 64.926 20 -0.21202 1062.363 20 3.81065 66.397 20 -0.21626 1063.84 20 2.12017 67.867 20 -0.2205 1065.31 20 2.18378 69.338 20 -0.21767 1066.781 20 2.7958 70.809 20 -0.22332 1068.252 20 3.78663 72.28 20 -0.22757 1069.722 20 3.77249 73.75 20 -0.22474 1071.193 20 3.71595 75.226 20 -0.22474 1072.664 20 1.01061 76.697 20 -0.22474 1074.14 20 2.15409 78.168 20 -0.22332 1075.609 20 3.09828 79.638 20 -0.22615 1077.081 20 3.74564 81.109 20 -0.22898 1078.552 20 3.73857 82.58 20 -0.22898 1080.022 20 3.73009 84.049 20 -0.23181 1081.493 20 3.72019 85.526 20 -0.23322 1082.964 20 3.71454 86.997 20 -0.23887 1084.44 20 3.70606 88.468 20 -0.24029 1085.911 20 3.70465 89.938 20 -0.24453 1087.381 20 3.70182 91.409 20 -0.24877 1088.851 20 3.69475 92.88 20 -0.24453 1090.323 20 3.68062 94.35 20 -0.24594 1091.793 20 3.67496 95.827 20 -0.25442 1093.264 20 3.6679 97.297 20 -0.25301 1094.74 20 3.66224 98.768 20 -0.25442 1096.21 20 3.65518 100.239 20 -0.25583 1097.681 20 3.64952 101.709 20 -0.26997 1099.152 20 3.64245 103.18 20 -0.27138 1100.622 20 3.63256 104.651 20 -0.27421 1102.093 20 3.62408 106.126 20 -0.28128 1103.564 20 3.62125 107.598 20 -0.28834 1105.04 20 3.61277 109.068 20 -0.28552 1106.511 20 3.60146 110.539 20 -0.29258 1107.981 20 3.59722 112.008 20 -0.294 1109.452 20 3.59298 113.48 20 -0.29682 1110.922 20 3.58874 114.95 20 -0.294 1112.393 20 3.58168 116.427 20 -0.29824 1113.864 20 3.58026 117.898 20 -0.3053 1115.34 20 3.57602 119.368 20 -0.30955 1116.811 20 3.56754 120.839 20 -0.30248 1118.282 20 3.5633 122.31 20 -0.30813 1119.752 20 3.55906 123.78 20 -0.30672 1121.223 20 3.55199 125.251 20 -0.31237 1122.694 20 3.54351 126.726 20 -0.3152 1124.164 20 1.63253 128.198 20 -0.31379 1125.64 20 0.79577 129.668 20 -0.31237 1127.111 20 0.79153 131.139 20 -0.3152 1128.582 20 0.7986 132.61 20 -0.31237 1130.052 20 0.79718 134.08 20 -0.32085 1131.523 20 0.7774 135.551 20 -0.31661 1132.994 20 0.7675 137.027 20 -0.31661 1134.464 20 0.77174 138.498 20 -0.31661 1135.939 20 0.7675 139.968 20 -0.3152 1137.411 20 0.76468 141.439 20 -0.3152 1138.882 20 0.75761 142.91 20 -0.31379 1140.353 20 0.76185 144.381 20 -0.3152 1141.823 20 0.75337 145.851 20 -0.3152 1143.294 20 0.97528 147.327 20 -0.31379 1144.765 20 3.48556 148.798 20 -0.3152 1146.241 20 3.47991 150.269 20 -0.31944 1147.712 20 3.48273 151.739 20 -0.31944 1149.182 20 1.1873 153.21 20 -0.32085 1150.653 20 0.74206 154.681 20 -0.31661 1152.124 20 0.73499 156.152 20 -0.32227 1153.594 20 2.13855 157.627 20 -0.32368 1155.065 20 3.40358 159.098 20 -0.32368 1156.541 20 3.41489 160.569 20 -0.32227 1158.012 20 3.41065 162.039 20 -0.32509 1159.482 20 3.38662 163.51 20 -0.32509 1160.953 20 3.3951 164.981 20 -0.32509 1162.424 20 3.3951 166.451 20 -0.33781 1163.893 20 3.39369 167.927 20 -0.33499 1165.365 20 3.39227 169.399 20 -0.34488 1166.841 20 3.38803 170.869 20 -0.34488 1168.312 20 3.39086 172.34 20 -0.34488 1169.782 20 3.38803 173.81 20 -0.34912 1171.253 20 3.37673 175.281 20 -0.35053 1172.724 20 3.36401 176.752 20 -0.35902 1174.195 20 3.37249 178.228 20 -0.36043 1175.665 20 3.36542 179.698 20 -0.35902 1177.141 20 3.35411 181.169 20 -0.36608 1178.612 20 3.32726 182.639 20 -0.37315 1180.083 20 3.31877 184.11 20 -0.36608 1181.553 20 3.31312 185.581 20 -0.37174 1183.023 20 3.31312 187.052 20 -0.37315 1184.495 20 3.30323 188.528 20 -0.37174 1185.965 20 3.30323 189.999 20 -0.37032 1187.441 20 3.30181 191.469 20 -0.37456 1188.91 20 3.29899 192.94 20 -0.36891 1190.381 20 3.30464 194.41 20 -0.37456 1191.851 20 3.30747 195.881 20 -0.37598 1193.322 20 3.29616 197.352 20 -0.37739 1194.793 20 3.29475 198.828 20 -0.37739 1196.263 20 3.29616 200.299 20 -0.3788 1197.74 20 3.29757 201.77 20 -0.38022 1199.21 20 3.29051 203.24 20 -0.36608 1200.681 20 3.28627 204.711 20 -0.37315 1202.152 20 3.2792 206.181 20 -0.3675 1203.622 20 3.28627 207.652 20 -0.36891 1205.093 20 3.2792 209.128 20 -0.36608 1206.564 20 3.29616 210.599 20 -0.36891 1208.04 20 3.28485 212.069 20 -0.36326 1209.511 20 3.28485 213.54 20 -0.36467 1210.981 20 3.28768 215.011 20 -0.37032 1212.452 20 3.28344 216.482 20 -0.3675 1213.923 20 3.2693 217.952 20 -0.36467 1215.395 20 3.27354 219.428 20 -0.37032 1216.866 20 3.26365 220.899 20 -0.3675 1218.342 20 3.28344 222.37 20 -0.3675 1219.812 20 3.26224 223.84 20 -0.37315 1221.283 20 3.2481 225.311 20 -0.37032 1222.754 20 3.25517 226.782 20 -0.37032 1224.224 20 3.26082 228.252 20 -0.37598 1225.695 20 3.25941 229.729 20 -0.38446 1227.166 20 3.27072 231.199 20 -0.39152 1228.642 20 3.28203 232.67 20 -0.39718 1230.113 20 3.26365 234.14 20 -0.39294 1231.583 20 3.25658 235.611 20 -0.39718 1233.054 20 3.25941 237.08 20 -0.39859 1234.525 20 3.25658 238.552 20 -0.39859 1235.995 20 3.2481 240.029 20 -0.39859 1237.466 20 3.26365 241.499 20 -0.40283 1238.942 20 3.258 242.97 20 -0.40425 1240.413 20 3.258 244.44 20 -0.40707 1241.883 20 3.25941 245.911 20 -0.40001 1243.354 20 3.24245 247.382 20 -0.41131 1244.825 20 3.23538 248.852 20 -0.4523 1246.295 20 3.2269 250.329 20 -0.41697 1247.766 20 3.21983 251.799 20 -0.41697 1249.242 20 3.21418 253.27 20 -0.41555 1250.713 20 2.95552 254.74 20 -0.39859 1252.183 20 2.57813 256.211 20 -0.40001 1253.654 20 2.32795 257.682 20 -0.3887 1255.125 20 2.49473 259.153 20 -0.39294 1256.596 20 2.39014 260.629 20 -0.39011 1258.066 20 3.17743 262.1 20 -0.42121 1259.542 20 3.84034 263.57 20 -0.42545 1261.013 20 3.83186 265.041 20 -0.41697 1262.483 20 3.83751 266.512 20 -0.42403 1263.954 20 3.83751 267.982 20 -0.41131 1265.425 20 3.83892 269.453 20 -0.41979 1266.895 20 3.8474 270.929 20 -0.42262 1268.366 20 3.85164 272.4 20 -0.44241 1269.842 20 3.86013 273.87 20 -0.45089 1271.313 20 3.86437 275.341 20 -0.4523 1272.784 20 3.86861 276.812 20 -0.44524 1274.254 20 3.87002 278.282 20 -0.40425 1275.725 20 3.86719 279.753 20 -0.41838 1277.196 20 3.87709 281.229 20 -0.36608 1278.667 20 3.87426 282.7 20 -0.41131 1280.143 20 3.87567 284.171 20 -0.37739 1281.613 20 3.87285 285.641 20 -0.26431 1283.084 20 3.87426 287.112 20 -0.25725 1284.554 20 3.87143 288.583 20 -0.25018 1286.025 20 3.87285 290.053 20 -0.24594 1287.496 20 3.87991 291.529 20 -0.24029 1288.966 20 3.87426 293 20 -0.23887 1290.443 20 3.87567 294.471 20 -0.24029 1291.913 20 3.87426 295.941 20 -0.23322 1293.384 20 3.86861 297.412 20 -0.23463 1294.855 20 3.8573 298.883 20 -0.2205 1296.325 20 3.85871 300.353 20 -0.21767 1297.796 20 3.83751 301.83 20 -0.21767 1299.267 20 3.8262 303.3 20 -0.20636 1300.743 20 3.78945 304.771 20 -0.20354 1302.214 20 3.78663 306.241 20 -0.20354 1303.684 20 3.81348 307.712 20 -0.20212 1305.155 20 3.78804 309.183 20 -0.2106 1306.625 20 3.77532 310.653 20 -0.22191 1308.094 20 3.77249 312.13 20 -0.22332 1309.567 20 3.7626 313.601 20 -0.22898 1311.042 20 3.7739 315.071 20 -0.23887 1312.512 20 3.77815 316.541 20 -0.24311 1313.983 20 3.77673 318.012 20 -0.25301 1315.454 20 3.76825 319.483 20 -0.25725 1316.926 20 3.73857 320.954 20 -0.26149 1318.396 20 3.7414 322.43 20 -0.26714 1319.867 20 3.73291 323.9 20 -0.27704 1321.343 20 3.66648 325.369 20 -0.27562 1322.814 20 3.64245 326.84 20 -0.28128 1324.284 20 3.6679 328.311 20 -0.28552 1325.755 20 3.6679 329.781 20 -0.2841 1327.226 20 3.68627 331.252 20 -0.28269 1328.697 20 3.26365 332.728 20 -0.28834 1330.167 20 2.46929 334.199 20 -0.29258 1331.641 20 1.10249 335.669 20 -0.294 1333.112 20 2.48484 337.14 20 -0.28693 1334.584 20 3.97885 338.611 20 -0.29824 1336.055 80 5.85026 340.081 20 -0.31944 1337.526 80 10.08071 341.552 20 -0.32368 1338.997 80 18.26881 343.028 20 -0.32651 1340.467 80 28.18414 344.499 20 -0.30672 1341.943 80 36.02736 345.97 20 -0.27845 1343.414 80 42.13063 347.442 20 -0.2629 1344.885 80 48.08973 348.913 20 -0.28552 1346.355 80 52.56611 350.383 20 -0.97811 1347.826 80 56.64532 351.854 20 -1.18164 1349.296 80 59.19236 353.33 80 1.25655 1350.767 80 60.78955 354.801 80 6.10892 1352.243 80 63.00866 356.271 80 12.29841 1353.714 80 64.6624 357.742 80 20.0865 1355.185 80 66.27656 359.213 80 28.52337 1356.656 80 67.94584 360.683 80 36.262 1358.126 80 69.15433 362.154 80 42.0642 1359.597 80 70.26248 363.63 80 46.7413 1361.067 80 71.08369 365.101 80 50.56185 1362.544 80 72.21728 366.572 80 54.12232 1364.014 80 73.13602 368.042 80 57.40434 1365.485 80 74.00953 369.513 80 59.97541 1366.955 80 74.87314 370.984 80 61.92879 1368.426 80 75.76362 372.454 80 64.06451 1369.896 80 76.72052 373.93 80 64.7995 1371.368 80 77.49085 375.401 80 66.61578 1372.844 80 78.16082 376.872 80 68.23842 1374.315 80 78.92832 378.342 80 69.19956 1375.785 80 79.71561 379.813 80 70.50842 1377.256 80 80.37428 381.283 80 71.06956 1378.727 80 81.0598 382.754 80 72.00243 1380.197 80 81.73826 385.595 80 72.99326 1381.667 80 82.52979 387.076 80 74.96643 1383.143 80 83.03863 388.547 80 76.26115 1384.614 80 83.53334 390.017 80 77.17 1386.085 80 84.09872 391.488 80 77.79615 1387.556 80 84.42381 392.958 80 78.0845 1389.026 80 84.73477 394.431 80 79.11914 1390.497 80 85.08813 395.902 80 79.85979 1391.967 80 85.48389 397.372 80 80.52128 1393.443 80 85.78072 398.843 80 81.15874 1394.914 80 85.87966 400.313 80 81.70999 1396.385 80 86.07754 401.784 80 82.45912 1397.856 80 86.31783 403.255 80 83.13757 1399.327 80 86.4309 404.731 80 83.64641 1400.797 80 86.61465 406.202 80 84.19766 1402.267 80 86.88321 407.672 80 85.10226 1403.743 80 87.05282 409.143 80 85.78072 1405.214 80 87.2083 410.612 80 86.27542 1406.685 80 87.40618 412.084 80 86.78427 1408.156 80 87.63233 413.553 80 87.36378 1409.627 80 87.75954 415.031 80 87.97156 1411.097 80 87.84435 416.502 80 88.49454 1412.567 80 87.9857 417.972 80 88.96097 1414.043 80 88.14118 419.443 80 89.46982 1415.514 80 88.35319 420.914 80 89.83731 1416.984 80 88.56521 422.384 80 90.26135 1418.456 80 88.72069 423.855 80 90.58644 1419.926 80 88.86203 425.331 80 90.9398 1421.397 80 89.04578 426.802 80 91.26489 1422.868 80 89.22953 428.272 80 91.53345 1424.343 80 89.37087 429.743 80 91.7172 1425.814 80 89.45568 431.214 80 91.92921 1427.285 80 89.55462 432.684 80 92.14123 1428.755 80 89.68183 434.155 80 92.40979 1430.226 80 89.76664 435.631 80 92.69248 1431.697 80 89.89385 437.102 80 93.05997 1433.168 80 90.03519 438.573 80 93.65362 1434.644 80 90.16241 440.043 80 93.22959 1436.114 80 90.36029 441.514 80 93.00343 1437.585 80 89.95039 442.985 80 93.14478 1439.055 80 90.04933 444.455 80 93.25786 1440.526 80 89.95039 445.931 80 93.38507 1441.997 80 89.86558 447.402 80 93.38507 1443.468 80 90.02106 448.873 80 93.14478 1444.944 80 89.90798 450.343 80 93.4416 1446.414 80 90.02106 451.814 80 93.68189 1447.885 80 90.14827 453.285 80 93.61122 1449.356 80 90.27548 454.755 80 93.8091 1450.826 80 90.65711 456.231 80 93.78083 1452.297 80 91.12355 457.702 80 93.99285 1453.767 80 91.67479 459.173 80 94.04939 1455.244 80 91.16595 460.642 80 94.10592 1456.714 80 90.51577 462.114 80 94.24727 1458.185 80 90.68538 463.585 80 94.48755 1459.655 80 90.71365 465.056 80 94.6713 1461.126 80 90.4875 466.532 80 94.72784 1462.596 80 89.92212 468.002 80 94.85505 1464.072 80 91.44864 469.473 80 94.9964 1465.544 80 92.73488 470.944 80 95.05293 1467.014 80 92.84796 472.414 80 95.13774 1468.485 80 92.93276 473.885 80 95.33562 1469.955 80 93.00343 475.356 80 95.4911 1471.427 80 93.08824 476.831 80 95.54764 1472.897 80 93.17305 478.302 80 95.71725 1474.368 80 93.22959 479.773 80 95.84446 1475.843 80 93.32853 481.244 80 96.57946 1477.314 80 93.45574 482.714 80 96.80561 1478.785 80 93.49814 484.185 80 96.94695 1480.255 80 93.49814 485.656 80 97.10243 1481.727 80 93.56881 487.132 80 97.22964 1483.197 80 93.66776 488.602 80 97.34272 1484.668 80 93.73843 490.073 80 97.38512 1486.144 80 93.7667 491.544 80 97.42753 1487.614 80 93.82323 493.015 80 97.45579 1489.085 80 93.87977 494.485 80 97.4982 1490.555 80 93.90804 495.956 80 97.56887 1492.026 80 93.92218 497.432 80 97.63954 1493.498 80 93.96458 498.903 80 97.66781 1494.968 80 93.99285 500.373 80 97.69608 1496.444 80 94.04939 501.844 80 97.76675 1497.915 80 94.12006 503.315 80 97.80916 1499.385 80 94.31794 504.785 80 97.87983 1500.857 80 94.45929 506.256 80 97.96464 1502.326 80 94.52996 507.732 80 98.04944 1503.797 80 94.64303 509.203 80 98.12011 1505.268 80 94.71371 510.672 80 98.12011 1506.744 80 94.75611 512.144 80 98.13425 1508.215 80 94.78438 513.615 80 98.17665 1509.685 80 94.84092 515.084 80 98.26146 1511.156 80 94.92572 516.555 80 98.33213 1512.626 80 94.95399 518.032 80 98.34627 1514.097 80 95.01053 519.503 80 98.3604 1515.568 80 95.0812 520.974 80 98.34627 1517.044 80 95.12361 522.444 80 98.4028 1518.515 80 95.15187 523.914 80 98.45934 1519.985 80 95.16601 525.386 80 98.50175 1521.456 80 95.15187 526.855 80 98.54415 1522.927 80 95.0388 528.332 80 98.57242 1524.397 80 95.0812 529.802 80 98.62896 1525.868 80 95.12361 531.273 80 98.71376 1527.344 80 95.13774 532.743 80 98.79857 1528.815 80 95.10947 534.215 80 98.85511 1530.285 80 95.16601 535.686 80 98.86924 1531.756 80 95.20841 537.156 80 98.92578 1533.227 80 95.29322 538.633 80 98.98232 1534.698 80 95.33562 540.103 80 99.01059 1536.168 80 95.36389 541.574 80 99.05299 1537.644 80 95.34976 543.044 80 99.09539 1539.115 80 95.37803 544.515 80 99.12366 1541.95 80 95.42043 545.986 80 99.1378 1543.431 80 95.36389 547.456 80 99.15193 1544.902 80 95.63245 548.933 80 99.19433 1546.372 80 95.68899 550.403 80 99.27914 1547.844 80 95.74552 551.874 80 99.32155 1549.315 80 95.78793 553.344 80 99.27914 1550.786 80 95.8586 554.815 80 99.15193 1552.258 80 95.92927 556.286 80 99.16607 1553.727 80 95.98581 557.756 80 99.20847 1555.198 80 96.05648 559.233 80 99.1802 1556.668 80 96.12715 560.703 80 99.12366 1558.146 80 96.18369 562.174 80 99.15193 1559.615 80 96.21196 563.645 80 99.15193 1561.086 80 96.25436 565.115 80 99.16607 1562.558 80 96.29677 566.586 80 99.20847 1564.027 80 96.32504 568.057 80 99.23674 1565.498 80 96.42398 569.533 80 99.23674 1566.97 80 96.46638 571.004 80 99.25087 1568.444 80 96.46638 572.474 80 99.25087 1569.915 80 96.46638 573.945 80 99.27914 1571.387 80 96.49465 575.415 80 99.30741 1572.856 80 96.52292 576.886 80 99.33568 1574.329 80 96.52292 578.357 80 99.43462 1575.798 80 96.52292 579.833 80 99.47702 1577.27 80 96.53705 581.304 80 99.47702 1578.745 80 96.48052 582.774 80 99.49116 1580.217 80 96.62186 584.245 80 99.51943 1581.686 80 96.56532 585.715 80 99.53356 1583.158 80 96.63599 587.186 80 99.53356 1584.627 80 96.60773 588.656 80 99.5477 1586.099 80 96.59359 590.133 80 99.5901 1587.568 80 96.62186 591.604 80 99.6325 1589.047 80 96.6784 593.074 80 99.66077 1590.515 80 96.69253 594.545 80 99.67491 1591.988 80 96.73494 596.016 80 99.73144 1593.457 80 96.76321 597.486 80 99.75971 1594.929 80 96.76321 598.957 80 99.77385 1596.398 80 96.73494 600.433 80 99.74558 1597.87 80 96.80561 601.904 80 99.74558 1599.345 80 96.81974 603.375 80 99.77385 1600.817 80 96.83388 604.845 80 99.80212 1602.286 80 96.86215 606.316 80 99.81625 1603.758 80 96.90455 607.787 80 99.80212 1605.228 80 96.94695 609.257 80 99.78798 1606.699 80 97.01763 610.734 80 99.75971 1608.169 80 97.03176 612.204 80 99.71731 1609.646 80 97.11657 613.675 80 99.71731 1611.116 80 97.18724 615.145 80 99.74558 1612.588 80 97.30032 616.616 80 99.75971 1614.057 80 97.31445 618.087 80 99.77385 1615.529 80 97.32858 619.558 80 99.78798 1616.998 80 97.34272 621.033 80 99.81625 1618.47 80 97.37099 622.504 80 99.83039 1619.945 80 97.39926 623.975 80 99.84452 1621.417 80 97.41339 625.446 80 99.85866 1622.886 80 97.45579 626.916 80 99.87279 1624.359 80 97.48406 628.387 80 99.88692 1625.828 80 97.4982 629.858 80 99.94346 1627.298 80 97.52647 631.334 80 100.05654 1628.769 80 97.56887 632.805 80 100.0424 1630.246 80 97.55474 634.275 80 100.07067 1631.716 80 97.55474 635.746 80 100.02827 1633.187 80 97.583 637.217 80 99.83039 1634.657 80 97.59714 638.687 80 99.77385 1636.129 80 97.63954 640.158 80 99.75971 1637.599 80 97.66781 641.634 80 99.77385 1639.071 80 97.66781 643.105 80 99.78798 1640.546 80 97.71021 644.575 80 99.83039 1642.017 80 97.71021 646.046 80 99.85866 1643.487 80 97.71021 647.517 80 99.85866 1644.958 80 97.75262 648.987 80 99.88692 1646.428 80 97.78089 650.458 80 99.92933 1647.9 80 97.78089 651.934 80 99.94346 1649.369 80 97.76675 653.405 80 99.92933 1650.847 80 97.75262 654.875 80 99.94346 1652.316 80 97.75262 656.346 80 99.92933 1653.788 80 97.76675 657.817 80 99.97173 1655.258 80 97.75262 659.287 80 99.97173 1656.728 80 97.86569 660.758 80 99.94346 1658.199 80 97.89396 662.234 80 99.9576 1659.671 80 97.86569 663.705 80 99.90106 1661.146 80 97.83743 665.176 80 99.90106 1662.616 80 97.82329 666.646 80 99.94346 1664.087 80 97.80916 668.117 80 100 1665.558 80 97.82329 669.588 80 99.90106 1667.028 80 97.82329 671.058 80 99.77385 1668.499 80 97.79502 672.534 80 99.02472 1669.97 80 97.86569 674.005 80 98.99645 1671.446 80 97.86569 675.476 80 98.99645 1672.917 80 97.87983 676.946 80 98.92578 1674.388 80 97.89396 678.417 80 98.47348 1675.858 80 97.92223 679.888 80 98.55828 1677.329 80 97.92223 681.358 80 98.57242 1678.799 80 97.89396 682.834 80 98.69963 1680.27 80 97.89396 684.305 80 98.91165 1681.746 80 97.92223 685.776 80 98.99645 1683.217 80 97.96464 687.246 80 98.96818 1684.687 80 97.97877 688.717 80 98.99645 1687.524 80 98.02117 690.188 80 99.02472 1689.003 80 97.87983 691.658 80 99.02472 1690.474 80 98.06358 693.135 80 98.99645 1691.946 80 98.03531 694.605 80 99.03886 1693.417 80 98.04944 696.076 80 99.05299 1694.887 80 98.04944 697.546 80 99.05299 1696.358 80 97.9929 699.017 80 99.10953 1697.829 80 97.96464 700.488 80 99.1802 1699.299 80 97.96464 701.958 80 99.1802 1700.77 80 98.04944 703.435 80 99.16607 1702.246 80 98.10598 704.906 80 99.20847 1703.717 80 98.09185 706.376 80 99.2226 1705.188 80 98.10598 707.847 80 99.23674 1706.658 80 98.13425 709.317 80 99.27914 1708.129 80 98.20492 710.788 80 99.29328 1709.6 80 98.16252 712.258 80 99.26501 1711.07 80 98.17665 713.734 80 99.12366 1712.547 80 98.19079 715.206 80 99.26501 1714.017 80 98.16252 716.676 80 99.20847 1715.488 80 98.13425 718.147 80 99.2226 1716.96 80 98.14838 719.618 80 99.53356 1718.429 80 98.13425 721.088 80 99.83039 1719.9 80 98.16252 722.559 80 99.80212 1721.37 80 98.13425 724.035 80 99.91519 1722.847 80 98.12011 725.506 80 99.9576 1724.318 80 98.14838 726.976 80 99.9576 1725.788 80 98.17665 728.447 80 99.98587 1727.258 80 98.20492 729.918 80 100.02827 1728.729 80 98.23319 731.388 80 100.05654 1730.201 80 98.19079 732.859 80 99.98587 1731.671 80 98.20492 734.335 80 99.68904 1733.147 80 98.20492 735.806 80 99.68904 1734.619 80 98.20492 737.277 80 99.70318 1736.088 80 98.23319 738.747 80 99.71731 1737.558 80 98.21906 740.218 80 99.67491 1739.03 80 98.21906 741.688 80 99.71731 1740.5 80 98.24733 743.159 80 99.77385 1741.97 80 98.21906 744.635 80 99.80212 1743.447 80 98.20492 746.106 80 99.83039 1744.918 80 98.21906 747.577 80 99.80212 1746.388 80 98.24733 749.046 80 99.84452 1747.86 80 98.27559 750.518 80 99.85866 1750.696 80 98.30386 751.989 80 99.83039 1752.175 80 98.16252 753.459 80 99.83039 1753.647 80 98.33213 754.935 80 99.83039 1755.118 80 98.33213 756.406 80 99.83039 1756.588 80 98.33213 757.877 80 99.84452 1758.059 80 98.33213 759.347 80 99.81625 1759.53 80 98.30386 760.818 80 99.81625 1761 80 98.30386 762.289 80 99.83039 1762.471 80 98.28973 763.759 80 99.81625 1763.948 80 98.30386 765.235 80 99.84452 1765.418 80 98.30386 766.706 80 99.84452 1766.888 80 98.318 768.177 80 99.85866 1768.359 80 98.28973 769.647 80 99.88692 1769.83 80 98.30386 771.118 80 99.87279 1771.3 80 98.30386 772.589 80 99.84452 1772.771 80 98.30386 774.06 80 99.84452 1774.247 80 98.34627 775.536 80 99.78798 1775.718 80 98.33213 777.006 80 99.74558 1777.188 80 98.30386 778.477 80 99.70318 1778.659 80 98.26146 779.948 80 99.68904 1780.13 80 98.24733 781.418 80 99.66077 1781.6 80 98.24733 782.889 80 99.64664 1783.071 80 98.24733 784.359 80 99.64664 1784.547 80 98.26146 785.836 80 99.64664 1786.018 80 98.26146 787.307 80 99.6325 1787.489 80 98.27559 788.777 80 99.60423 1788.959 80 98.30386 790.248 80 99.57597 1790.43 80 98.30386 791.719 80 99.60423 1791.9 80 98.26146 793.189 80 99.61837 1793.371 80 98.26146 794.66 80 99.60423 1794.847 80 98.26146 796.136 80 99.61837 1796.318 80 98.24733 797.607 80 99.5901 1797.789 80 98.318 799.077 80 99.60423 1799.259 80 98.26146 800.548 80 99.60423 1800.73 80 98.27559 802.019 80 99.60423 1803.672 80 98.27559 803.489 80 99.6325 1805.148 80 98.27559 804.96 80 99.6325 1806.618 80 98.28973 806.436 80 99.61837 1808.089 80 98.318 807.907 80 99.64664 1809.559 80 98.30386 809.378 80 99.68904 1811.03 80 98.34627 810.848 80 99.70318 1812.501 80 98.34627 812.319 80 99.67491 1813.972 80 98.33213 813.79 80 99.68904 1815.448 80 98.3604 815.26 80 99.73144 1816.919 80 98.28973 816.736 80 99.71731 1818.389 80 98.30386 818.207 80 99.70318 1819.86 80 98.318 819.676 80 99.68904 1821.33 80 98.33213 821.148 80 99.67491 1822.801 80 98.34627 822.619 80 99.66077 1824.272 80 98.34627 824.09 80 99.66077 1825.748 80 98.33213 825.56 80 99.66077 1827.219 80 98.318 827.036 80 99.6325 1828.689 80 98.33213 828.507 80 99.61837 1830.16 80 98.34627 829.978 80 99.6325 1831.631 80 98.34627 831.448 80 99.60423 1833.101 80 98.3604 832.919 80 99.60423 1834.572 80 98.37454 834.39 80 99.57597 1836.048 80 98.34627 835.861 80 99.56183 1837.519 80 98.34627 837.337 80 99.5477 1838.989 80 98.3604 838.807 80 99.57597 1840.46 80 98.3604 840.278 80 99.57597 1841.931 80 98.4028 841.748 80 99.56183 1843.401 80 98.4028 843.219 80 99.56183 1844.877 80 98.37454 844.69 80 99.51943 1846.348 80 98.37454 846.161 80 99.53356 1847.819 80 98.45934 847.637 80 99.5477 1849.289 80 98.45934 849.108 80 99.51943 1850.76 80 98.48761 850.577 80 99.49116 1852.231 80 98.47348 852.048 80 99.49116 1855.173 80 98.4028 853.519 80 99.49116 1858.121 80 98.37454 854.99 80 99.50529 1861.062 80 97.51233 856.461 80 99.47702 1864.002 20 54.99159 857.937 80 99.44876 1866.95 20 13.78535 859.407 80 99.46289 1869.891 20 6.52447 860.878 80 99.56183 1872.838 20 5.83188 862.349 80 99.61837 1875.778 20 5.55909 865.184 80 99.61837 1878.72 20 5.4135 866.665 80 99.42049 1881.661 20 5.29195 868.137 80 99.57597 1884.601 20 5.19583 869.608 80 99.5901 1887.55 20 5.11385 871.078 80 99.5901 1890.491 20 3.60995 872.549 80 99.56183 1893.438 20 2.92866 874.02 80 99.56183 1896.378 20 2.88343 875.49 80 99.57597 1899.321 20 2.82407 876.961 80 99.60423 1902.261 20 2.77318 878.437 80 99.60423 1905.202 20 2.72654 879.908 80 99.56183 1908.151 20 2.6799 881.378 80 99.56183 1911.091 20 2.63043 882.849 80 99.46289 1914.038 20 2.58944 884.32 80 99.46289 1916.979 20 2.55127 885.79 80 99.47702 1919.921 20 2.51876 887.261 80 99.36395 1922.861 20 2.47636 888.737 80 99.37808 1925.802 20 2.4495 890.208 80 99.33568 1928.751 20 2.41558 891.678 80 99.32155 1931.692 20 2.38731 893.149 80 99.30741 1934.638 20 2.36894 894.62 80 99.2226 1937.579 20 2.33784 896.09 80 99.20847 1940.521 20 2.32088 897.561 80 98.96818 1943.461 20 2.29403 899.037 80 98.99645 1946.402 20 2.28696 900.508 80 99.02472 1949.351 20 2.26293 901.979 80 99.08126 1952.292 20 2.25021 903.449 80 99.06712 1955.239 20 2.22759 904.92 80 99.08126 1958.179 20 2.21063 906.391 80 99.08126 1961.121 20 2.18943 907.861 80 99.09539 1964.062 20 2.1654 909.338 80 99.16607 1967.002 20 2.14561 910.808 80 99.19433 1969.951 20 2.11593 912.279 80 99.25087 1972.892 20 2.10038 913.749 80 99.25087 1975.839 20 2.09614 915.22 80 98.99645 1978.779 20 2.07211 916.691 80 98.37454 1981.723 20 2.04385 918.161 80 98.318 1984.663 20 2.02123 919.638 80 98.79857 1987.604 20 2.00144 921.108 20 97.28618 1990.552 20 1.97741 922.579 20 94.04939 1993.492 20 1.96187 924.05 20 68.40238 1996.439 20 1.94914 925.52 20 39.27971 1999.381 20 1.92512 926.991 20 17.2483 2002.323 20 1.92512 928.462 20 8.88917 2003.792 20 1.90533 929.938 20 6.31811 2006.735 20 1.89967 931.408 20 5.60856 2009.675 20 1.88837 932.879 20 5.13788 2012.622 20 1.87706 934.35 20 4.89477 2015.563 20 1.8601 935.82 20 4.69971 2018.503 20 1.84738 937.291 20 4.63752 2021.452 20 1.83748 938.762 20 4.56402 2024.393 20 1.82193 940.238 20 4.49618 2027.333 20 1.80497 941.709 20 4.41702 2030.274 20 1.79649 943.179 20 4.34635 2033.222 20 1.78236 944.65 20 4.16402 2036.163 20 1.77529 946.121 20 4.15978 2039.103 20 1.75974 947.591 20 4.20501 2042.051 20 1.74137 949.062 20 4.1626 2044.993 20 1.73571 950.538 20 4.13292 2047.934 20 1.73006 952.009 20 4.09334 2050.874 20 1.70462 953.479 20 4.04953 2053.823 20 1.69472 954.95 20 4.0255 2056.763 20 1.69755 956.421 20 3.99299 2059.704 20 1.67776 957.891 20 3.95341 2062.653 20 1.67069 959.362 20 2.22618 2065.593 20 1.6608 960.838 20 1.87282 2068.534 20 1.64667 962.309 20 1.84455 2071.475 20 1.64243 963.78 20 1.83748 2074.422 20 1.62546 965.25 20 1.80639 2077.363 20 1.62122 966.721 20 1.78942 2080.303 20 1.6085 968.192 20 1.7654 2083.253 20 1.60568 969.662 20 1.74702 2086.194 20 1.60568 971.137 20 1.72158 2089.134 20 1.59013 972.608 20 1.70179 2092.075 20 1.58872 974.08 20 1.69048 2095.023 20 1.57882 975.55 20 1.67069 2097.964 20 1.58165 977.021 20 1.64808 2100.904 20 1.57741 978.492 20 1.63112 2103.853 20 1.56751 979.962 20 1.61557 2106.793 20 1.56186 981.439 20 1.59578 2109.734 20 1.56327 982.909 20 1.57882 2112.675 20 1.55479 984.38 20 1.56045 2115.624 20 1.54631 985.85 20 1.54348 2118.564 20 1.54348 987.321 20 1.52652 2121.505 20 1.53218 988.791 20 1.50674 2124.452 20 1.52794 990.262 20 1.49119 2127.392 20 1.52087 991.739 20 1.48412 2130.333 20 1.5138 993.21 20 1.4714 2133.273 20 1.50249 994.68 20 1.4714 2136.222 20 1.50815 996.151 20 1.4615 2139.162 20 1.49401 997.621 20 1.4615 2142.103 20 1.48836 999.092 20 1.44737 2145.052 20 1.48412 Figure 6 A) Example of a sensor dynamic characterization in soil; the red percentage values are the RH values displayed by the commercial sensor. B) Normalised static capacitance in soil (■ upsweep curve, ● downsweep curve). A) Example of a sensor dynamic characterization in soil; the red percentage values are the RH values displayed by the commercial sensor. B) Normalised static capacitance in soil (■ upsweep curve, ● downsweep curve). A) Capacitance in soil vs Time A) Capacitance in soil vs Time Time [s] Capacitance [pF] Time [s] Capacitance [pF] 1.654 15.863 501.95 20.994 3.13 15.628 503.421 20.995 4.6 15.59 504.892 20.997 6.071 15.566 506.363 20.998 7.541 15.502 507.839 20.999 9.012 15.468 509.31 21 10.483 15.388 510.778 21.002 11.953 15.286 512.251 21.003 13.43 15.259 513.721 21.004 14.9 15.222 515.192 21.006 16.371 15.206 516.663 21.007 17.842 15.204 518.139 21.009 19.314 15.154 519.61 21.007 20.785 15.146 521.08 21.007 22.255 15.141 522.551 21.012 23.732 15.125 524.021 21.013 25.202 15.107 525.492 21.015 26.671 15.142 526.963 21.017 28.142 15.164 528.439 21.019 29.613 15.429 529.91 21.021 31.083 16.009 531.38 21.023 32.554 16.284 532.851 21.024 34.03 16.739 534.32 21.025 35.501 16.73 535.792 21.027 36.971 16.723 537.263 21.027 38.442 16.709 538.739 21.029 39.913 16.678 540.21 21.032 41.383 16.68 541.68 21.033 42.854 16.703 543.151 21.034 44.33 16.713 544.622 21.029 45.801 16.705 546.092 21.027 47.272 16.607 547.563 20.997 48.742 16.727 549.039 20.987 50.213 16.734 550.51 20.933 51.684 16.729 551.981 18.614 53.156 16.726 553.451 15.846 54.632 16.724 554.922 15.408 56.103 16.721 556.393 15.272 57.573 16.717 557.863 15.164 59.044 16.715 559.339 15.272 60.515 16.712 560.81 15.265 61.986 16.709 562.281 15.266 63.456 16.707 563.751 15.283 64.932 16.705 565.222 19.487 66.402 16.702 566.693 22.421 67.874 16.7 568.163 22.66 69.344 16.697 569.64 22.72 70.815 16.695 571.109 22.829 72.286 16.693 572.581 22.97 73.756 16.69 574.052 23.035 75.233 16.687 575.522 23.089 76.703 16.684 576.993 23.135 78.174 16.682 578.464 23.178 79.644 16.679 579.94 23.215 81.115 16.676 581.41 23.248 82.586 16.674 582.881 23.28 84.056 16.673 584.352 23.31 85.533 16.67 585.822 23.336 87.003 16.669 587.293 23.362 88.474 16.67 588.764 23.389 89.944 16.667 590.24 23.412 91.415 16.665 591.711 23.433 92.886 16.664 593.181 23.453 94.356 16.662 594.652 23.472 95.833 16.66 596.123 23.49 97.304 16.659 597.593 23.509 98.774 16.656 599.064 23.526 100.245 16.655 600.54 23.542 101.715 16.652 602.011 23.559 103.186 16.65 603.481 23.574 104.656 16.649 604.952 23.589 106.132 16.647 606.423 23.603 107.604 16.646 607.894 23.617 109.074 16.645 609.364 23.629 110.544 16.643 610.84 23.64 112.016 16.642 612.311 23.654 113.486 16.64 613.782 23.666 114.957 16.639 615.252 23.676 116.433 16.637 616.723 23.688 117.904 16.636 618.194 23.698 119.374 16.634 619.664 23.709 120.845 16.633 621.14 23.719 122.316 16.632 622.611 23.729 123.787 16.63 624.082 23.739 125.257 16.63 625.552 23.749 126.733 16.629 627.023 23.757 128.204 16.627 628.494 23.766 129.675 16.626 629.964 23.775 131.145 16.625 631.44 23.783 132.616 16.624 632.911 23.791 134.087 16.622 634.382 23.799 135.557 16.621 635.852 23.806 137.033 16.619 637.323 23.812 138.504 16.618 638.794 23.817 139.975 16.617 640.264 23.83 141.445 16.615 641.74 23.838 142.916 16.613 643.211 23.845 144.387 16.612 644.682 23.853 145.857 16.611 646.153 23.859 147.333 16.61 647.623 23.866 148.804 16.608 649.094 23.872 150.274 16.607 650.565 23.879 151.745 16.606 652.041 23.885 153.216 16.605 653.512 23.892 154.687 16.604 654.982 23.897 156.157 16.602 656.453 23.903 157.634 16.601 657.924 23.908 159.104 16.6 659.394 23.914 160.575 16.599 660.865 23.924 162.046 16.598 662.341 23.929 163.516 16.597 663.812 23.934 164.987 16.596 665.282 23.939 166.457 16.595 666.753 23.944 167.934 16.594 668.224 23.949 169.404 16.593 669.694 23.953 170.875 16.592 671.165 23.957 172.345 16.591 672.641 23.963 173.816 16.59 674.112 23.968 175.287 16.589 675.582 23.973 176.758 16.588 677.053 23.978 178.234 16.588 678.524 23.982 179.704 16.586 679.993 23.986 181.175 16.585 681.465 23.991 182.646 16.585 682.941 23.995 184.117 16.585 684.412 23.999 185.587 16.583 685.883 24.003 187.058 16.583 687.353 24.008 188.534 16.582 688.824 24.013 190.005 16.582 690.294 24.017 191.475 16.581 691.765 24.021 192.946 16.581 693.241 24.025 194.417 16.58 694.712 24.029 195.887 16.579 696.182 24.033 197.358 16.578 697.653 24.037 198.834 16.578 699.124 24.041 200.305 16.579 700.595 24.044 201.775 16.578 702.065 24.048 203.246 16.578 703.542 24.052 204.717 16.577 705.012 24.056 206.187 16.574 706.482 24.059 207.658 16.542 707.954 24.063 209.134 16.49 709.424 24.066 210.605 16.276 710.895 24.07 212.076 16.381 712.366 24.073 213.546 16.526 713.841 24.076 215.017 16.47 715.312 24.079 216.487 16.465 716.783 24.082 217.958 16.492 718.254 24.085 219.434 15.71 719.724 24.089 220.905 15.102 721.195 24.091 222.376 14.221 722.666 24.095 223.846 14.429 724.142 24.098 225.317 14.88 725.612 24.101 226.787 14.745 727.083 24.105 228.259 14.801 728.554 24.108 229.735 18.457 730.024 24.111 231.205 17.893 731.495 24.113 232.676 17.965 732.966 24.117 234.147 18.022 734.442 24.119 235.617 18.078 735.913 24.122 237.088 18.122 737.383 24.125 238.559 18.159 738.854 24.128 240.035 18.212 740.325 24.13 241.505 18.376 741.795 24.131 242.976 18.451 743.266 24.133 244.447 18.476 744.742 24.137 245.918 18.494 746.213 24.139 247.388 18.51 747.683 24.143 248.859 18.529 749.154 24.146 250.335 18.545 750.625 24.148 251.806 18.558 752.095 24.151 253.276 18.569 753.566 24.153 254.747 18.58 755.042 24.156 256.218 18.593 756.513 24.159 257.688 18.603 757.984 24.162 259.159 18.612 759.454 24.164 260.635 18.624 760.925 24.166 262.106 18.632 762.396 24.168 263.576 18.639 763.866 24.166 265.047 18.645 765.343 24.163 266.518 18.651 766.813 24.174 267.988 18.656 768.284 24.177 269.459 18.661 769.755 24.18 270.935 18.666 771.225 24.183 272.406 18.671 772.696 24.185 273.877 18.675 774.166 24.187 275.347 18.678 775.643 24.189 276.818 18.683 777.113 24.192 278.288 18.688 778.584 24.194 279.759 18.692 780.055 24.024 281.236 18.696 781.525 20.468 282.706 18.698 782.996 15.572 284.177 18.701 784.467 15.64 285.647 18.705 785.943 15.37 287.118 18.708 787.413 15.196 288.589 18.713 788.884 15.204 290.059 18.71 790.355 14.506 291.536 18.715 791.826 14.708 293.006 18.718 793.296 15.257 294.477 18.721 794.767 15.996 295.946 18.721 796.243 24.033 297.418 18.724 797.714 25.822 298.889 18.726 799.184 26.741 300.36 18.728 800.655 27.372 301.836 18.73 802.125 27.857 303.306 18.732 803.596 28.259 304.777 18.733 805.067 28.616 306.248 18.735 806.543 28.956 307.718 18.736 808.014 29.206 309.189 18.737 809.484 29.41 310.659 18.74 810.955 29.6 312.136 18.742 812.426 29.786 313.606 18.743 813.896 29.949 315.077 18.745 815.367 30.085 316.548 18.747 816.843 30.191 318.019 18.748 818.314 30.292 319.489 18.749 819.785 30.382 320.96 18.751 821.255 30.465 322.436 18.752 822.726 30.556 323.907 18.753 824.197 30.616 325.377 18.755 825.667 30.671 326.848 18.757 827.142 30.727 328.319 18.759 828.614 30.786 329.789 18.76 830.085 30.78 331.26 18.76 831.555 30.722 332.736 18.76 833.026 30.746 334.205 18.757 834.497 30.771 335.676 18.758 835.967 30.797 337.148 18.759 837.443 30.816 338.619 18.76 838.914 30.836 340.089 18.76 840.385 30.856 341.56 18.762 841.855 30.87 343.036 18.763 843.326 30.877 344.507 18.764 844.797 30.894 345.978 18.765 846.267 30.914 347.448 18.765 847.744 30.928 348.919 18.767 849.214 30.944 350.39 18.771 850.685 30.977 351.86 18.772 852.155 30.988 353.337 18.723 853.626 31.008 354.807 18.673 855.096 31.025 356.278 18.535 856.568 31.028 357.748 17.909 858.043 31.048 359.219 15.507 859.515 31.06 360.69 15.037 860.985 31.079 362.16 15.068 862.455 31.092 363.637 15.306 863.927 31.104 365.107 15.421 865.397 31.174 366.578 15.544 866.868 31.207 368.048 15.628 868.344 31.222 369.519 17.345 869.815 31.236 370.99 19.873 871.285 31.25 372.461 20.11 872.756 31.262 373.937 20.222 874.227 31.258 375.407 20.301 875.697 31.262 376.878 20.363 877.168 31.267 378.349 20.406 878.644 31.274 379.82 20.463 880.115 31.282 381.29 20.499 881.585 31.29 382.761 20.528 883.056 31.294 384.237 20.555 884.527 31.3 385.708 20.579 885.997 31.308 387.178 20.598 887.468 31.33 388.649 20.619 888.944 31.343 390.12 20.64 890.415 31.356 391.59 20.659 891.886 31.371 393.061 20.676 893.356 31.385 394.537 20.691 894.827 31.395 396.008 20.703 896.298 31.405 397.478 20.716 897.768 31.407 398.949 20.728 899.244 31.411 400.42 20.739 900.715 31.414 401.89 20.749 902.186 31.423 403.361 20.751 903.656 31.431 404.837 20.761 905.127 31.438 406.308 20.771 906.598 31.442 407.779 20.78 908.068 31.446 409.249 20.788 909.545 31.448 410.72 20.795 911.015 31.45 412.19 20.801 912.486 31.463 413.661 20.813 913.956 31.474 415.137 20.82 915.427 31.475 416.608 20.826 916.898 31.472 418.079 20.806 918.369 31.476 419.549 20.818 919.845 31.48 421.021 20.842 921.315 31.485 422.491 20.847 922.786 31.499 423.961 20.852 924.257 31.51 425.438 20.858 925.727 31.513 426.908 20.862 927.198 31.516 428.379 20.866 928.669 31.516 429.849 20.87 930.145 31.515 431.32 20.872 931.616 31.515 432.791 20.877 933.086 31.513 434.261 20.88 934.557 31.515 435.738 20.884 936.028 31.518 437.208 20.887 937.498 31.513 438.679 20.89 938.969 31.511 440.149 20.892 940.445 31.51 441.62 20.894 941.916 31.511 443.091 20.896 943.386 31.505 444.562 20.901 944.856 31.451 446.038 20.904 946.328 31.346 447.509 20.907 947.798 31.312 448.979 20.909 949.269 30.981 450.448 20.911 950.745 26.196 451.921 20.915 952.216 20.639 453.391 20.918 953.687 17.579 454.862 20.921 955.157 16.341 456.337 20.923 956.628 16.335 457.809 20.925 958.099 15.967 459.279 20.929 959.569 15.869 460.75 20.931 961.045 15.832 462.221 20.931 962.516 22.334 463.691 20.936 963.987 24.668 465.162 20.939 965.457 25.958 466.638 20.941 966.928 26.874 468.109 20.944 968.399 27.415 469.579 20.947 969.869 35.901 471.05 20.949 971.346 37.418 472.521 20.952 972.816 37.702 473.992 20.954 974.287 37.821 475.462 20.957 975.757 37.876 476.938 20.96 977.228 37.907 478.409 20.962 978.699 37.931 479.88 20.965 980.17 37.881 481.35 20.967 981.646 37.969 482.821 20.969 983.116 38.082 484.292 20.971 984.587 38.096 485.762 20.973 986.058 38.103 487.238 20.976 987.528 38.114 488.709 20.978 988.999 38.126 490.179 20.98 990.47 38.124 491.65 20.981 991.946 38.127 493.121 20.982 993.416 38.129 494.592 20.984 994.885 38.137 496.062 20.986 996.358 38.202 497.538 20.988 997.828 38.206 499.009 20.99 999.299 38.204 500.48 20.992 1000.769 38.213 B) Normalised static capacitance in soil (■ upsweep curve, ● downsweep curve) Relative Humidity [%] Normalised Capacitance (Upsweep) [-] Standard Deviation (Upsweep) [-] Normalised Capacitance (Downsweep) [-] Standard Deviation (Downsweep) [-] 37 1.01 0.0139 1 0 42 1.21 0.0345 1.26 0.0622 55 1.59 0.209 1.67 0.318 63 1.89 0.29 1.91 0.311 68 2.28 0.251 2.45 0.389 73 2.74 0.244 2.74 0.244 B) Normalised static capacitance in soil (■ upsweep curve, ● downsweep curve) Figure 7 Dynamic response of a sensor inkjet-printed on a silverberry leaf. Dynamic response of a sensor inkjet-printed on a silverberry leaf. Figure 7 Time [s] Capacitance [pF] Time [s] Capacitance [pF] 1.682 0.01048 437.226 0.00994 3.148 0.01044 438.696 0.00993 4.621 0.01045 440.165 0.0099 6.089 0.01046 441.636 0.00994 7.561 0.01049 443.108 0.00993 9.031 0.01047 444.578 0.00995 10.502 0.01042 446.049 0.00991 11.972 0.01046 447.525 0.00988 13.445 0.01044 448.995 0.00992 14.918 0.01045 450.466 0.00992 16.39 0.01044 451.937 0.00994 17.86 0.01047 453.408 0.00992 19.331 0.01041 454.877 0.0099 20.802 0.01045 456.348 0.0099 22.273 0.01046 457.824 0.0099 23.745 0.0105 459.297 0.00992 25.221 0.01042 460.767 0.00993 26.689 0.01043 462.236 0.00993 28.162 0.01041 463.708 0.00991 29.631 0.01021 465.178 0.00987 31.101 0.01637 466.65 0.00989 32.573 0.02219 468.126 0.00993 34.043 0.02219 469.596 0.00987 35.519 0.02189 471.067 0.00995 36.99 0.02112 472.537 0.00984 38.46 0.02068 474.007 0.00994 39.932 0.02015 475.479 0.0099 41.402 0.01929 476.95 0.00989 42.872 0.01862 478.426 0.00988 44.344 0.01788 479.897 0.00989 45.82 0.0174 481.366 0.0099 47.291 0.0173 482.837 0.00985 48.761 0.01671 484.308 0.00991 50.23 0.01628 485.779 0.0098 51.701 0.01583 487.251 0.00989 53.172 0.01551 488.727 0.00987 54.644 0.01516 490.196 0.00988 56.119 0.01474 491.667 0.00992 57.59 0.01446 493.137 0.00992 59.062 0.01405 494.607 0.00989 60.53 0.01374 496.079 0.00987 62.002 0.0135 497.55 0.0099 63.472 0.01337 499.026 0.00984 64.942 0.01322 500.496 0.00987 66.419 0.01303 501.966 0.00988 67.89 0.01289 503.438 0.00984 69.361 0.01274 504.909 0.00934 70.831 0.01261 506.378 0.0174 72.302 0.01252 507.849 0.02357 73.772 0.01243 509.327 0.02332 75.244 0.01239 510.797 0.02333 76.72 0.01228 512.266 0.02315 78.19 0.01227 513.737 0.02283 79.661 0.01221 515.209 0.02232 81.132 0.01215 516.679 0.02175 82.603 0.01206 518.15 0.02137 84.074 0.01202 519.626 0.02045 85.543 0.01199 521.097 0.01965 87.019 0.01194 522.567 0.01898 88.491 0.0119 524.038 0.01782 89.961 0.01183 525.509 0.01689 91.432 0.01185 526.98 0.01602 92.902 0.01179 528.45 0.01533 94.372 0.01174 529.927 0.01518 95.845 0.01174 531.399 0.01535 97.321 0.01172 532.867 0.01522 98.791 0.01166 534.338 0.01503 100.263 0.01171 535.809 0.01473 101.732 0.01166 537.279 0.0142 103.202 0.01165 538.75 0.01381 104.674 0.0116 540.227 0.01358 106.143 0.01159 541.697 0.01327 107.62 0.01159 543.168 0.01316 109.091 0.01151 544.638 0.01302 110.561 0.01153 546.108 0.01286 112.033 0.01151 547.58 0.01279 113.502 0.01149 549.051 0.01269 114.974 0.01147 550.527 0.01258 116.444 0.01144 551.996 0.01247 117.921 0.01144 553.468 0.0124 119.392 0.01143 554.938 0.01223 120.862 0.01139 556.409 0.01217 122.333 0.0114 557.88 0.01213 123.803 0.01141 559.351 0.01203 125.275 0.01139 560.827 0.01196 126.745 0.01139 562.297 0.01193 128.221 0.01133 563.767 0.01182 129.692 0.01133 565.238 0.01181 131.162 0.01137 566.709 0.01177 132.633 0.01131 568.18 0.01171 134.104 0.01128 569.65 0.01166 135.573 0.01132 571.127 0.01164 137.044 0.01132 572.597 0.01162 138.521 0.01127 574.069 0.01153 139.99 0.01127 575.539 0.01151 141.464 0.01124 577.01 0.01146 142.933 0.01128 578.48 0.01148 144.402 0.0113 579.95 0.01144 145.873 0.01123 581.427 0.01137 147.346 0.01122 582.897 0.01139 148.823 0.01127 584.369 0.01132 150.291 0.01125 585.839 0.0113 151.762 0.01119 587.308 0.01131 153.232 0.01118 588.781 0.01129 154.702 0.0112 590.251 0.01125 156.174 0.01119 591.728 0.01121 157.645 0.0112 593.198 0.01124 159.12 0.01121 594.668 0.01124 160.592 0.01119 596.138 0.01118 162.064 0.01112 597.609 0.01113 163.532 0.01117 599.079 0.01119 165.004 0.01122 600.551 0.01113 166.474 0.01114 602.026 0.01109 167.945 0.01113 603.498 0.01114 169.423 0.01111 604.969 0.01108 170.894 0.0111 606.439 0.01108 172.363 0.01114 607.909 0.01107 173.833 0.01111 609.381 0.01107 175.303 0.01108 610.851 0.01101 176.775 0.01108 612.328 0.01104 178.244 0.01108 613.798 0.01097 179.721 0.0111 615.269 0.01098 181.192 0.01104 616.739 0.011 182.663 0.01108 618.209 0.01096 184.133 0.01103 619.68 0.01096 185.604 0.01108 621.15 0.01096 187.075 0.01105 622.627 0.01094 188.546 0.01104 624.098 0.01096 190.022 0.01111 625.569 0.01091 191.491 0.01105 627.039 0.01087 192.963 0.01104 628.511 0.01084 194.434 0.01104 629.98 0.01087 195.904 0.01105 631.453 0.01086 197.376 0.01103 632.929 0.01086 198.845 0.01105 634.398 0.01077 200.322 0.01099 635.868 0.01083 201.793 0.01103 637.34 0.01088 203.262 0.01101 638.81 0.01081 204.734 0.01105 640.281 0.01081 206.205 0.01104 641.752 0.01079 207.675 0.011 643.227 0.0108 209.146 0.01101 644.698 0.01079 210.622 0.01102 646.169 0.01077 212.093 0.01092 647.639 0.01076 213.565 0.01097 649.111 0.01074 215.033 0.01098 650.581 0.01071 216.504 0.01094 652.051 0.01074 217.975 0.01095 653.528 0.01075 219.446 0.01095 654.999 0.01073 220.923 0.01095 656.469 0.01071 222.393 0.01099 657.94 0.01068 223.862 0.01092 659.411 0.01071 225.332 0.01093 660.881 0.01069 226.805 0.01095 662.351 0.01068 228.276 0.01094 663.827 0.01069 229.746 0.01095 665.298 0.01069 231.222 0.01094 666.769 0.01066 232.692 0.01087 668.242 0.01062 234.164 0.01093 669.711 0.01059 235.634 0.01094 671.18 0.01067 237.104 0.0109 672.652 0.01063 238.575 0.01089 674.128 0.01064 240.045 0.0109 675.598 0.01064 241.521 0.01094 677.07 0.01063 242.995 0.01092 678.539 0.01062 244.463 0.01089 680.011 0.01064 245.934 0.01089 681.482 0.01057 247.403 0.01085 682.951 0.0106 248.877 0.01087 684.429 0.0106 250.347 0.0109 685.899 0.01061 251.822 0.01088 687.369 0.01054 253.292 0.01083 688.84 0.01056 254.764 0.01089 690.312 0.01057 256.234 0.0109 691.782 0.01056 257.705 0.01087 693.253 0.01053 259.175 0.01085 694.729 0.01058 260.645 0.01085 696.199 0.01052 262.122 0.01086 697.671 0.01049 263.594 0.01085 699.14 0.01054 265.064 0.01089 700.611 0.01052 266.533 0.01081 702.082 0.01051 268.005 0.01081 703.553 0.0105 269.475 0.01087 705.028 0.01052 270.945 0.01087 706.499 0.01047 272.424 0.01084 707.97 0.01051 273.895 0.01088 709.441 0.01049 275.363 0.00912 710.912 0.01051 276.835 0.00365 712.381 0.01048 278.306 0.00466 713.852 0.01044 279.776 0.00489 715.329 0.01052 281.246 0.00489 716.8 0.01043 282.723 0.00488 718.269 0.01046 284.194 0.00487 719.741 0.01043 285.664 0.0049 721.212 0.01047 287.134 0.00487 722.682 0.01039 288.604 0.00489 724.153 0.01045 290.076 0.00491 725.629 0.01045 291.546 0.00492 727.1 0.01043 293.022 0.00486 728.571 0.01048 294.495 0.00491 730.04 0.01038 295.963 0.00492 731.511 0.01043 297.435 0.00491 732.981 0.0104 298.907 0.00487 734.453 0.0104 300.38 0.00492 735.929 0.01044 301.846 0.00491 737.4 0.01041 303.324 0.00488 738.869 0.01044 304.795 0.0049 740.34 0.01042 306.266 0.00492 741.811 0.01041 307.735 0.00491 743.281 0.01042 309.206 0.00489 744.752 0.01039 310.677 0.00489 746.228 0.01038 312.146 0.00485 747.699 0.01039 313.625 0.00492 749.17 0.01039 315.095 0.00492 750.641 0.01035 316.568 0.00483 752.112 0.01037 318.035 0.0049 753.581 0.01034 319.512 0.00485 755.053 0.01034 320.983 0.00488 756.53 0.01036 322.455 0.00491 758 0.01032 323.924 0.00492 759.47 0.01034 325.395 0.00488 760.941 0.01034 326.866 0.00491 762.413 0.01033 328.336 0.00489 763.882 0.01035 329.807 0.0049 765.352 0.01031 331.278 0.0049 766.829 0.01028 332.748 0.00494 768.3 0.01037 334.223 0.00488 769.771 0.01036 335.695 0.00489 771.242 0.01034 337.165 0.00487 772.712 0.01031 338.634 0.0049 774.183 0.01031 340.106 0.00493 775.652 0.01032 341.579 0.00489 777.129 0.01032 343.048 0.00488 778.6 0.01034 344.524 0.00488 780.071 0.01028 345.996 0.0049 781.542 0.01033 347.464 0.0049 783.013 0.01029 348.936 0.00492 784.483 0.01032 350.408 0.00486 785.954 0.01028 351.878 0.00491 787.43 0.0103 353.347 0.00496 788.901 0.01028 354.824 0.00489 790.37 0.01028 356.294 0.00492 791.842 0.01029 357.764 0.00492 793.312 0.01031 359.236 0.00492 794.783 0.0103 360.707 0.0049 796.252 0.01025 362.178 0.00487 797.73 0.01029 363.649 0.00489 799.2 0.01024 365.125 0.00491 800.672 0.01028 366.596 0.0049 802.142 0.01024 368.065 0.0049 803.612 0.01022 369.536 0.00476 805.084 0.01021 371.007 0.00446 806.553 0.01024 372.48 0.00447 808.03 0.01021 373.947 0.00257 809.501 0.01027 375.424 0.0079 810.97 0.01027 376.895 0.01002 812.442 0.01025 378.365 0.01 813.913 0.01023 379.837 0.00999 815.383 0.01026 381.306 0.00998 816.854 0.01027 382.776 0.00999 818.329 0.01023 384.247 0.00994 819.801 0.01023 385.724 0.00997 821.272 0.01022 387.195 0.01 822.743 0.01021 388.666 0.01004 824.212 0.01019 390.135 0.00999 825.682 0.01019 391.606 0.00994 827.154 0.0102 393.078 0.00994 828.631 0.01018 394.548 0.00992 830.1 0.0102 396.024 0.00999 831.572 0.0102 397.495 0.00998 833.042 0.01018 398.964 0.00993 834.513 0.01022 400.435 0.00997 835.984 0.0102 401.907 0.00999 837.454 0.01017 403.376 0.00996 838.93 0.01019 404.847 0.00997 840.402 0.01022 406.323 0.00997 841.874 0.01022 407.794 0.00992 843.343 0.01021 409.266 0.00995 844.813 0.01022 410.735 0.00998 846.284 0.01014 412.206 0.00996 847.755 0.01019 413.676 0.00996 849.23 0.01019 415.147 0.00991 850.701 0.01019 416.624 0.00995 852.171 0.01017 418.094 0.00994 853.642 0.01013 419.565 0.00996 855.113 0.01018 421.035 0.00995 856.584 0.01015 422.506 0.00995 858.054 0.01012 423.977 0.00994 859.531 0.01016 425.448 0.00995 861 0.01012 426.924 0.00992 862.472 0.0102 428.396 0.00995 863.944 0.0101 429.867 0.00996 865.413 0.01017 431.337 0.00994 866.884 0.01012 432.807 0.00994 868.354 0.01015 434.278 0.00991 869.831 0.01013 435.747 0.00987 871.302 0.01013 ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the FET program within the Horizon 2020 Research and Innovation Program of the European Commission, under the HyPhOE FET-open project (grant agreement no. 800926).
https://openalex.org/W4362531769	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Data_2_from_Breast_Cancer_Chemoprevention_Use_and_Views_of_Australian_Women_and_Their_Clinicians/22533914/1/files/39997178.pdf	English	null	Supplementary Data 2 from Breast Cancer Chemoprevention: Use and Views of Australian Women and Their Clinicians	null	2,023	cc-by	1,927	Translating Evidence into Practice: Optimising and Facilitating Prevention and Screening in Women with a Strong Family History of Breast Cancer 1. Do you consent to participating in this research? Yes 2. What is your gender? Male Female Other 20 – 29 30 – 39 40 – 49 50 – 59 60+ GP Breast Surgeon General Surgeon I would rather not say % public % private 3. What is your age? OR UNIQUE CLINICIAN NUMBER 4. What percentage of your time is spent in the following practice types? Please ensure percentages equal 100%. 5. What is your profession? UNIQUE CLINICIAN NUMBER RISK-REDUCING MEDICATIONS 6. Before this study, had you ever heard of breast cancer risk-reducing medications/chemoprevention? These can be prescribed to women without breast cance 6. Before this study, had you ever heard of breast cancer risk-reducing 6. Before this study, had you ever heard of breast cancer risk-reducing medications/chemoprevention? These can be prescribed to women with y y g medications/chemoprevention? These can be prescribed to women without b Translating Evidence into Practice: 1. Do you consent to participating in this research? 1. Do you consent to participating in this research? Yes 2. What is your gender? Male Female Other 20 – 29 30 – 39 40 – 49 50 – 59 60+ I would rather not say 3. What is your age? OR 3. What is your age? 3. What is your age? 20 – 29 30 – 39 40 – 49 50 – 59 60+ % public % private 4. What percentage of your time is spent in the following practice types? Please ensure percentages equal 100%. % public % private 4. What percentage of your time is spent in the following practice types? Please ensure percentages equal 100%. 5. What is your profession? No Please skip to the last page and enter your details for your chance to win an Apple Ipad. No Please skip to the last page and enter your details for your chance to win an Apple Ipad. Deﬁnition of risk-reducing medications Medications that reduce the risk of breast cancer may be considered by women who don’t have breast cancer but are at an increased risk. These medicines are also known as chemoprevention. The most common risk-reducing medication is tamoxifen. Other risk-reducing medications include raloxifene, anastrazole and exemestane. These tablets are taken once a day, usually for five years. 7. Have you ever discussed risk-reducing medications with your patients and/or prescribed them to your patients? Tick all that apply. 7. I have initiated discussion with patients Patients have initiated discussion with me I have written first prescription I have written ongoing prescriptions I have never discussed or prescribed risk-reducing medications with patients 8. 9. Thinking about discussing or prescribing risk-reducing medications with your patients, how much of a barrier are each of the following? 9. Thinking about discussing or prescribing risk-reducing medications with your patients, how much of a barrier are each of the following? g I have insufficient knowledge of risk-reducing medications It is difficult to measure whether the medication is working I find it hard to access tools/resources to help me estimate patients’ breast cancer risk I find it hard to access good information/resources for my patients e.g. patient information sheets There is no evidence that they reduce mortality 1. 2. 3. 4. 5. Somewhat a barrier Not at all a barrier Not applicable Strong barrier Somewhat a barrier Not at all a barrier Not applicable Strong barrier Somewhat a barrier Not at all a barrier Not applicable Strong barrier It is not my role to discuss risk-reducing medications There are no incentives/rewards for discussing risk-reducing medications with patients Lack of time during consultations There are no procedures (e.g. a checklist that facilitates discussion) that encourage me to discuss risk-reducing medications 10. 11. 12. 13. I have difficulty identifying patients suitable for risk-reducing medication I have inadequate training and confidence in breast cancer risk assessment I have difficulty explaining to patients the pros and cons of risk-reducing medications I am not confident in providing advice/information to patients about risk-reducing medications 6. 7. 8. 9. No Please skip to the last page and enter your details for your chance to win an Apple Ipad. I have insufficient knowledge of risk-reducing medications It is difficult to measure whether the medication is working I find it hard to access tools/resources to help me estimate patients’ breast cancer risk I find it hard to access good information/resources for my patients e.g. patient information sheets There is no evidence that they reduce mortality 1. 2. 3. 4. 5. Somewhat a barrier Not at all a barrier Not applicable Strong barrier Somewhat a barrier Not at all a barrier Not applicable Strong barrier I have difficulty identifying patients suitable for risk-reducing medication I have inadequate training and confidence in breast cancer risk assessment I have difficulty explaining to patients the pros and cons of risk-reducing medications I am not confident in providing advice/information to patients about risk-reducing medications 6. 7. 8. 9. Somewhat a barrier Not at all a barrier Not applicable Strong barrier It is not my role to discuss risk-reducing medications There are no incentives/rewards for discussing risk-reducing medications with patients Lack of time during consultations There are no procedures (e.g. a checklist that facilitates discussion) that encourage me to discuss risk-reducing medications 10. 11. 12. 13. Somewhat a barrier Not at all a barrier Not applicable Strong barrier 9a. Which one of these factors (1 to 22) is the greatest barrier? 9b. Are there any other barriers that discourage you from discussing or prescribing risk-reducing medications with patients? If no, please leave blank. Patients don’t ask me about risk-reducing medications I don’t routinely assess breast cancer risk with my patients There are other things I wish to achieve in most consultations that interfere with my ability to discuss risk-reducing medications I forget to discuss risk-reducing medications with patients 19. 20. 21. 22. Somewhat a barrier Not at all a barrier Not applicable Strong barrier Medication side effects I don’t believe they decrease the risk of breast cancer I’m concerned I might increase the patient’s worry about breast cancer I don’t think patients want to discuss taking medications for prevention of breast cancer I feel uncomfortable prescribing a ‘cancer drug’ to healthy women 14. 15. 16. 17. 18. Somewhat a barrier Not at all a barrier Not applicable Strong barrier 9a. Which one of these factors (1 to 22) is the greatest barrier? No Please skip to the last page and enter your details for your chance to win an Apple Ipad. 9b Are there any other barriers that discourage you from discussing or prescribing risk reducing Patients don’t ask me about risk-reducing medications I don’t routinely assess breast cancer risk with my patients There are other things I wish to achieve in most consultations that interfere with my ability to discuss risk-reducing medications I forget to discuss risk-reducing medications with patients 19. 20. 21. 22. Somewhat a barrier Not at all a barrier Not applicable Strong barrier Medication side effects I don’t believe they decrease the risk of breast cancer I’m concerned I might increase the patient’s worry about breast cancer I don’t think patients want to discuss taking medications for prevention of breast cancer I feel uncomfortable prescribing a ‘cancer drug’ to healthy women 14. 15. 16. 17. 18. 9a. Which one of these factors (1 to 22) is the greatest barrier? 9b. Are there any other barriers that discourage you from discussing or prescribing risk-reducing medications with patients? If no, please leave blank. Thinking about discussing or prescribing risk-reducing medications with your patients, how much of a facilitator are each of the following? S h 10. Thinking about discussing or prescribing risk-reducing medications with your patients, how much of a facilitator are each of the following? 10. 10. Thinking about discussing or prescribing risk reducing medications with your patients, how much of a facilitator are each of the following? If the patient is diagnosed with atypical hyperplasia that increases their risk of breast cancer If the patient is diagnosed with lobular carcinoma in situ (LCIS) that increases their risk of breast cancer If a patient has a strong family history of breast cancer The beneficial effects of risk-reducing medications e.g. better blood lipid profile and improvements in bone density with tamoxifen If I had better tools to help me identify patients who were suitable Knowing some risk-reducing medications are PBS funded Sometimes it is easier to discuss risk-reducing medications than bilateral mastectomy Support from my peers Support from specialists Clear guidelines/recommendations e.g. RACGP and Cancer Australia guidelines If it were endorsed as part of my professional role by the relevant college or other peak body If my medical software prompted me to discuss risk-reducing medications If I knew my colleagues discuss it with their patients I expect positive outcomes for women who take risk-reducing medications 10a. Which one of these factors (1 – 14) is the greatest facilitator? 10b. No Please skip to the last page and enter your details for your chance to win an Apple Ipad. Are there any other facilitators that encourage you to discuss risk-reducing medications with patients? If no, please leave blank. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Somewhat a facilitator Not at all a facilitator Not applicable Strong facilitator 10. 10a. Which one of these factors (1 – 14) is the greatest facilitator? 10b. Are there any other facilitators that encourage you to discuss risk-reducing medications with patients? If no, please leave blank. 11. What do you think should be the role of each clinician type below with regard to discussing and prescribing risk-reducing medications. Tick all that apply. 12. In which of these circumstances do you think it is appropriate to discuss risk-reducing medications? Tick all that apply. Clinician type 11a. GP role: 11b. Breast/General Surgeon role: 11c. Familial Cancer Clinic/Genetic Specialist role: Initiate discussion of risk-reducing medications Write ﬁrst prescription Write ongoing prescriptions In a consultation where breast cancer risk happens to come up. During a consultation specifically planned to focus on breast cancer risk. During a consultation focused on general preventive healthcare. There is no circumstance under which I would discuss risk-reducing medications. Clinician type 11a. GP role: 11b. Breast/General Surgeon role: 11c. Familial Cancer Clinic/Genetic Specialist role: Initiate discussion of risk-reducing medications Write ﬁrst prescription Write ongoing prescriptions Clinician type 12. In which of these circumstances do you think it is appropriate to discuss risk-reducing medications? Tick all that apply. In a consultation where breast cancer risk happens to come up. During a consultation specifically planned to focus on breast cancer risk. During a consultation focused on general preventive healthcare. There is no circumstance under which I would discuss risk-reducing medications. 12. In which of these circumstances do you think it is appropriate to discuss risk-reducing medications? Tick all that apply. n which of these circumstances do you think it is appropriate to discuss risk-reducing medications? Tick all that apply. 12. 13. Have you heard of iPrevent, a web-based tool to help clinicians and patients collaboratively assess and manage breast cancer risk? (www.petermac.org/iprevent) 13. Please enter your details on the last page for your chance to win an Apple iPad. – 6 – THIS PAGE IS INTENTIONALLY BLANK – 7 – For your chance to win an Apple iPad please provide your email address: Please return completed survey in the reply paid envelope. Thank you for completing the survey. Please return completed survey in the reply paid envelope. © New Data Solutions 19665
https://openalex.org/W4311694922	https://periodicos.udesc.br/index.php/ensinarmode/article/download/22831/15045	Portuguese	null	Núcleo de extensão e prática profissional na área da moda: formação complementar e inclusão social	Revista de Ensino em Artes, Moda e Design	2,022	cc-by	9,202	1 Projeto financiado pela Pró-Reitoria de Extensão do IFRN – Campus Caicó. 2 Professora do Instituto Federal do Rio Grande do Norte – Campus Caicó. Mestre em Engenharia de Produção (UFRN) e Graduada em Design de Moda (UFC). Lattes: http://lattes.cnpq.br/6771346194407250; ORCID: https://orcid.org/0000-0003-2530-1144; E-mail: livia.solino@ifrn.edu.br. 3 Professora do Instituto Federal do Rio Grande do Norte – Campus Caicó. Mestre em Design (UFRN), Especialista em Design de Moda (SENAI CETIQT) e Graduada em Engenharia Têxtil (UFRN). Lattes: http:// lattes.cnpq.br/4588211284857742; E-mail: moally.soares@ifrn.edu.br. 4 Professor do Instituto Federal do Rio Grande do Norte – Campus Caicó. Mestre em Design (UFCG), Espe- cialista em Comunicação, Semiótica e Linguagens Visuais (UBC) e Graduado em Design de Moda (IFRN). Lattes: http://lattes.cnpq.br/3950194171500432; ORCID: https://orcid.org/0000-0003-0710-6142; E-mail: ital- odantasdesign@hotmail.com. DOI:10.5965/25944630712023e2831 NÚCLEO DE EXTENSÃO E PRÁTICA PROFISSIONAL NA ÁREA DA MODA: FORMAÇÃO COMPLEMENTAR E INCLUSÃO SOCIAL1 Extension core and professional practice in the fashion area: complementary training and social inclusion Núcleo de extensión y práctica profesional en el área de la moda: formación complementaria e inclusión social Lívia Juliana Silva Solino de Souza2 Moally Janne de Brito Soares3 Ítalo José de Medeiros Dantas4 1 Projeto financiado pela Pró-Reitoria de Extensão do IFRN – Campus Caicó. 2 Professora do Instituto Federal do Rio Grande do Norte – Campus Caicó. Mestre em Engenharia de Produção (UFRN) e Graduada em Design de Moda (UFC). Lattes: http://lattes.cnpq.br/6771346194407250; ORCID: https://orcid.org/0000-0003-2530-1144; E-mail: livia.solino@ifrn.edu.br. 3 Professora do Instituto Federal do Rio Grande do Norte Campus Caicó Mestre em Design (UFRN) DOI:10.5965/25944630712023e2831 Núcleo de extensión y práctica profesional en el área de la moda: formación complementaria e inclusión social Tendo isso em mente, este artigo visa expor e discutir as ações institucionais e o impacto do NEPP entre os anos de 2021 e 2022 no cenário sociocultural local. Para tanto, empregou- -se o método de pesquisa-ação, com verificação qualitativa dos objetivos. De tal maneira, ofertou-se um curso de técnicas de bordado, importante elemento regional, revitalizando o fazer local. Aliado a isso, forneceu-se um cenário para o desenvolvimento de uma coleção de moda pelos alunos da graduação, empregando tal motivo artesanal em sua configura- ção, conduzindo uma parceria com as alunas de bordado de modo a elas mostrarem seus trabalhos. A coleção, portanto, tinha a intenção de servir como vitrine para os trabalhos das mulheres em situação de vulnerabilidade social. Com isso, as oficinas tiveram duração de quatro (4) dias. Nesse entremeio, as alunas inscritas no curso do bordado surpreenderam pela forma rápida que aprenderam a bordar. Palavras-chaves: Extensão; Bordado; Ensino de moda. Palavras-chaves: Extensão; Bordado; Ensino de moda. Keywords: Extension; Embroidery; Fashion Teaching. Núcleo de extensión y práctica profesional en el área de la moda: formación complementaria e inclusión social Lívia Juliana Silva Solino de Souza2 Moally Janne de Brito Soares3 Ítalo José de Medeiros Dantas4 4 Professor do Instituto Federal do Rio Grande do Norte – Campus Caicó. Mestre em Design (UFCG), Espe- cialista em Comunicação, Semiótica e Linguagens Visuais (UBC) e Graduado em Design de Moda (IFRN). Lattes: http://lattes.cnpq.br/3950194171500432; ORCID: https://orcid.org/0000-0003-0710-6142; E-mail: ital- odantasdesign@hotmail.com. RESUMO RESUMO O Núcleo de Extensão e Prática Profissional (NEPP) é uma iniciativa do Instituto Federal do Rio Grande do Norte (IFRN) como forma de interligar os saberes acadêmicos com os diferentes atores da sociedade. No campus Caicó, interior do supramencionado estado, o NEPP foi fundado em 2017, passando a ofertar cursos de formação continuada para mulheres em situação de vulnerabilidade social, de modo a engendrarem em um ofício, adentrando no mercado de trabalho. Ademais, o núcleo ainda permite o desenvolvimento da prática profissional dos alunos do curso superior de tecnologia em Design de Moda. Tendo isso em mente, este artigo visa expor e discutir as ações institucionais e o impacto do NEPP entre os anos de 2021 e 2022 no cenário sociocultural local. Para tanto, empregou- -se o método de pesquisa-ação, com verificação qualitativa dos objetivos. De tal maneira, ofertou-se um curso de técnicas de bordado, importante elemento regional, revitalizando o fazer local. Aliado a isso, forneceu-se um cenário para o desenvolvimento de uma coleção de moda pelos alunos da graduação, empregando tal motivo artesanal em sua configura- ção, conduzindo uma parceria com as alunas de bordado de modo a elas mostrarem seus trabalhos. A coleção, portanto, tinha a intenção de servir como vitrine para os trabalhos das mulheres em situação de vulnerabilidade social. Com isso, as oficinas tiveram duração de quatro (4) dias. Nesse entremeio, as alunas inscritas no curso do bordado surpreenderam pela forma rápida que aprenderam a bordar. O Núcleo de Extensão e Prática Profissional (NEPP) é uma iniciativa do Instituto Federal do Rio Grande do Norte (IFRN) como forma de interligar os saberes acadêmicos com os diferentes atores da sociedade. No campus Caicó, interior do supramencionado estado, o NEPP foi fundado em 2017, passando a ofertar cursos de formação continuada para mulheres em situação de vulnerabilidade social, de modo a engendrarem em um ofício, adentrando no mercado de trabalho. Ademais, o núcleo ainda permite o desenvolvimento da prática profissional dos alunos do curso superior de tecnologia em Design de Moda. Abstract The Extension and Professional Practice Center (NEPP) is an initiative of the Federal In- stitute of Rio Grande do Norte (IFRN) as a way of linking academic knowledge with the different actors in society. On the Caicó campus, in the interior of the state, the NEPP was founded in 2017, offering continuing education courses for women in situations of social vulnerability, to engender a profession, entering the job market. In addition, the nucleus still allows the development of the professional practice of students of the higher technology course in Fashion Design. This paper aims to expose and discuss the institutional actions and the impact of NEPP between 2021 and 2022 on the local sociocultural scenario. For that, the action research method was used, with qualitative verification of the objectives. In this way, a course on embroidery techniques was offered, an important regional element, revitalizing local work. Allied to this, a scenario was provided for the development of a fash- ion collection by undergraduate students, employing such a handcrafted motif in its configu- ration, using embroidery students as labor. The collection, therefore, was intended to serve as a showcase for the work of women in situations of social vulnerability. As a result, the workshops lasted four (4) days. In the meantime, the students enrolled in the embroidery course were surprised by the quick way they learned to embroider. 2 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Palabras clave: Extensión; Bordado; Enseñanza de la moda. 1 INTRODUÇÃO 1 INTRODUÇÃO A cidade de Caicó no Rio Grande do Norte é referência em produção de produ- tos têxteis como vestuário, cama, mesa e banho, rendas, bordados, tanto artesanais, como confeccionados industrialmente, sendo, também, um importante polo comercial da região Seridó. Onde recebe diariamente consumidores em buscados mais diversos produtos e serviços, sobretudo no que diz respeito à área têxtil e confecção de peças de vestuário. O bordado de Caicó ganhou no ano de 2018 o Selo de Indicação Geográfica, que foi concedido pelo Instituto Nacional de Propriedade Industrial (INPI) (INPI, 2020; PE- DUZZI, 2020). Este selo garante aos consumidores a autenticidade do bordado caicoense, tornando-o um elemento ímpar na cultura local. Nesse contexto, observou-se que o borda- do que gerou esse selo é mais comumente aplicado em produtos para o lar, sendo pouco explorado na confecção de vestuário, principalmente em produtos carregados de informa- ção de moda e/ou tendência. De tal forma, entende-se que a formalização desse selo para o bordado local contribui diretamente na criação de valor, e o aproveitamento desse fato por este projeto pode trazer à tona a utilização de uma técnica local consolidada e valida- da, fazendo com que essas pessoas envolvidas entrem em uma rede existente de renda e criação. Pensando nisso, o Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte (IFRN) – campus Caicó, oferta, anualmente, vagas nos cursos técnicos em vestuário e têxtil, bem como no curso de graduação em Design de Moda, de modo a ter indivíduos formados e preparados para atender as demandas locais. Em 2017, fundou-se o Núcleo de Extensão e Prática Profissional (NEPP), com projetos voltados para pesquisa de tendência e mercado, fomentando a economia criativa local. Nos anos seguintes, tomou lugar ações direcionadas para formação complementar de mulheres em situação de vulne- rabilidade social (MENDES; MEIRELES, 2020). Sendo assim, o NEPP é uma iniciativa do IFRN que busca promover ações que possam fazer com que os discentes do Curso Superior de Tecnologia (CST) em Design de Moda trabalhem em conjunto com as atividades propostas no projeto não fiquem na teoria. Além disso, o projeto aqui discutido trabalha também na perspectiva de inserção social, a partir do entendimento acerca da importância da extensão para a conexão efetiva entre sociedade e instituição de ensino. Resumen El Centro de Extensión y Práctica Profesional (NEPP) es una iniciativa del Instituto Federal de Rio Grande do Norte (IFRN) como una forma de vincular el conocimiento académico con los diferentes actores de la sociedad. En el campus de Caicó, en el interior del mencionado estado, se fundó en 2017 el NEPP, que ofrece cursos de educación continua para mujeres en situación de vulnerabilidad social, con el fin de generar una profesión, ingresando al mercado laboral. Además, el núcleo aún permite el desarrollo de la práctica profesional de los estudiantes del curso de tecnología superior en Diseño de Moda. Con eso en mente, este artículo tiene como objetivo exponer y discutir las acciones institucionales y el impacto de la NEPP entre 2021 y 2022 en el escenario sociocultural local. Para ello, se utilizó el mé- todo de investigación acción, con verificación cualitativa de los objetivos. De esta manera, se ofreció un curso sobre técnicas de bordado, elemento regional importante, dinamizador del trabajo local. Aliado a esto, se brindó un escenario para el desarrollo de una colección de moda por parte de estudiantes de pregrado, empleando en su configuración un motivo tan artesanal, utilizando como mano de obra a estudiantes de bordado. La colección, por tanto, pretendía servir como escaparate del trabajo de mujeres en situación de vulnerabili- dad social. Como resultado, los talleres tuvieron una duración de cuatro (4) días. Mientras tanto, las alumnas inscritas en el curso de bordado quedaron sorprendidas por la rapidez con la que aprendieron a bordar. Palabras clave: Extensión; Bordado; Enseñanza de la moda. 3 REAMD, Florianópolis, v. 7, n. 1, e2831, p. 01-19, fev./ mai. 2023 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social 1 INTRODUÇÃO Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social 1 INTRODUÇÃO Com esse conceito em mente, a extensão teve seu surgimento na Inglaterra durante o século XIX, com a intenção de direcionar novos caminhos para a sociedade e promover a educação continuada. Na atualidade, ela vem surgindo como instrumento a ser usado pelas Universidades para a efetivação de compromisso social. A Extensão Uni- Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social versitária possui um papel bastante importante no que se diz respeito às contribuições que podem trazer à sociedade (RODRIGUES et al., 2013). Assim sendo, a extensão aparece como uma oportunidade de colocar em prática os conhecimentos aprendidos em sala de aula, tratando esse processo como uma rede de compartilhamento permeado por atores discentes, docentes e sociedade. Portanto, enten- Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social versitária possui um papel bastante importante no que se diz respeito às contribuições que podem trazer à sociedade (RODRIGUES et al., 2013). Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social versitária possui um papel bastante importante no que se diz respeito às contribuições que podem trazer à sociedade (RODRIGUES et al., 2013). versitária possui um papel bastante importante no que se diz respeito às contribuições que podem trazer à sociedade (RODRIGUES et al., 2013). Assim sendo, a extensão aparece como uma oportunidade de colocar em prática os conhecimentos aprendidos em sala de aula, tratando esse processo como uma rede de compartilhamento permeado por atores discentes, docentes e sociedade. Portanto, enten- de-se que é importante que a universidade apresente uma concepção do que a extensão tem em relação com à comunidade em geral dentro do processo de ensino, pois, aquele que está na condição de aprender, acaba ampliando seu conhecimento quando há esse contato (RODRIGUES et al., 2013). Diante deste cenário, verificou-se a oportunidade de criar e capacitar um grupo de mulheres que possam bordar produtos de moda, uma vez que as artesãs que bordam produtos para o lar não costumam aceitar trabalhos orientados aos artigos do vestuário. 2 O PAPEL DO DESIGNER DE MODA NA SOCIEDADE Ao passar do século XXI é notória a influência da moda como força motriz de ascensão social e geração de empregos, em específico aos diferentes tipos de trabalho que essa indústria oferece. De acordo com Pereira (2004, p. 59) “a mobilidade e a ascen- são social são processos que estão ligados a aspectos hierarquizantes característicos das sociedades modernas. Entre estes aspectos, o capital físico é um dos mais valorizados”. No entanto, embora se observe esse mais amplo acesso ao consumo e produção de moda, vale a pena enfatizar as dimensões por trás desse fomento ao emprego, à medida que se observa um processo de precarização que traz à tona uma problemática sobre a subvalo- rização da mão de obra trabalhista (STAMPA; 2018; LOURENÇO; LORE; STAMPA, 2019), em especial a artesanal, bem como ambientes inóspitos e trabalhos análogos à escravidão (BERLIM, 2021). De tal maneira, a moda se torna de fato entendida como um fenômeno social, cultural e econômico para os indivíduos, independentes de raça, gênero, classe social, ida- de etc. É neste contexto que o desenvolvimento de habilidades na área de moda contribui na geração e oportunidades de pluralização social (LIPOVETSKY, 2001; PEREIRA, 2004). Pensando assim, produtos de moda podem divulgar conceitos, discursos ou até mesmo uma causa. Neste sentido, os produtos desenvolvidos ganham espaço não só nos gos- tos do público, mas também possuem o poder de contribuir no desenvolvimento de uma sociedade mais justa e igualitária, no entanto, por vezes, existe mais uma mobilização no discurso do que ações propriamente ditas, e o designer tem papel fundamental no caminho desta construção (POSNER, 2015). O papel do designer de moda, neste contexto, é atuar em toda a cadeia so- cial criativo-produtiva de moda, propondo produtos e serviços desenvolvidos e produzidos, principalmente, pelas indústrias têxtil e de confecção (vestuário, cama, mesa e banho, de- coração), calçados e bolsas, joias e bijuterias e até mesmo pela indústria de cosméticos e beleza (maquiagens e esmaltes), disseminando e democratizando as informações de moda (GUSTAVSEN; YATES, 2013). Gustavsen e Yates (2013) dividem a indústria da moda em três grandes áreas, onde o Designer pode atuar: criação, comercialização e comunicação. No que diz respeito a área de criação, o profissional deve propor novos produtos, agrupados em formato de coleção e idealizados de acordo um tema, para serem ofertados a um público específico. 1 INTRODUÇÃO Além disso, a necessidade dessa ação aparece na medida em que se vem ampliando a utilização de tipologias artesanais nos artefatos de moda e que, por isso, os designers, empresas, e pequenas marcas que desenvolvam peças com tais tipologias artesanais, vol- tadas às vocações regionais, irão necessitar cada vez mais de mão de obra qualificada (CORREIA; 2022; SANTOS; HELD, 2022). Na perspectiva do discente, a prática profissional exercida dentro de sua forma- ção consiste em uma atividade na qual é vinculada o ensino, a pesquisa e a extensão. Eles são considerados indicadores de uma formação articulada e integral de sujeitos para atuar no mundo em constantes mudanças e desafios, regida pelos princípios de oportunidades de igualdade para todos, flexibilidade, aprendizado entre teoria e prática e o acompanha- mento total de um supervisor ao estudante durante todo período de seu desenvolvimento da prática (IFRN, 2019). Por entender o contínuo impacto destas práticas para o cenário local, em 2021/2022, a proposta do NEPP focou novamente nas mulheres em situação de vulnerabi- lidade social e econômica, de modo que pudessem ter uma capacitação profissionalizante, por meio de cursos relacionados à economia criativa ofertados pelo projeto. No entanto, desta vez optou-se por oferecer cursos de bordado regional, no qual o foco era o desenvol- vimento de produtos de moda. Na execução desses cursos, teve-se colaboração de artesãs que trabalham há um bom tempo com bordados, bem como dos discentes de Design de Moda que compunham a equipe do projeto no respectivo ano. Pensando nisso, este artigo tem por objetivo expor e discutir sobre as ações so- ciais e o impacto do Núcleo de Extensão e Prática Profissional do departamento de Moda do IFRN – Campus Caicó entre os anos de 2020 e 2021 no cenário sociocultural local. Com isso em mente, empregou-se os métodos de pesquisa-ação, com uma abordagem qualitativa dos dados, focando-se especialmente no relato de experiência das práticas pro- fissionais. 5 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social 2 O PAPEL DO DESIGNER DE MODA NA SOCIEDADE O artesanato, categoria em que o bordado se encontra, é um legado que foi transmitido ao longo dos anos e através das gerações. No entanto, essa prática artesa- nal do bordado tradicional local brasileiro vem sendo desvalorizada, especialmente pelos mais jovens, em detrimento aos elementos artesanais de outras regiões do mundo. Michetti REAMD, Florianópolis, v. 7, n. 1, e2831, p. 01-19, fev./ mai. 2023 6 6 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social (2012, p. 345) fala que “O “bordado da bisavó” não pode ser apenas retomado enquanto fol- clore. Mais uma vez, o elemento popular que fundamenta o discurso da tradição, valorizada nesse mercado, deve sistematizado, apurado, acrescido de conteúdo de moda”. O artesa- nato é uma prática de grande valor artístico e cultural, e a sua valorização é uma medida contra a extinção dessa atividade que possui valores simbólicos e de identidade cultural. Nesse contexto, a moda pode ser inserida através de elementos de diferenciação gerando uma demanda crescente por produtos artesanais (SILVA 2009) Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social (2012, p. 345) fala que “O “bordado da bisavó” não pode ser apenas retomado enquanto fol- clore. Mais uma vez, o elemento popular que fundamenta o discurso da tradição, valorizada nesse mercado, deve sistematizado, apurado, acrescido de conteúdo de moda”. O artesa- nato é uma prática de grande valor artístico e cultural, e a sua valorização é uma medida contra a extinção dessa atividade que possui valores simbólicos e de identidade cultural. Nesse contexto, a moda pode ser inserida através de elementos de diferenciação gerando uma demanda crescente por produtos artesanais (SILVA, 2009). Segundo Almeida (2017), o pensamento em caráter social é fundamental na aproximação do design com as comunidades artesanais. O design pode atuar como ferra- menta para potencializar o trabalho dos artesãos. De acordo com França (2005), o artesa- nato voltou a ser valorizado no Brasil com o apoio de diversos projetos, ONGs e designers. Promovendo o resgate por meio do enaltecimento das nossas tradições culturais e abrindo caminhos para a recuperação da nossa capacidade de criação. 2.1 O PAPEL DO NÚCLEO DE EXTENSÃO E PRÁTICA PROFISSIONAL PARA A COMUNIDADE DE CAICÓ E REGIÃO O Núcleo de Extensão e Prática Profissional (NEPP - Nuance) do Curso Superior de Tecnologia em Design de Moda do IFRN campus Caicó tem como um dos seus objetivos propagar os conhecimentos adquiridos ao longo do curso com o intuito de desenvolver as habilidades profissionais dos alunos. Ele também busca proporcionar o retorno do conheci- mento acadêmico ofertado pelo IFRN à sociedade, através do convívio do grupo de alunos e docentes com a comunidade externa, da oferta deformação complementar, da inclusão social e da valorização das vocações locais (MENDES; MEIRELES, 2020). Em artigos anteriores, publicado por Mendes e Meireles (2020), expôs-se as ações voltadas para os anos de 2017, 2018 e 2019. No primeiro ano, pôde-se observar ações direcionadas a pesquisa de tendências e mercado, instruindo os varejistas locais so- bre a utilização da informação de Moda para construção de produtos mercadologicamente mais efetivos. De acordo com Mendes e Meireles (2020, p. 173), “identificou-se 6 (seis) ma- crotendências globais de consumo e comportamento com traços de desdobramento na rea- lidade local. O material desenvolvido foi apresentado e disponibilizado aos lojistas através de um seminário”. Embora tenha sido concluído com êxito, os coordenadores do projeto entenderam, por análise de alcance, o impacto social do projeto como ainda relativamente pequeno. Nos anos seguintes, em 2018 e 2019, as ações do projeto foram modificadas, desta vez abrangendo as mulheres em situação de vulnerabilidade social que viviam, espe- Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social cialmente, no entorno da instituição de ensino. Pensando nisso, desenvolveu-se um projeto orientado para a oferta de cursos de formação complementar e continuada na área de cos- tura, de modo a esses indivíduos aprenderem um novo ofício, perpassando sua situação socioeconômica (Figura 1). Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social cialmente, no entorno da instituição de ensino. Pensando nisso, desenvolveu-se um projeto orientado para a oferta de cursos de formação complementar e continuada na área de cos- tura, de modo a esses indivíduos aprenderem um novo ofício, perpassando sua situação socioeconômica (Figura 1). cialmente, no entorno da instituição de ensino. Pensando nisso, desenvolveu-se um projeto orientado para a oferta de cursos de formação complementar e continuada na área de cos- tura, de modo a esses indivíduos aprenderem um novo ofício, perpassando sua situação socioeconômica (Figura 1). 2.1 O PAPEL DO NÚCLEO DE EXTENSÃO E PRÁTICA PROFISSIONAL PARA A COMUNIDADE DE CAICÓ E REGIÃO Figura 1 – Projeto NEPP 2019 – Oficinas de costura para mulheres em situação de vulnerabilidade socioeconômica Fonte: Acervo NEPP (2019) Figura 1 – Projeto NEPP 2019 – Oficinas de costura para mulheres em situação de vulnerabilidade socioeconômica Fonte: Acervo NEPP (2019) De acordo com Mendes e Meireles (2020, p. 175), a “experiência foi bastante exitosa, pois estimulou o convívio entre alunos de diversos níveis de conhecimento, a troca de experiência e saberes empíricos e populares e o trabalho em equipe, habilidade indis- pensável para profissionais da indústria criativa”. Aliado a isso, desenvolveu-se ainda uma coleção de moda, contando com os alunos do curso CST em Design de Moda para elabo- ração do processo criativo e produtivo. Pensando nessa continuidade de discussão, este artigo apresenta os relatos do NEPP nos anos posteriores, tendo em vista a modificação para um novo nicho. 2.2 O BORDADO E A SUA INFLUÊNCIA EM CAICÓ (RIO GRANDE DO NORTE) Tendo em mente o papel do bordado para este trabalho, em específico ele como um veículo de ensino e profissionalização de mulheres em situação de vulnerabilidade so- cial, observou-se a necessidade de conduzir um resgate histórico do seu desenvolvimento, até chegar na região Seridó do estado do Rio Grande do Norte. Segundo SEBRAE (2013), o bordado é uma forma de criar desenhos ornamen- tais à mão ou à máquina em um tecido, utilizando-se de ferramentas, como agulhas e fios têxteis, de maneira que os fios utilizados formem o desenho desejado. O bordado, além de 8 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social ter papel ornamental na peça, servia para distinguir os trajes com monogramas das iniciais dos nomes próprios ou palavras e símbolos referentes à atividade profissional, assim como desenhos ornamentais (BRAGA; PRADO, 2011). Dessa forma, identificando a família a qual o indivíduo pertencia. Tendo isso em mente, pode-se observar que o bordado agrega valor aos produtos de vestuário, trazendo elementos culturais, criando uma peça com sig- Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social ter papel ornamental na peça, servia para distinguir os trajes com monogramas das iniciais dos nomes próprios ou palavras e símbolos referentes à atividade profissional, assim como desenhos ornamentais (BRAGA; PRADO, 2011). Dessa forma, identificando a família a qual o indivíduo pertencia. Tendo isso em mente, pode-se observar que o bordado agrega valor aos produtos de vestuário, trazendo elementos culturais, criando uma peça com sig- nificado que pode refletir a personalidade de quem usa. A inserção do bordado na moda se dá pela necessidade de desenvolver produ- tos com valor agregado, podendo citar uma produção sustentável e a configuração de pe- ças exclusivas. Segundo Almeida (2013), a moda brasileira articula no campo simbólico dos seus artefatos traços comuns e reconhecíveis no país e mobiliza esses elementos como portadores de uma identidade regional. Leitão (2007) observa que designers de moda que trabalham com o artesanato buscam “procurar raízes”, “valorizar a cultura popular”, “positi- var nossa natureza” e fazer uso do que há de mais “autenticamente brasileiro”. Pensando na evolução do núcleo para uma nova tipologia artesanal, chegou-se ao ensino de bordado. 2.2 O BORDADO E A SUA INFLUÊNCIA EM CAICÓ (RIO GRANDE DO NORTE) Historicamente, o bordado é uma tradição familiar, segundo Braga e Prado (2011), era essencial que toda mulher possuísse conhecimentos das técnicas de costura e bordado. Esses ensinamentos eram passados dentro do ambiente familiar como uma forma de recreação em grupos de costura, mães passavam os conhecimentos para as filhas, fazendo surgir a necessidade de se reproduzir produtos de utilidade doméstica e expressar a capacidade criativa e produtiva como forma de trabalho. Segundo relatos, a prática do exercício do bordado teria chegado à região do Seridó, principalmente na cidade de Caicó, por meio das esposas dos colonizadores portu- gueses que foram ali se fixando no final do século XVIII e no início do século XIX (BATISTA, 1988). O bordado foi trazido, segundo as fontes históricas, da Ilha da Madeira, em Portugal. Daí em diante, o bordado foi sendo desenvolvido e transmitido para outras mulheres na cidade (ARAÚJO, 2013, p. 28). A prática foi difundida, inicialmente, entre as mulheres das camadas sociais que possuíam um poder aquisitivo maior, visto que a matéria-prima era de difícil acesso, tanto com relação ao local de venda quanto ao seu preço, devido a ser considerada “cara” e nem todas as pessoas podiam comprar os tecidos e as linhas apropriadas. Inicialmente, as linhas eram trazidas da Ilhada Madeira, com certo tempo, esse ma- terial já podia ser adquirido na cidade de Recife, em Pernambuco. Posteriormente, com a procura por linha de qualidade para dar um bom efeito nos bordados, iniciou- -se a comercialização da linha na cidade de Caicó (ARAÚJO, 2013, p. 28). A prática foi difundida, inicialmente, entre as mulheres das camadas sociais que possuíam um poder aquisitivo maior, visto que a matéria-prima era de difícil acesso, tanto com relação ao local de venda quanto ao seu preço, devido a ser considerada “cara” e nem todas as pessoas podiam comprar os tecidos e as linhas apropriadas. Inicialmente, as linhas eram trazidas da Ilhada Madeira, com certo tempo, esse ma- terial já podia ser adquirido na cidade de Recife, em Pernambuco. Posteriormente, com a procura por linha de qualidade para dar um bom efeito nos bordados, iniciou- -se a comercialização da linha na cidade de Caicó (ARAÚJO, 2013, p. 28). A prática foi difundida, inicialmente, entre as mulheres das camadas sociais que possuíam um poder aquisitivo maior, visto que a matéria-prima era de difícil acesso, tanto com relação ao local de venda quanto ao seu preço, devido a ser considerada “cara” e nem todas as pessoas podiam comprar os tecidos e as linhas apropriadas. Inicialmente, as linhas eram trazidas da Ilhada Madeira, com certo tempo, esse ma- terial já podia ser adquirido na cidade de Recife, em Pernambuco. Posteriormente, com a procura por linha de qualidade para dar um bom efeito nos bordados, iniciou- -se a comercialização da linha na cidade de Caicó (ARAÚJO, 2013, p. 28). 3 METODOLOGIA DO PROJETO DE EXTENSÃO De acordo com a sua natureza, o projeto tem a classificação do tipo aplicado, e a abordagem qualitativa (GIL, 2008). Pode-se classificar este trabalho de extensão como uma dimensão da pesquisa-ação, pois existe uma interação social entre o investigador, o objeto de estudo e o participante do objeto que está sendo estudado, ou trabalhado, contri- buindo para essa realização do exercício. Para alcançar os objetivos do projeto NEPP 2021/2022 foi idealizada uma se- quência de etapas como uma forma de comunicar aos participantes a respeito das ativida- des que foram sugeridas. Inicialmente, foram escolhidos os participantes para fazer parte da equipe da execução dos cursos, compostos pelos alunos do curso Superior de Tecnolo- gia em Design de Moda do IFRN, campus Caicó e das bordadeiras seridoenses. Em segui- da, mulheres em situação de vulnerabilidade social foram convidadas para participar como alunas nas oficinas de bordados. Após isso, foram coletados dados materiais e imateriais no dia a dia local, por meio de pesquisa de campo, avaliações das vocações locais e o incentivo nas dinâmicas que serviram para inspirar o desenvolvimento do material didático e das práticas do curso. Foi a partir das pesquisas que selecionaram e discutiram qual seria os temas pertinentes no qual seriam ministrados. Para desenvolvimento dos cursos, partiu-se de um princípio de elaboração das ementas e das apostilas. Sendo assim, para o planejamento das aulas, idealizou-se ativida- des práticas de bordado. A execução das oficinas que compõem o curso durante os 4 dias e a confecção de produtos de moda (organizados em uma coleção pelos discentes do CST em Design de Moda), que correspondam a valores estéticos globais contemporâneos e que ao mesmo tempo atendam ao interesse do público local. 2.2 O BORDADO E A SUA INFLUÊNCIA EM CAICÓ (RIO GRANDE DO NORTE) O que o bordado traz de herança para a região Seridoense são histórias carre- gadas de força, que foram acolhidas pela população, e que despertam o interesse daqueles que querem aprender as técnicas, sendo assim, o bordado ganhou força na região, sendo presente até hoje. Este atual projeto trouxe a proposta das oficinas de bordado visando va- 9 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social lorizar ainda mais essa arte milenar e passá-la para mais pessoas, ensinando as técnicas de bordado ao público geral, sendo difundido e ministrado por bordadeiras caicoenses. Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social lorizar ainda mais essa arte milenar e passá-la para mais pessoas, ensinando as técnicas de bordado ao público geral, sendo difundido e ministrado por bordadeiras caicoenses. 4 OFERTA DE CURSO NA ÁREA DE BORDADO PARA FORMAÇÃO COMPLEMENTAR E INCLUSÃO SOCIAL Para o NEPP de 2021/2022 optou-se por propor uma nova gama de ações para a comunidade. Com o desenrolar da ênfase em costura no curso ofertado, notou-se a que- da pelo núcleo na repetição de conteúdo, não havendo mais interesse da sociedade por 10 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social esse curso. Por isso, decidiu-se por, ao invés de ofertar cursos de costura, voltar-se para uma tipologia artesanal significativa para a região, o bordado. No entanto, não havendo ex- pertise nem na coordenação do projeto, nem entre os discentes, recorreu-se as bordadei- ras da região. Tal necessidade se mostrou como uma possível estratégia para chegar até essa comunidade por vezes distante da realidade institucional e, de tal maneira, mostrar o trabalho feito pelo Núcleo de Extensão e Prática Profissional para Caicó e Região, ressal- tando sua importância. Duas bordadeiras da cidade apresentaram interesse, tornando-se ministrantes das oficinas, acompanhadas pelos discentes voluntários no projeto. esse curso. Por isso, decidiu-se por, ao invés de ofertar cursos de costura, voltar-se para uma tipologia artesanal significativa para a região, o bordado. No entanto, não havendo ex- pertise nem na coordenação do projeto, nem entre os discentes, recorreu-se as bordadei- ras da região. Tal necessidade se mostrou como uma possível estratégia para chegar até essa comunidade por vezes distante da realidade institucional e, de tal maneira, mostrar o trabalho feito pelo Núcleo de Extensão e Prática Profissional para Caicó e Região, ressal- tando sua importância. Duas bordadeiras da cidade apresentaram interesse, tornando-se ministrantes das oficinas, acompanhadas pelos discentes voluntários no projeto. Para seguir uma didática que fosse de fácil compreensão e por se ter um tempo limitado a 4 oficinas de 4 horas cada (Quadro 1), resolveu-se que o primeiro dia seria para fazer uma introdução geral ao tema e ensinar os primeiros pontos do bordado aos alunos inscritos (Figura 2 – A), que foram eles: ponto corrente, ponto atrás, ponto cheio e ainda foi iniciado a técnica do ponto rococó, com demonstração por parte das ministrantes convida- das. Quadro 1 – Síntese dos conteúdos ministrados nos quatro dias de oficinas de bordado Oficina Conteúdo 1 • Introdução geral; • Primeiros pontos: corrente, atrás, cheio e rococó. 2 • Ponto rococó aplicado: desenvolvimento do desenho de uma folha. 3 • Aplicação dos pontos de bordado: trabalho a partir de um risco maior. 4 OFERTA DE CURSO NA ÁREA DE BORDADO PARA FORMAÇÃO COMPLEMENTAR E INCLUSÃO SOCIAL 4 • Aplicação dos pontos de bordado: trabalho a partir de um risco maior; • Finalização das oficinas e compartilhamento dos trabalhos desenvolvidos. Fonte: Elaborado pelos autores Quadro 1 – Síntese dos conteúdos ministrados nos quatro dias de oficinas de bordado Quadro 1 – Síntese dos conteúdos ministrados nos quatro dias de oficinas de bordado Fonte: Elaborado pelos autores No primeiro encontro entre a turma de alunos das oficinas e as professoras con- vidadas, houve um breve momento de apresentação conduzido pelos voluntários do projeto NEPP. Logo após as apresentações e boas-vindas, as ministrantes começaram a introduzir o conteúdo do curso (Figura 2 – B). 11 REAMD, Florianópolis, v. 7, n. 1, e2831, p. 01-19, fev./ mai. 2023 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Figura 2 – Primeiro dia de oficina de bordado – NEPP 2021/2022 Fonte: Acervo NEPP (2022) B A Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Figura 2 – Primeiro dia de oficina de bordado – NEPP 2021/2022 Figura 2 – Primeiro dia de oficina de bordado – NEPP 2021/2022 Fonte: Acervo NEPP (2022) B A Fonte: Acervo NEPP (2022) B A B A Fonte: Acervo NEPP (2022) As ministrantes, junto com os alunos tutores voluntários do NEPP, optaram por iniciar a aula de “Introdução ao bordado” pela apresentação de alguns resultados, ou seja, mostrando diversos bordados já feitos, criados anteriormente pelas próprias ministrantes, de modo que despertasse o olhar dos alunos à dimensão estética de tal tipologia artesanal, à beleza do bordado já pronto. Essa estratégia foi empregada como forma de auxiliá-los a entender que, para chegar aquele resultado, seria necessário passar por algumas etapas, que são elas: o risco e os pontos. Ao final do primeiro dia, alguns alunos já tinham conseguido desenvolver alguns pontos de bordados e todos se mostraram animados, afirmando interesse em participar do grupo on-line do WhatsApp, que foi criado com o propósito de comunicação, troca de experiências, compartilhamento de vídeos aula e o processo de criação de cada um deles. Ressalta-se que a ferramenta de comunicação supramencionada se mostrou como um importante diferencial para acompanhar o processo de desenvolvimento da prática de bor- dado dos alunos das oficinas, de modo que, mesmo quando não estavam em sala de aula, elas costumavam compartilhar fotografias de quando estavam estudando alguns pontos (Figura 3 – A). Na segunda oficina abordou-se a técnica da folha. Nela foi utilizado o ponto rococó, um ponto considerado “complicado” por algumas bordadeiras, em especial as que foram convidadas para serem ministrantes, o que despertou uma série de dúvidas por parte das alunas participantes da oficina. Fonte: Elaborado pelos autores Para elucidar a técnica, a professora decidiu fazer a demonstração do ponto por grupos pequenos, para que assim eles pudessem ver a técnica bem de perto. Em seguida cada aluno tentou reproduzir, o que facilitou o entendimento dos estudantes. Neste dia, foi desenvolvido um desenho padrão para os membros das oficinas treinarem a técnica de rococó (Figura 3 – B). 12 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social A terceira oficina foi ministrada apenas por uma das bordadeiras convidadas, onde a tutora levou um desenho base maior, para que os alunos pudessem treinar em um risco mais detalhado. O objetivo dessa prática foi que os alunos utilizassem, em um mes- mo desenho, todos os pontos apresentados desde a primeira aula. Por requerer um tempo maior de prática, neste dia não se conseguiu concluir o exercício, ficando acertado dos alunos treinarem em casa e concluir o desenho na aula subsequente (Figura 3 – C). Na quarta oficina, desenvolveu-se a continuidade com o risco que as alunas ti- nham iniciado no encontro anterior. Para completar o desenho proposto, elas passaram as 4 horas de oficina bordando, e mesmo assim não foi possível finalizar a atividade dentro do horário limite previsto anteriormente. Por esse motivo, as alunas levaram os materiais para casa para poderem concluir toda a arte. Entendemos que esse processo de ganhar intimi- dade com a técnica e ir fazendo “ao seu tempo” é muito importante e é o que irá garantir que as aulas adquiram confiança e não desistam de aprender e desenvolver essa habilidade (Figura 3 – D). Figura 3 – Oficinas de Bordado para inclusão social – NEPP 2021/2022 REAMD, Florianópolis, v. 7, n. 1, e2831, p. 01-19, fev./ mai. 2023 Figura 3 – Oficinas de Bordado para inclusão social – NEPP 2021/2022 Fonte: Acervo NEPP (2022) A B C D Fonte: Acervo NEPP (2022) A B C D A A B D Fonte: Acervo NEPP (2022) C D D C Fonte: Acervo NEPP (2022) 13 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Para a conclusão das aulas das oficinas, a equipe do projeto, de voluntários a coordenadores, convidou as bordadeiras que participaram do projeto para contribuírem no desenvolvimento de uma coleção de moda, aplicando o que foi aprendido nas oficinas ao bordar as peças da coleção “Trama Seridoense: Caicó de Encantos Mil” que foi desenvol- vida pelos discentes do CST em Design de Moda, também vinculados ao projeto do NEPP. 5 PROPOSTA DE COLEÇÃO DE MODA PARA VALORIZAÇÃO DOS SABERES LOCAIS E DIVULGAÇÃO DO TRABALHO DAS OFICINAS Em paralelo com as oficinas de bordado, o NEPP conduzia um segundo projeto, voltado para o desenvolvimento da prática profissional dos discentes do CST em Design de Moda, orientado ao planejamento, desenvolvimento e execução de uma coleção de vestuá- rio (Figura 4). De tal maneira, tinha-se como princípio a aplicação da tipologia artesanal do bordado na configuração estética da coleção, de modo a servir como uma forma de divulga- ção para a sociedade do trabalho dos dois públicos do projeto: discentes do próprio CST e mulheres em situação de vulnerabilidade econômica, que participaram das oficinas de bor- dado. Após a conclusão das oficinas, os alunos interessados em apresentar seus trabalhos de bordado à sociedade, dedicaram-se a bordar as peças criadas pelos alunos do CST. A criação do tema, desenhos e modelagens da coleção de moda, tiveram como inspiração um monólogo de Francisco Maguila, um artista da região Seridó do estado do Rio Grande do Norte – localização geográfica do município de Caicó. A obra escolhida cha- ma-se “Filho de Joana e Pedro”, que foi composta com base na vivência de Francisco com seus pais. Assim como as peças, os bordados que complementam a imagem de moda da coleção também foram inspirados em obras literárias de personalidades seridoenses. Para tanto foram escolhidos os artistas seguintes: Djalma Mota, Dodora Medeiros, Patrícia Gur- gel e Edcarlos Medeiros. A finalidade era de valorizar a cultura local e reconhecer o trabalho grandioso que esse poetas fazem e representam para a região (Figura 5). 14 REAMD, Florianópolis, v. 7, n. 1, e2831, p. 01-19, fev./ mai. 5 PROPOSTA DE COLEÇÃO DE MODA PARA VALORIZAÇÃO DOS SABERES LOCAIS E DIVULGAÇÃO DO TRABALHO DAS OFICINAS 2023 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Figura 4 – Reunião de planejamento da coleção de vestuário, modelagem e costura das peças pelos dis- centes do CST em Design de Moda – NEPP 2021/2022 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Figura 4 – Reunião de planejamento da coleção de vestuário, modelagem e costura das peças pelos dis- centes do CST em Design de Moda – NEPP 2021/2022 Fonte: Acervo NEPP (2022) Figura 5 – Coleção final e bordada – NEPP 2021/2022 Fonte: Acervo NEPP (2022) Figura 4 – Reunião de planejamento da coleção de vestuário, modelagem e costura das peç centes do CST em Design de Moda – NEPP 2021/2022 Figura 4 – Reunião de planejamento da coleção de vestuário, modelagem e costura das peças pelos dis- centes do CST em Design de Moda – NEPP 2021/2022 Fonte: Acervo NEPP (2022) Figura 5 Coleção final e bordada NEPP 2021/2022 Fonte: Acervo NEPP (2022) Figura 5 – Coleção final e bordada – NEPP 2021/2022 Fonte: Acervo NEPP (2022) Fonte: Acervo NEPP (2022) Fonte: Acervo NEPP (2022) Figura 5 – Coleção final e bordada – NEPP 2021/2022 Fonte: Acervo NEPP (2022) O presente projeto desenvolveu um evento para apresentação da coleção, que ganhou o título de “Trama Seridoense: Caicó de Eventos Mil”. Ele contou com a participa- ção das alunas e professoras das oficinas dos bordados, que protagonizaram os bordados das peças. A abertura da exposição aconteceu às 17h do dia 19 de maio de 2022 (Figura Figura 5 – Coleção final e bordada – NEPP 2021/2022 Figura 5 – Coleção final e bordada – NEPP 2021/2022 Fonte: Acervo NEPP (2022) Fonte: Acervo NEPP (2022) Fonte: Acervo NEPP (2022) O presente projeto desenvolveu um evento para apresentação da coleção, que ganhou o título de “Trama Seridoense: Caicó de Eventos Mil”. Ele contou com a participa- ção das alunas e professoras das oficinas dos bordados, que protagonizaram os bordados das peças. A abertura da exposição aconteceu às 17h do dia 19 de maio de 2022 (Figura 15 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social 6), na casa do artesão, estrutura cultural local, e permaneceu disponível para visitação nos dias 20 e 21 de maio do mesmo ano. 5 PROPOSTA DE COLEÇÃO DE MODA PARA VALORIZAÇÃO DOS SABERES LOCAIS E DIVULGAÇÃO DO TRABALHO DAS OFICINAS Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social 6), na casa do artesão, estrutura cultural local, e permaneceu disponível para visitação nos dias 20 e 21 de maio do mesmo ano. 6), na casa do artesão, estrutura cultural local, e permaneceu disponível para visitação nos dias 20 e 21 de maio do mesmo ano. Figura 6 – Exposição da coleção na Casa do Artesão – NEPP 2021/2022 Fonte: Acervo NEPP (2022) 6), na casa do artesão, estrutura cultural local, e permaneceu disponível para visitação nos dias 20 e 21 de maio do mesmo ano. Figura 6 – Exposição da coleção na Casa do Artesão – NEPP 2021/2022 Figura 6 – Exposição da coleção na Casa do Artesão – NEPP 2021/2022 Figura 6 – Exposição da coleção na Casa do Artesão – NEPP 2021/2022 Figura 6 – Exposição da coleção na Casa do Artesão – NEPP 2021/2022 Fonte: Acervo NEPP (2022) Fonte: Acervo NEPP (2022) Fonte: Acervo NEPP (2022) De tal maneira, esse evento foi empregado como uma forma de vitrine para os designers e bordadeiras das oficinas, onde se preparou etiquetas para as roupas, com to- das as informações de que as criou e bordou, incluindo contatos para possíveis trabalhos. Sendo assim, esta foi a estratégia pensada para que a informação sobre os atores envolvi- dos na criação chegasse as demais pessoas da sociedade. 16 Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Núcleo de extensão e prática profissional na área da moda: Formação complementar e inclusão social Assim sendo, o objetivo maior da exposição, além de receber os artistas home- nageados na coleção, era que a sociedade caicoense pudesse vir prestigiar esse momento tão importante para a interligação entre sociedade, instituição de ensino, cultura, artesanato e moda. Assim sendo, o objetivo maior da exposição, além de receber os artistas home- nageados na coleção, era que a sociedade caicoense pudesse vir prestigiar esse momento tão importante para a interligação entre sociedade, instituição de ensino, cultura, artesanato e moda. 6 CONSIDERAÇÕES FINAIS O ambiente institucional serve como uma porta de entrada para o fomento de ações que interligam o fazer acadêmico, em especial da prática profissional dos alunos de graduação, com atividades que contribuem diretamente para a solução de problemáticas sociais. Nesse sentido, este artigo teve como objetivo expor e discutir sobre as ações con- duzidas pelo NEPP durantes os anos de 2021 e 2022, focando no caso de mulheres em situação de vulnerabilidade social. De tal forma, conduziu-se o projeto dentro de duas dimensões: a proposição de oficinas de bordado como estratégia para um novo saber-fazer e o desenvolvimento de uma coleção de moda para divulgação do trabalho local. Por relatos ouvidos durante o desenvolvimento, observou-se que o projeto proporcionou momentos construtivos, aulas produtivas, cercadas por uma constante troca de conhecimentos, um espaço colaborativo, tanto por parte das professoras convidadas, como dos alunos participantes inscritos nas ofi- cinas. Assim como os pontos de um bordado, o projeto entrelaçou histórias de bordadeiras, mulheres em situação de vulnerabilidade social e estudantes de moda. Assim sendo, o conhecimento adquirido em todo o processo criativo da coleção nos últimos meses, aliado a pesquisa realizada aqui abordada, demonstraram que as ten- dências podem surgir de diversas fontes, dentre elas, a cultura popular. Pois, entende-se que ela é o grande palco onde a moda atua, apoderando-se dos saberes e fazeres locais, os ressignificando e recriando5. REFERÊNCIAS REAMD, Florianópolis, v. 7, n. 1, e2831, p. 01-19, fev./ mai. 2023 17 ALMEIDA, A.J.M. A identidade nacional e a cultura popular no design de moda brasi- leiro. 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https://openalex.org/W3012806260	https://labanimres.biomedcentral.com/track/pdf/10.1186/s42826-020-00038-0	English	null	Characterization of aged male BALB/ccenp mice as a model of dementia	Laboratory animal research	2,020	cc-by	8,251	Abstract Dementia is defined as cognitive impairment in more than one cognitive area and leads to an abnormal degree of impairment in the ability to remember past events. Among mice models of dementia the most used strains are SAMP8 and C57BL/6. There is no reference to characterizing a model of dementia in naturally aged mice of the BALB/c strain, or to the minimum age at which these animals can be used. The aim of this study was the characterization of aged male BALB/ccenp mice as a model of dementia from the evaluation of behavioural, pathological and biochemical markers. One hundred and twenty mice were used and 10 of these were analysed from 8 to 9 months of age, and every 4 months, in a comparative way to young control animals from 4 to 5 months. At the age of 12–13 months there was cognitive impairment in the animals from the Y-maze and object recognition tests and this impairment was maintained at 16–17 months of age. An increase in oxidative damage to proteins in the brains of aged animals was also found in relation to young animals; as well as a decrease in the concentration of triglycerides. At the age of 16–17 months, a significant decrease in the size of the thymus and brain was obtained. We consider that it’s a very useful option to use animals 12–13 months of age where there are symptoms of cognitive deficiency, histopathological and biochemical elements characteristic of dementia. Keywords: Dementia, Alzheimer’s type dementia, Animal models, Aging © The Author(s). 2020 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. * Correspondence: nashelly.esquivel@biocen.cu †Nashelly Esquivel and Yenela García contributed equally to this work. 1Laboratorio de Antianémicos y Nutracéuticos, Centro Nacional de Biopreparados (BioCen). Beltrán main road km 1 1/2, Mayabeque, Cuba Full list of author information is available at the end of the article Esquivel et al. Laboratory Animal Research (2020) 36:7 https://doi.org/10.1186/s42826-020-00038-0 Esquivel et al. Laboratory Animal Research (2020) 36:7 https://doi.org/10.1186/s42826-020-00038-0 Laboratory Animal Research (2020) 36:7 Esquivel et al. Laboratory Animal Research https://doi.org/10.1186/s42826-020-00038-0 Open Access Introduction innate immunity in the central nervous system, mainly by microglial cells, that is a key element in the neurode- generative process [5]. Dementia is defined as cognitive impairment character- ized by loss of intellectual ability of sufficient severity to interfere either with occupational functioning and usual social activities [1]. Cognitive areas involved in dementia includes aphasia (language deterioration), apraxia (motor difficulties), agnosia (failure to recognize objects despite intact sensory capacity), or a disturbance in executive functioning [2]. Around 80% cases of dementia world- wide are represented by Alzheimer’s disease (AD) and they are characterized by different stages of cognitive and functional impairments [3]. Development of animal models of dementia is challen- ging as there is no single animal model that can explain all the cognitive, behavioural, biochemical and histo- pathological abnormalities of different types of this pathology [6]. An ideal animal model should mimic the human disease and reproduce complexities of human behaviour in rodents. Several animal models of AD and cognitive impairment have been developed, which can be broadly classified according to diverse pathophysio- logical bases [7]. To develop animal models of dementia generally use transgenic animals or the bilateral stereo- taxic administration into the ventricles of the brain of different chemical substances. However, the major dis- advantage of these models is that the acute phase of the disease doesn’t reproduce the pathology as it evolves in humans, where the development of disease occurs over months and years. Added to these disadvantages to prove some hypotheses, is the high cost of transgenic AD is characterized histopathologically by extracellular amyloid-beta plaques and intraneural fibrillar tangles due to tau protein hyperphosphorylation, oxidative stress and synaptic and neuronal losses [4]. The gradual devel- opment of AD is due to the action of different patho- physiological events, among which the activation of * Correspondence: nashelly.esquivel@biocen.cu †Nashelly Esquivel and Yenela García contributed equally to this work. 1Laboratorio de Antianémicos y Nutracéuticos, Centro Nacional de Biopreparados (BioCen). Beltrán main road km 1 1/2, Mayabeque, Cuba Full list of author information is available at the end of the article * Correspondence: nashelly.esquivel@biocen.cu †Nashelly Esquivel and Yenela García contributed equally to this work. 1Laboratorio de Antianémicos y Nutracéuticos, Centro Nacional de Biopreparados (BioCen). Beltrán main road km 1 1/2, Mayabeque, Cuba Full list of author information is available at the end of the article Experimental design When the animals were 4–5 months of age a sample of 10 animals was randomly selected and they were consid- ered the control group. From this first experimental time, groups of 10 animals were randomly selected every four months until animal showed cognitive impairment. Body weight was measured in all the experimental times, and cognitive function was evaluated by the application of different behavioral tests. In addition percent survival and the percentage of animals that showed an adequate state of health were determined from the total of ani- mals that started the experiments in each experimental time. In front of this situation some studies report the use of economic models, non-invasive and that reproduce part of the pathogenesis of the disease. Aged animals are used routinely in drug development due to age related cognitive decline and behavioural alterations which mimic not only the neurochemical and morphological alterations but also the cholinergic hypofunction that is similar to pathophysiology of AD [9–11]. Another ad- vantage of this model is that they develop without any central neurochemical manipulations and occurs a slow progression of the disease associated to the natural aging process [12]. Rodents have a leading role in animal models of dementia and among the most used strains by researchers studying cognitive disorders are C57BL/6 [13] and SAMP8 [14]. In the case of the BALB/c strain there are few studies where it was used aged mice as a model of dementia [15]. Esquivel et al. Laboratory Animal Research (2020) 36:7 Page 2 of 11 Page 2 of 11 Esquivel et al. Laboratory Animal Research (2020) 36:7 animals that increase the price of neurobiological re- search [8]. Total of possibles alternations = Total of entries - 2. Object recognition task: it’s based on the natural in- stinct of rodents to explore novel objects and evaluate non-spatial memory [21]. The task was performed under the conditions described by García and Esquivel [20]. Animals and housing conditions Young BALB/ccenp male mice (20–24 g, n = 120) were purchased from Centro Nacional para la Producción de Animales de Laboratorio (CENPALAB, La Habana, Cuba). All experimental mice were given space with a controlled consistent temperature (21 ± 3 °C) and light- ing environment (12 h/12 h light/dark cycle). Mice were fed with the EAO1004cenp diet for rodents ad libitum and drinking water. The animals were adapted for a week to the experimental conditions prior to the begin- ning of the experiments and then mice were individual identified by ears perforation nomenclature. Animals were placed in polypropylene cages with wood chip bed- ding (Sournid, Spain), at the rate of 10 animals per cage. All experimental protocols related to the use of animals were approved by the Institutional Animal Care and Use Committee at Centro Nacional de Biopreparados. The percentange of time that mice spent recognizing the novel object (NO) or familiar object (FO), relative to the total object recognition time or Preference Index (PI), was calculated by the following expression: IP NO % ð Þ ¼ Time Exp:NO Time Exp:FO þ Time Exp:NO 100 Time Exp. FO: Time of exploration of FO Time Exp. NO: Time of exploration of NO IP NO % ð Þ ¼ Time Exp:NO Time Exp:FO þ Time Exp:NO 100 Time Exp. FO: Time of exploration of FO Time Exp. NO: Time of exploration of NO Behavioral tasks The animals were transferred to the place where the be- havioral tests were carried out 24 h before the beginning of the assays. At the end of each task, the surfaces of the mazes were disinfected with 70% ethanol solution. Y-maze task (spontaneous alternation): it allows evalu- ating the spatial memory of short-term work [19] and in this study it was used the methodology described by García and Esquivel [20]. In the spontaneous alternation task, it was considered that the mice made alternations when they sequentially visited the three arms, without repeating any. We also counted the number of repeated entries to the same arm (perseveration) and incorrect entries to the arms for each animal. Finally, spontaneous alternation was calculated by the following expression: There is no reference to the characterization of a de- mentia model in aged mice of the BALB/c strain, even though it’s widely used in behavioural studies related to stress, fear and cognitive function [16–18]. On the other hand, among the models of aged mice described for dementia there is variability about the minimum age at which behavioural, pathological or biochemical symp- toms of the disease take place [13–15]. In all of these models there is great variety about the minimal age at which behavioural, pathological or biochemical symp- toms of the disease appear. Therefore it’s important to characterize a model where these aspects of dementia are explained and centralized. The aim of this study was the characterization of aged male BALB/ccenp mice as a model of dementia from the evaluation of behavioural, pathological and biochemical markers. Spontaneous alternation % ð Þ ¼ Alternations Total of possible alternations 100 Spontaneous alternation % ð Þ Total of possibles alternations = Total of entries - 2. Hematological and biochemical determinations It was also obtained brain homogenates in Tris buffer at pH 7.6 containing 0.32 mM sucrose (Sigma- Aldrich, Merck, Darmstadt, Germany), 10 mM Tris (Sigma-Aldrich, Merck, Darmstadt, Germany) and 1 mM EDTA (Sigma-Aldrich, Merck, Darmstadt, Germany) and differential centrifugation was performed to obtain the post-mitochondrial fraction, according to the method de- scribed by Uwe and Von Hagen [22]. The supernatant was dispensed in aliquots of 1 mL, which were stored at −80 °C. In these homogenates the oxidative damage to lipids and proteins was measured. (Sartorius, Göttingen, Germany). The relative weight of the organ was calculated in relation to the body weight of the animal before sacrifice (also was analized weight organs in the esperimental times of 8–9 and 12–13 months of age). All mice organs, except the thymus, were fixed in phosphate buffered 10% formalin solution (Sigma-Aldrich, Merck, Darmstadt, Germany). After in- clusion in paraffin, the organs were cut and stained with hematoxylin/eosin (Sigma-Aldrich, Merck, Darmstadt, Germany) according to conventional techniques. The histological evaluation of organs and tissues was carried out with an optical microscope (Meiji Techno, Tokyo, Japan). Statistical analysis l One-way analysis of variance (ANOVA) was used to identify significant differences between all experimental groups (GraphPad Prism 5 for Windows, Release 5.03, Standard Version, San Diego, CA, USA). These levels were verified by Tukey’s post-hoc analysis. All values were expressed as the means ± standard deviation (SD) and a P value < 0.05 was considered significant. Percent survival and body weight The oxidative damage to the lipids was measured from the thiobarbituric acid reactive substances (TBARS) method according to Okawa et al. [23]. The TBARS were determined from the molar extinction coefficient of the malondialdehyde (MDA) (ξ = 155 mM - 1 cm - 1 mL). The concentration of proteins in the tissue hom- ogenate was determined by the modified Lowry method for biological tissues [24]. In addition, to quantify the oxidative damage to proteins, it was used the method described by Resnick and Parker [25]. The amount of proteins with carbonyl groups was determined from the maximum absorption measured at 360 nm; and the molar extinction coefficient of hydrazine (ξ = 22.000 mM - 1 cm - 1 mL). On the other hand, the concentration of proteins in the samples was calculated from different concentrations of bovine serum albumin (0.2 to 2.0 mg/ mL) that were read at 280 nm. Finally, the result was expressed as the relationship between the molar concen- tration of the carbonyl groups and the concentration of proteins present in the tissue homogenate. Percent survival of male BALB/ccenp mice at the age of 16–17 months was 93.33%. However, 51 animals were sacrificed (42.50% of the total) due to a deteriorated health state when the mice reached the age of 8–9 months. The sum of the natural deaths and the animals that were sacrificed represented a real survival of 50.83%. The body weight of male BALB/ccenp mice showed sta- tistically significant differences from the age of 8–9 months, whose weight was significantly greater than that of the control group from 4 to 5 months (Fig. 1). Among Fig. 1 Body weight of male BALB/ccenp mice up to 16–17 months of age. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age) Hematological and biochemical determinations Ten animals from 8 to 9 months and 12–13 months of age and 10 control animals from 4 to 5 months of age were used. The animals were anesthetized in a ketamine and xylazine (Sigma-Aldrich, Merck, Darmstadt, Germany) mix Esquivel et al. Laboratory Animal Research (2020) 36:7 Page 3 of 11 Esquivel et al. Laboratory Animal Research Page 3 of 11 and approximately 100 μL of blood was extracted by the retro-orbital plexus, which was added the ethylenediamine- tetraacetic acid (EDTA) anticoagulant solution (Sigma-Al- drich, Merck, Darmstadt, Germany). This sample was analyzed in the optical microscope (Olympus, Tokyo, Japan), with the objective of 100 × immersions, and 100 cells were observed to calculate the percentages corre- sponding to the nuclear polymorphs (NPM) and the lym- phocytes. The rest of the blood was then collected without EDTA (Sigma-Aldrich, Merck, Darmstadt, Germany) to ob- tain the serum. In the serum sample collected, the concen- tration of triglycerides was measured with the Monotriglitest reagent kit (HELFA® Diagnostics, La Habana, Cuba). It was also obtained brain homogenates in Tris buffer at pH 7.6 containing 0.32 mM sucrose (Sigma- Aldrich, Merck, Darmstadt, Germany), 10 mM Tris (Sigma-Aldrich, Merck, Darmstadt, Germany) and 1 mM EDTA (Sigma-Aldrich, Merck, Darmstadt, Germany) and differential centrifugation was performed to obtain the post-mitochondrial fraction, according to the method de- scribed by Uwe and Von Hagen [22]. The supernatant was dispensed in aliquots of 1 mL, which were stored at −80 °C. In these homogenates the oxidative damage to lipids and proteins was measured. and approximately 100 μL of blood was extracted by the retro-orbital plexus, which was added the ethylenediamine- tetraacetic acid (EDTA) anticoagulant solution (Sigma-Al- drich, Merck, Darmstadt, Germany). This sample was analyzed in the optical microscope (Olympus, Tokyo, Japan), with the objective of 100 × immersions, and 100 cells were observed to calculate the percentages corre- sponding to the nuclear polymorphs (NPM) and the lym- phocytes. The rest of the blood was then collected without EDTA (Sigma-Aldrich, Merck, Darmstadt, Germany) to ob- tain the serum. In the serum sample collected, the concen- tration of triglycerides was measured with the Monotriglitest reagent kit (HELFA® Diagnostics, La Habana, Cuba). Behavioral tasks As a result of the tasks that were applied to evaluate the cognitive function, it was obtained in the Y maze that there were significant differences in relation to the num- ber of alternations obtained in the different experimental times (Fig. 2a). The differences were found between the groups of animals 8–9, 12–13 and 16–17 months of age, in relation to young animals group. In addition, signifi- cant differences were found in the number of errors (incorrect entries to the arms of the Y maze) where the group of 12–13 months of age differed statistically from the rest of the groups. The percentage of alternation was significantly lower in the animals from 12 to 13 and 16–17 months of age than in the animals from 8 to 9 and 4–5 months of age (Fig. 2b). One of the hematological parameters analyzed was the population of lymphocytes that in the group of 12–13 month of age decreased significantly in relation to the young animals (Fig. 4a). On the other hand, the popula- tion of nuclear polymorphs (Fig. 4b) in animals 12–13 months of age was significantly higher than in animals from 4 to 5 months of age. The concentration of trigly- cerides in the serum showed a significant decrease in the group of 12–13 month of age in comparison with the animals of 4–5 months of age (Fig. 4c). Also in these male BALB/ccenp mice at the age of 12– 13 months, the oxidative damage to the lipids in the brain homogenate had an increase nevertheless those there wasn’t a significant difference in relation to the animals of 4–5 months of age (Fig. 5a). On the other hand, oxidative damage to proteins showed a significant increase in animals of 12–13 compared to animals of 4–5 months of age (Fig. 5b). The other behavioral task used to evaluate if the natur- ally aged BALB/ccenp mice had cognitive impairment was the object recognition. In this task there were ob- tained significant differences between the PI by FO and NO in each of the ages of the animals evaluated, except for mice of 16–17 months of age (Fig. 3). In the mice of 4–5 and 8–9 months of age PI by NO was significantly It was found that weight of brain in mice of 16–17 months of age was significantly lower than in the control mice (Fig. 6a). Histological studies Histological analyzes were performed in 5 young animals (4–5 months of age) and 5 aged animals (16–17 months of age). The animals were sacrificed by cervical disloca- tion and the brain, thymus, spleen, liver, kidneys and lungs were colected. Organs were rinsed with cold 0.9% NaCl solution (Sigma-Aldrich, Merck, Darmstadt, Germany) (2–8 °C) to eliminate rest of blood, dried with filter paper and were weighted using an analytic balance Fig. 1 Body weight of male BALB/ccenp mice up to 16–17 months of age. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age) Fig. 1 Body weight of male BALB/ccenp mice up to 16–17 months of age. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age) Page 4 of 11 Esquivel et al. Laboratory Animal Research (2020) 36:7 Esquivel et al. Laboratory Animal Research (2020) 36:7 Esquivel et al. Laboratory Animal Research the aged mice groups there were no statistically signifi- cant differences. higher than by FO. However, in group of 12–13 months of age PI was significantly higher for FO than for NO. Behavioral tasks In the case of the thymus, the weight was Fig. 2 Y maze task of male BALB/ccenp mice up to16–17 months of age. a Number of alternations and wrongs when that the animal visit each arm. b Percentage of alternation. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age). + indicates P < 0.05 compared to group of 8–9 months of age. ^ indicates P < 0.05 compared to group of 12–13 months of age Fig. 2 Y maze task of male BALB/ccenp mice up to16–17 months of age. a Number of alternations and wrongs when that the animal visit each arm. b Percentage of alternation. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age). + Fig. 2 Y maze task of male BALB/ccenp mice up to16–17 months of age. a Number of alternations and wrongs when that the animal visit each arm. b Percentage of alternation. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age). + indicates P < 0.05 compared to group of 8–9 months of age. ^ indicates P < 0.05 compared to group of 12–13 months of age Esquivel et al. Laboratory Animal Research (2020) 36:7 Page 5 of 11 Esquivel et al. Laboratory Animal Research Fig. 3 PI (preference index) for FO (familiar object) and NO (novel object) in the object recognition task of male BALB/ccenp mice up to 16–17 months of age. Data shown are the means±SD. * indicates P < 0.05 compared to FO at each group ig. 3 PI (preference index) for FO (familiar object) and NO (novel object) in the object recognition task of male BALB/ccenp m months of age. Data shown are the means±SD. * indicates P < 0.05 compared to FO at each group Fig. 3 PI (preference index) for FO (familiar object) and NO (novel object) in the object recognition t months of age. Data shown are the means±SD. * indicates P < 0.05 compared to FO at each group Fig. 3 PI (preference index) for FO (familiar object) and NO (novel object) in the object recognition task of male BALB/ccenp mice up to 16–17 months of age. Data shown are the means±SD. Behavioral tasks * indicates P < 0.05 compared to FO at each group significant differences between the experimental groups (Fig. 6f). significantly lower in the animals at the age of 16–17 months than in the controls group (4–5 months of age) (Fig. 6b). On the other hand thymus weight was 22% lower than the weight in the control animals. As a result of the comparison between the groups for weight of spleen, lungs and kidneys, was obtained a significant in- crease in the animals of 16–17 months of age in relation to control group (Fig. 6c-g). Liver weight didn’t show Histological studies When the mice were 8–9 months of age the aggres- siveness reached the greatest dimensions during the ex- perimental period. In consequence of this situation mice were housed individually. In accordance with several studies, an increase in mice body weight until 8–9 months of age was observed and after this experi- mental time body weight then remains constant (Fig. 1) [27–29]. Few liver glycogen was observed in the liver of 20% of young animals. However, the most relevant alter- ations occurred in 60% of the aged animals and were the discrete perivascular lymphocytic infiltration (Fig. 8a) and focal lipidosis (Fig. 8b). Steatosis was present in 60% of the animals in the group of 16–17 months of aged. The results obtained in the Y-maze test showed the superiority for the control group in the number of alter- nations (Fig. 2a) in relation to animals of 8–9, 12–13 and 16–17 months of age. Significant decrease in the percentage of alternation (Fig. 2b) was found in the ani- mals from 12 to 13 and from 16 to 17 months of age. These results suggest that aged animals, from 12 to 13 months of age, show a decrease in the ability to orient themselves in the space and they don’t show the natural instinct to explore new places that is reported for young male mice of 9 weeks of age without being subjected to any drug or treatment [19, 30]. Therefore, based on the mechanism of memory that is evaluated in the Y-maze test, it can be affirmed that male BALB/ccenp mice since aged of 12–13 months show cognitive impairment that appears in dementia. The findings observed in the kidney and in the brain were only in the group of animals of 16–17 months of age. In the case of kidney, the most important damage presented was a glomerular degeneration, characterized by a decrease in its capillaries (Fig. 9a). In the case of brain, in 40% of animals aged 16–17 months, an area with degeneration of the granular layer of the cerebellum was observed (Fig. 10). On the other hand, the morpho- logical characteristics of the hippocampus were normal for both young control animals and those aged 16–17 months of age. The results obtained in the object recognition test (Fig. Histological studies Among the histological studies, the morphology of the lung was evaluated and inflammatory infiltrative alter- ations and aberrations of cell growth were observed. The most visible finding was the thickening of the alveolar Fig. 4 Hematological parameters and triglyceride concentration in male BALB/ccenp mice up to 12–13 months of age. a Lymphocytes. b Nuclear polymorphs. c Triglycerides. Data (n = 10) shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age) Fig. 4 Hematological parameters and triglyceride concentration in male BALB/ccenp mice up to 12–13 months of age. a Lymphocytes. b Nuclear polymorphs. c Triglycerides. Data (n = 10) shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age) Esquivel et al. Laboratory Animal Research (2020) 36:7 Esquivel et al. Laboratory Animal Research Page 6 of 11 (2020) 36:7 Fig. 5 Oxidative damage in brain homogenate of male BALB/ccenp mice up 12–13 months of age. a TBARS (thiobarbituric acid reactive substances) as an indicator of damage to lipids. b Carbonylated proteins as an indicator of damage to proteins. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age). + indicates P < 0.05 compared to group of 8–9 months of age Fig. 5 Oxidative damage in brain homogenate of male BALB/ccenp mice up 12–13 months of age. a TBARS (thiobarbituric acid reactive substances) as an indicator of damage to lipids. b Carbonylated proteins as an indicator of damage to proteins. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age). + indicates P < 0.05 compared to group of 8–9 months of age that the male BALB/c are the most aggressive strain of mice and those with greater difficulty to adapt to group housing [26]. septa, characterized by a minimal infiltration of lympho- cytes and few macrophages; it was presented in 40% of the animals of the group aged of 16–17 months of age and in 20% of those of the young group. In addition, in 40% of the aged animals, anaplasias with a focal charac- ter were observed in the lung, well delimited, rounded, characterized by cellular polymorphism, presence of mi- totic details in the nuclei of some cells of the periphery and poor vascularization (Fig. 7). Histological studies 3) allow us to affirm that when the animals are 12–13 and 16–17 months of age, the motivation to explore new objects isn’t activated correctly, which in- dicates the appearance of cognitive impairment [31]. The fact that in this work the cognitive impairment of BALB/ccenp aged mice is demonstrated with the application of two behavioural task, Y maze and ob- ject recognition test (Figs. 2 and 3) that are related to Discussion Percent survival obtained at the end of the experimental procedure and the number of mice sacrificed due to their deteriorated state of health, were due to the fre- quent fights produced between the animals of the same cage. Although there isn’t reference about the survival percentage of BALB/c aged mice. However the occur- rence of frequent brawls in the cages is due to the fact Esquivel et al. Laboratory Animal Research (2020) 36:7 Esquivel et al. Laboratory Animal Research Page 7 of 11 Fig. 6 Weight of the organs relative to the body weight of male BALB/ccenp mice up to 16–17 months of age. a Brain weight. b Thymus weight. c Spleen weight. d Lungs weight. e Kidneys weight. f Liver weight. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age). + indicates P < 0.05 compared to group of 8–9 months of age. ^ indicates P < 0.05 compared to group of 12– 13 months of age Fig. 6 Weight of the organs relative to the body weight of male BALB/ccenp mice up to 16–17 months of age. a Brain weight. b Thymus weight. c Spleen weight. d Lungs weight. e Kidneys weight. f Liver weight. Data shown are the means±SD. * indicates P < 0.05 compared to young control group (4–5 months of age). + indicates P < 0.05 compared to group of 8–9 months of age. ^ indicates P < 0.05 compared to group of 12– 13 months of age hippocampal functionality [32], demonstrates the use- fulness of this animal model for the study of demen- tia and AD. myeloid progenitors to the detriment of lymphoid. In the case of the lymphoid fraction of the animals from 12 to 13 months of age (Fig. 4a), it supposes that these decreases are related to the thymus involution that occurs during aging, and in particular, the progressive loss of the thymic epithelial space, where the thymopoiesis takes place [34]. Contrary with the reported by literature about the changes in the population of NPM (Fig. 4b) [33], it was found that in the mice from 12 to 13 months of age there was a significant increase compared to the young control animals. Discussion However according to Shaw et al., [34] the bal- ance between the generation of myeloid lineages (mono- cytes and granular leukocytes, such as NPM, neutrophils, basophils and eosinophils) and lymphoid (T, B and NK lymphocytes), characteristic of young individuals, is lost with aging (Fig. 4a, b), showing a tendency towards In the case of the thymus weight, this behaviour has been corroborated in naturally aged mice, specifically BALB/c, where the involution of the organ started from 12 weeks of age (3 months). These authors also found that at 35 weeks (8–9 months) the weight decreased by 45% in relation to the weight of the organ when the animal was 6 weeks old [35]. Esquivel et al. Laboratory Animal Research (2020) 36:7 Page 8 of 11 Fig. 7 Damages presented in lung of BALB/ccenp male mice of 16–17 months of age a Focal lesion with tumor appearance present in the lungs of mice of 16–17 months of age. b Lungs of control group (4–5 months of age). HE 100× Fig. 7 Damages presented in lung of BALB/ccenp male mice of 16–17 months of age a Focal lesion with tumor appearance present in the lungs of mice of 16–17 months of age. b Lungs of control group (4–5 months of age). HE 100× In agreement with studies in CBA/C a mice and in rats, our data reveal a strong increase in spleen weight (Fig. 6c) until the age of 16–17 months [36]. Some stud- ies have shown age-related white pulp atrophy in the spleen of Sprague–Dawley rats which correlate with a decrease of greater than 80% in lymphocytes number with aging [37]. The results obtained in this work with relation to weight of thymus and spleen (Fig. 6b, c) may explain the decrease in the lymphocyte population obtained in aged mice (Fig. 4a) because in these organs take place lymphocyte production. The result obtained in this work about serum triglycer- ides concentration (Fig. 4c) is agree with those obtained by Araki et al. [38]. According to these authors, in aged ani- mals the mobilization of lipids deteriorates with age due to the decrease in the gene expression of Apos. This leads in the long term to an excessive accumulation of lipids in tis- sues such as the liver, resulting in a higher incidence of age-related diseases and in particular dementia. Discussion Morever, the focal lipidosis in liver obtained in BALB/ccenp mice at 16–17 month of age (Fig. 8b) suggest the presence of an in- crease of circulating lipids that is very possible to include Fig. 8 Damages presented in liver of BALB/ccenp male mice of 16–17 months of age. a Perivascular lymphocytic infiltration. HE 40×. b Macro focal and micro vesicular lipidosis. c Liver of control group (4–5 months of age). HE 200× Fig. 8 Damages presented in liver of BALB/ccenp male mice of 16–17 months of age. a and micro vesicular lipidosis. c Liver of control group (4–5 months of age). HE 200× Fig. 8 Damages presented in liver of BALB/ccenp male mice of 16–17 months of age. a Perivascular lymphocytic infiltration. HE 40×. b Macro focal and micro vesicular lipidosis. c Liver of control group (4–5 months of age). HE 200× Fig. 8 Damages presented in liver of BALB/ccenp male mice of 16–17 months of age. a Perivascular lymphocytic infiltration. HE 40×. b Macro focal and micro vesicular lipidosis. c Liver of control group (4–5 months of age). HE 200× Esquivel et al. Laboratory Animal Research (2020) 36:7 Page 9 of 11 Fig. 9 Damages presented in kidney of BALB/ccenp male mice of 16–17 months of age. a Degeneration of the glomeruli. HE 200X. b Kidney of control group (4–5 months of age). HE 200× Fig. 9 Damages presented in kidney of BALB/ccenp male mice of 16–17 months of age. a Degeneration of the glomeruli. HE 200X. b Kidney of control group (4–5 months of age) HE 200× ig. 9 Damages presented in kidney of BALB/ccenp male mice of 16–17 months of age. a Degeneration of the glomeruli. HE ontrol group (4–5 months of age). HE 200× vulnerable to attack by lipid membranes, proteins, RNA and nuclear DNA [39]. triglycerides. In the liver, aging causes an increase in lipid accumulation (micro and macro vesicular lipidosis) as a result of multiple alterations in lipid metabolism, among which are a lower β-oxidation and/or an increase in de novo synthesis [36]. Therefore, steatosis found in patho- logical studies is likely to be associated with the renal aging process and not with AD. In the case of the finding of the decrease in brain size in mice aged 16–17 months (Fig. Discussion The fact that the cerebellum suffers the effects of this neurodegenerative pathology may be a side effect of the disease, or on the contrary, have a more specific im- portance in the degeneration process, or in its clinical consequences. many study references were found in mice, Jacobs et al. [42] demonstrated that transgenic mouse models carry- ing AD mutations (AbetaPP / PS1, APPswe / PS1dE9) have evidenced the role for the cerebellum in the patho- physiology of sporadic AD and its clinical manifesta- tions. Early onset AD, especially from presenilin 1 mutations, exhibits cerebellar motor phenomena such as ataxia, as well as myoclonus or extrapyramidal symp- toms. The fact that the cerebellum suffers the effects of this neurodegenerative pathology may be a side effect of the disease, or on the contrary, have a more specific im- portance in the degeneration process, or in its clinical consequences. Acknowledgements g To professors Onelia Bárbara González Navarro and José Suarez Alba of the Preclinical Research Division in the Center for Genetic Engineering and Biotechnology (CIGB, Cuba) for their contribution in the processing of organs for histological studies. Similar to our results, a significant increase in kidney weight (Fig. 6e) has previously been observed with aging in rodents that may be a result of a fibroblast accumula- tion as noted previously in renal tissue [44]. Lim et al. [44] reported that associated with aging in mice there is an increase in mesangial expansion and tubule intersti- tial fibrosis that is accompanied by inflammation, apop- tosis and oxidative stress that together impair renal function. This deterioration in renal function was reflected through the glomerular degeneration found in the histology of this organ in BALB/ccenp aged mice in our study (Fig. 9a). It has long been believed that kidney function is linked to brain activity. Clinical studies dem- onstrate that patients with a low glomerular filtration rate or structural or functional renal damage (chronic kidney disease) are more prone to cognitive impairment and AD, and the degree of cognitive impairment is closely related to kidney disease progression and renal failure. However, the mechanisms behind this linkage are unclear. For these reasons this is another element Authors’ contributions Nashelly Esquivel Crespo and Yenela García Hernández contributed equally to this work. Nashelly Esquivel Crespo and Yenela García Hernández designed and performed experiment, organized and analyzed data, wrote and correct the manuscript. Claudio Rodríguez Martínez, Bestraida Lores Cintra and Mariví Gutiérrez Machado managed general research and drafting. All authors read and approved this final manuscript. Authors’ information h ll l Nashelly Esquivel: Research and especialist in the Laboratorio de Antianémicos y Nutracéuticos. Centro Nacional de Biopreparados. Abbreviations The significant increase in the weight of the lungs (Fig. 6d) obtained in aged animals to 16–17 months may be related to the tumor lesions observed in lung hist- ology in aged mice (Fig. 7). Lung disease in midlife may increase the risk of dementia. Many studies have showed that cognitive function is impaired in patients with lung disease with or without hypoxemia. Cerebral disturbance is noted in patients with lung disease and may be related to hypoxia in the brain [43]. Abbreviations AD: Alzheimer’s disease; ANOVA: One-way analysis of variance; EDTA: Ethylenediaminetetraacetic acid; FO: Familiar object; MDA: Malondialdehyde; NO: Novel object; PI: Preference index; SD: Standard deviation; TBARS: Thiobarbituric acid reactive substances AD: Alzheimer’s disease; ANOVA: One-way analysis of variance; EDTA: Ethylenediaminetetraacetic acid; FO: Familiar object; MDA: Malondialdehyde; NO: Novel object; PI: Preference index; SD: Standard deviation; TBARS: Thiobarbituric acid reactive substances Conclusions The models of aged mice described for dementia present variability about the minimum age at which behavioral, pathological or biochemical symptoms of the disease take place [13, 14]. In spite of this variability it’s more advantageous to use aged mice with the minimum pos- sible age because with very old animals the experiment becomes more expensive. In addition, the use of animals at the minimum age at which symptoms of dementia ap- pear has the advantage that allows reducing the occur- rence of fights among the groups that affect the health and welfare of animals. On the other hand at very ad- vanced ages, animals are less motivates to participate in behavioral tests and other pathologies associated with the aging process appear that could affect the usefulness of the animal model. Therefore, we consider that it’s a very useful option to use animals 12–13 months of age where there are symptoms of cognitive deficiency, histo- pathological and biochemical elements characteristic of dementia and which were described earlier in this research. The cerebellum has an important function in the ner- vous system since it is responsible for the modulation of the cognitive and emotional part, in addition to its func- tion in motor coordination. However, it is the first of these functions that has allowed in different studies the role of the cerebellum in neurodegenerative and neuro- psychiatric disorders as in AD [41]. It is known that the cerebellum contributes to the cognitive and neuro- psychiatric deficit in AD and in dementia in general. However, it is not clear in the literature whether a cerebellar lesion may be the cause or consequence of dementia [42]. Discussion 6a), there isn’t evidence about the weight behaviour of this organ in aged mice, but it has been found post-mortem that the size of the brains of people with AD is lower than that of healthy people of the same age [40]. The decrease in brain weight is associated with the neuronal death described in AD [5], therefore this result contribute to collect experimental evidence to use aged BALB/ccenp mice as model of dementia. The increase in oxidative damage to proteins in the brain homogenate of male BALB/ccenp mice of 12–13 months of age (Fig. 5) corresponds to the literature that describes it as a physiological event associated with the aging process [5] and is an element that supports the usefulness of these animals as a model of AD. In the pathophysiology of AD it’s known that oxidative stress plays an important role. The excess of free radicals and the special susceptibility of the nervous tissue to oxidative damage make it more The degeneration found in the granular layer of the cerebellum of aged animals (Fig. 10) corresponds to the structural changes that have been described in the brain in the presence of dementia and specifically of AD in humans and nonhuman primates [41]. Although not Fig. 10 Degeneration of cerebellum’s granular layer of BALB/ccenp male mice of 16–17 months of age. HE 200× Fig. 10 Degeneration of cerebellum’s granular layer of BALB/ccenp male mice of 16–17 months of age. HE 200× Page 10 of 11 Page 10 of 11 Esquivel et al. Laboratory Animal Research (2020) 36:7 Esquivel et al. Laboratory Animal Research (2020) 36:7 that supports the usefulness of BALB/ccenp aged mice of 12–13 month of age as an AD model. that supports the usefulness of BALB/ccenp aged mice of 12–13 month of age as an AD model. that supports the usefulness of BALB/ccenp aged mice of 12–13 month of age as an AD model. many study references were found in mice, Jacobs et al. [42] demonstrated that transgenic mouse models carry- ing AD mutations (AbetaPP / PS1, APPswe / PS1dE9) have evidenced the role for the cerebellum in the patho- physiology of sporadic AD and its clinical manifesta- tions. Early onset AD, especially from presenilin 1 mutations, exhibits cerebellar motor phenomena such as ataxia, as well as myoclonus or extrapyramidal symp- toms. Availability of data and materials Availability of data and materials The data that support the findings of this study are available on request from the corresponding author on reasonable request. The data that support the findings of this study are available on request from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. References Inbred mouse strain differences in the establishment of long-term fear memory. Behav Brain Res. 2003;140:97–106. 44. Lim JH, Kim EN, Kim MY, Chung S, Shin SJ, et al. Age-associated molecular changes in the kidney in aged mice. Oxidative Med Cell Longev. 2012;2012:1–10. 17. Brinks V, van der Mark M, de Kloet R, Oitzl M. Emotion and cognition in high and low stress sensitive mouse strains: a combined neuroendocrine and behavioral study in BALB/c and C57BL/6J mice. Front Behav Neurosci. 2007;1(8):1–12. https://doi.org/10.3389/neuro.08.008.2007. 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https://openalex.org/W3031470308	https://europepmc.org/articles/pmc7319350?pdf=render	English	null	Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites	PLOS computational biology/PLoS computational biology	2,020	cc-by	14,857	OPEN ACCESS Citation: Perlinska AP, Stasiulewicz A, Nawrocka EK, Kazimierczuk K, Setny P, Sulkowska JI (2020) Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites. PLoS Comput Biol 16(5): e1007904. https:// doi.org/10.1371/journal.pcbi.1007904 Editor: Rebecca C. Wade, Heidelberg Institute for Theoretical Studies (HITS gGmbH), GERMANY Received: October 7, 2019 Accepted: April 23, 2020 Published: May 26, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pcbi.1007904 PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY RESEARCH ARTICLE Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites Agata P. PerlinskaID1,2☯, Adam StasiulewiczID2,3☯, Ewa K. NawrockaID2,4, Krzysztof KazimierczukID2, Piotr SetnyID2, Joanna I. Sulkowska2,4* Agata P. PerlinskaID1,2☯, Adam StasiulewiczID2,3☯, Ewa K. NawrockaID2,4, Krzysztof KazimierczukID2, Piotr SetnyID2, Joanna I. Sulkowska2,4* 1 College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Warsaw, Poland, 2 Centre of New Technologies, University of Warsaw, Warsaw, Poland, 3 Faculty of Pharmacy, Medical University of Warsaw, Warsaw, Poland, 4 Faculty of Chemistry, University of Warsaw, Warsaw, Poland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * jsulkowska@cent.uw.edu.pl ☯These authors contributed equally to this work. * jsulkowska@cent.uw.edu.pl Abstract S-adenosylmethionine (SAM) is one of the most important enzyme substrates. It is vital for the function of various proteins, including large group of methyltransferases (MTs). Intrigu- ingly, some bacterial and eukaryotic MTs, while catalysing the same reaction, possess sig- nificantly different topologies, with the former being a knotted one. Here, we conducted a comprehensive analysis of SAM conformational space and factors that affect its vastness. We investigated SAM in two forms: free in water (via NMR studies and explicit solvent simulations) and bound to proteins (based on all data available in the PDB and on all-atom molecular dynamics simulations in water). We identified structural descriptors—angles which show the major differences in SAM conformation between unknotted and knotted methyltransferases. Moreover, we report that this is caused mainly by a characteristic for knotted MTs compact binding site formed by the knot and the presence of adenine-binding loop. Additionally, we elucidate conformational restrictions imposed on SAM molecules by other protein groups in comparison to conformational space in water. Introduction S-adenosylmethionine (SAM or AdoMet) is an ubiquitous molecule and the second most widely used enzyme substrate after ATP [1]. It is utilized in many different chemical reactions, including transfer of the methyl group. It acts as methyl donor for a variety of methyltrans- ferases (MTs), involved in methylation of small molecules [2], proteins [3], DNA [4], and RNA [5]. Interestingly, SAM binds not only to proteins but also to RNA—it is a substrate for riboswitches [6]. The diversity of molecules capable of binding SAM is great not only in a variety of per- formed functions, but also in their structure and topology. In the case of SAM-dependent methyltransferases, the proteins are divided into five classes, each with a different fold, includ- ing a knotted one [7]. The set of knotted methyltransferases (MTs) is the largest group of all known knotted proteins, which structures comprise now about 2% of PDB’s deposits. Interest- ingly, all of the known structures of knotted MTs possess the same type of the knotted region, which is a deep trefoil knot [8, 9]. The knot core is located in the middle of the protein chain and spans short part of the sequence (about 45 amino acids in the native conformation), thus is more compact than in most knotted proteins, and has long tails (Fig 1A). For example in family of knotted carbonic anhydrase—the knot core spans along almost all of the protein structure, but the active site is formed by only a small fraction of the protein (e.g. in protein with PDB id: 2hfx, length of the knot core is 232 aa). Similar situation is found in members of UCH family (e.g in the case of the protein with PDB id: 3kw5, the knot core covers 215 aa). The length of all of the knot cores in the full sequences of proteins can be found in the KnotProt. Competing interests: The authors have declared that no competing interests exist. Such type of knot cannot be untied by random thermal fluctuations [10]. Moreover, sur- prisingly—despite low sequence similarity (less than 20%) between different members of this class, the knotted region is conserved [10]. Even more surprising, the ligand binding site is par- tially formed by the knot core [11]. However, not only residues from the knotted region are responsible for ligand binding. differences between analogous, yet topologically different enzymes, as well as for future rational drug design. differences between analogous, yet topologically different enzymes, as well as for future rational drug design. Author summary The topology of a folded polypeptide chain has great impact on the resulting protein function and its interaction with ligands. Interestingly, topological constraints appear to affect binding of one of the most ubiquitous substrates in the cell, S-adenosylmethionine (SAM), to its target proteins. Here, we demonstrate how binding sites of specific proteins restrict SAM conformational freedom in comparison to its unbound state, with a special interest in proteins with non-trivial topology, including an exciting group of knotted methyltransferases. Using a vast array of computational methods combined with NMR experiments, we identify key structural features of knotted methyltransferases that impose unorthodox SAM conformations. We compare them with the characteristics of standard, unknotted SAM binding proteins. These results are significant for understanding Copyright: © 2020 Perlinska 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 input files for the MD simulation and the trajectory are available from the figshare service (https://doi.org/10.6084/m9. figshare.9944423). 1 / 25 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Funding: This work was supported by European Molecular Biology Organization installation grants (\#2057 to JIS, \#3051 to PS), Polish Ministry for Science and Higher Education (\#0003/ID3/2016/ 64 to JIS), National Science Centre (2018/29/N/ NZ1/02896 to APP). The contribution of EKN and KK is a part of "Methods of non-stationary signal processing for more sensitive NMR spectroscopy" project carried out within the FIRST TEAM programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Fig 1. Structures of knotted and unknotted analogous tRNA methyltransferases. (A) Knotted methyltransferase with a trefoil knot depicted with rainbow coloring (TrmD, PDB ID: 4yvg), (B) unknotted methyltransferase (Trm5, PDB ID: 2zzm). S-adenosylmethionine is shown with sticks i Fig 1. Structures of knotted and unknotted analogous tRNA methyltransferases. (A) Knotted methyltransferase with a trefoil knot depicted with rainbow coloring (TrmD, PDB ID: 4yvg), (B) unknotted methyltransferase (Trm5, PDB ID: 2zzm). S-adenosylmethionine is shown with sticks representation. https://doi.org/10.1371/journal.pcbi.1007904.g001 structural and chemical characteristics of the binding sites. These differences are a valuable input, for drug design in particular, since there are several knotted MTs essential for patho- genic bacteria, e.g. Haemophilus influenzae [17] or Pseudomonas aeruginosa [18]. Assessing the differences in the binding modes of SAM will allow designing novel antibiotics with poten- tially low toxicity and minor adverse effects. The selectivity of the new compound is crucial, since it has to bind and block the knotted (bacterial) active site and not the unknotted (eukary- otic) one. To date, there have been some attempts to exploit differences between the knotted TrmD and unknotted Trm5 proteins (Fig 1) [19, 20] but none resulted in a design of a selective inhibitor. Nonetheless, there is a constant need for a deeper understanding of SAM binding modes and conformational variability among different proteins, which is a determining factor for rational drug design. Working towards novel antibiotics is an exceptionally important task because of increasing antimicrobial resistance, and huge decline in the development of new antibacterial drugs since 2000 [21, 22]. Moreover, the presence of analogous proteins that are knotted and unknotted raises a ques- tion if there is a specific reason for existing such distinct proteins that perform exactly the same function? Even though, we know of analogous proteins created by convergent evolution [7, 23], knotted proteins do not seem to be potent candidates for the evolutionary selection. Especially, since knots are not frequently present in proteins, their appearance is considered statistically rare [10, 24, 25], and also there are examples of knotted proteins whose folding is slower than of their unknotted counterparts [26, 27] in the bulk. An in vivo study has shown that molecular chaperones can significantly speed up the knotting of a MT [28], and another study suggested important role of the ribosome [29, 30]. Nevertheless, knotted structures present challenges to current theories of protein folding [15, 15, 29, 31–35]. Introduction As the majority of knotted MTs are homodimers, the site is formed also by the amino acids from the other chain. Here, it is worth to mention that over 70% of knotted proteins are enzymes [12]. Also their enzymatic active sites are contained at least partially within the knotted core. On the top of this it was shown that the structure of the knotted core is crucial for the protein’s dimerization and activity [11, 13]. All of the above suggest the importance of the knotted region in these pro- teins, however its exact function remains unknown [14, 15]. Knotted methyltransferases are not the only group of knotted proteins that bind SAM. Knotted SAM synthases possess the same type of knot but, unlike in methyltransferases, it encompasses most of the protein (about 260 amino acids) and is shallow. Also, the conforma- tion of SAM differs, which suggests that the presence of its bent form observed in knotted MTs might be related to the position and the compactness of the knot. Our study aims at better understanding existence and function of knots in proteins from a new perspective—ligand binding. It is known that knotted MTs bind ligands in a different fashion than other SAM-dependent MTs [14, 16], however the reason for this distinction is not clear. To most of the proteins, SAM binds in an extended conformation, whereas to the knotted ones in a bent form. Different conformations of the ligand imply differences in both PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 2 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 1. Structures of knotted and unknotted analogous tRNA methyltransferases. (A) Knotted methyltransferase with a trefoil knot depicted with rainbow coloring (TrmD, PDB ID: 4yvg), (B) unknotted methyltransferase (Trm5, PDB ID: 2zzm). S-adenosylmethionine is shown with sticks representation. https://doi.org/10.1371/journal.pcbi.1007904.g001 Results and discussion We performed a comprehensive overview of the conformational space available for SAM in two different environments—in water, and bound to proteins that were divided based on their topology into knotted and unknotted ones. In order to compare and describe the ana- lyzed conformations, we considered two descriptors (Fig 2). The first one is the O4’-C1’- N9-C8 dihedral angle (glycosidic dihedral angle) that shows the spatial relation between ade- nine and ribose moieties. It is often used in characterization of the nucleotide conformations [37, 38] and differentiates between syn and anti conformations. We further divide anti con- formations into anti1 and anti2 (Fig 2B) to better describe the conformation of SAM bound in proteins. The second measure we use is the SD-O4’-N9 angle that we found to best differ- entiate between SAM conformations adopted in knotted (bent) and unknotted (extended) MTs (Fig 2C). These angles enable the characterization of two features of SAM, that show its overall conformation. Even though the role of the knot is still 3 / 25 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites undiscovered, its presence may bring some advantage to the protein, otherwise its conservation would be unfavorable [12, 36]. Despite the nature of this advantage remains unknown, in the case of MTs it may be related to the ligand binding and protein function since the knot is an essential part of the active site. By performing a comprehensive analysis of all available struc- tures of SAM-dependent methyltransferases, we are providing additional novel insights into the role of the knot in SAM binding. Because of its omnipresence in the three domains of life, and considerable medical signifi- cance, SAM is a key subject of scientific interest. In this project, we focused on understanding the variety of SAM conformations and factors that affect them. First, in order to determine how vast is SAM’s conformational space, we carried out NMR experiment and combined it with Molecular Dynamics (MD) simulation of SAM in water. Then, we analyzed the confor- mations of SAM bound to different proteins, with great emphasis on knotted and unknotted MTs, to establish: (1) the binding mode of SAM, (2) how the topology affects SAM’s confor- mation, (3) the part of the knotted protein that predetermines ligand conformation. Compared results of both parts elucidate the impact of binding site structure on SAM conformation by showing the differences in the restrictions put on the ligand by different types of proteins. SAM in water In search of all of the accessible conformations of the ligand in its free form in water we used both theoretical and experimental approach. We combined 2D ROESY data set with conform- ers generated using Molecular Dynamics (MD) simulations. NMR spectroscopy. We determined the conformation of SAM in solvent by conducting NMR experiments. Due to the pyramidal inversion process of a methyl group located on a sulfur atom, SAM in solution is present in two epimeric forms as (-)-SAM and (+)-SAM. We observed peak at 2.94 ppm for 25˚C that probably belongs to the second SAM epimer ((+)-SAM), present in a smaller amount than the dominant (-)-SAM epimer in the solution [39]. We based our analysis on the dominating (-)-SAM form. Peaks with chemical shifts of 6.05, 8.25, 8.39 ppm probably belong to (+)-SAM enantiomer or to degradation products of SAM. As a result, we obtained 39 interproton distances of SAM with respective error values. Pre- vious studies examined mostly the conformation of the ribose and its spatial relationship with adenine and reported no more than 14 distances [40, 41]. Our data provide a high resolution input for the calculations aimed at conformation prediction, including information of the rela- tive position between all of the moieties of SAM (Table 1), which was not available before. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 4 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 2. Structure and conformations of SAM. (A) Chemical structure of SAM with glycosidic dihedral angle (O4’-C1’-N9-C8) shown with green line and the SD-O4’-N9 angle with dashed circles, (B) syn, anti1, anti2 conformations about the glycosidic angle (arrows show O4’, C1’, N9 and C8 positions), (C) bent (green) and extended (cyan) conformations (arrows show SD, O4’ and N9 positions). https://doi.org/10.1371/journal.pcbi.1007904.g002 Fig 2. Structure and conformations of SAM. (A) Chemical structure of SAM with glycosidic dihedral angle (O4’-C1’-N9-C8) shown with green line and the SD-O4’-N9 angle with dashed circles, (B) syn, anti1, anti2 conformations about the glycosidic angle (arrows show O4’, C1’, N9 and C8 positions), (C) bent (green) and extended (cyan) conformations (arrows show SD, O4’ and N9 positions). https://doi.org/10.1371/journal.pcbi.1007904.g002 https://doi.org/10.1371/journal.pcbi.1007904.g002 MD simulation. To investigate the conformations of SAM in its free form, we performed 1μs long simulation of SAM in water using AMBER99 force field with improved parameters for the ligand [42]. SAM in water Principal Component Analysis (PCA) of the trajectory shows that the rota- tion around the glycosidic angle has the biggest contribution to the ligand’s flexibility as it interchanges between syn and anti conformations. The syn conformation is more frequently present in the trajectory, which agrees with the recent results [41]. Conformation about the glycosidic angle does not correspond to the specific conformation of the methionine moiety as it is a flexible part of the molecule. Regardless of whether SAM is in syn or anti conformation the methionine moiety samples similar space. Overall, the simulation indicates great confor- mational freedom of the molecule, without any significant preference for adopting a specific conformation. We utilized conformers generated with MD in further analysis of ligand’s con- formation in water. Fitting conformations to NMR data. In order to refine the conformational distribution of SAM in its free form, we fitted the interproton distances measured with NMR to the PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 5 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Table 1. Interproton distances in SAM measured with NMR in 25˚C. Atom1 Atom2 Distance [Å] Error [Å] H3’ H5’ 2.10 0.14 H3’ H5” 2.26 0.20 H4’ H5’ 2.29 0.16 H4’ H5” 2.46 0.17 Hβ Hγ’ 2.57 0.18 Hα Hβ 2.70 0.19 H2’ H5” 2.78 0.19 H8 H1’ 2.82 0.19 Hγ’ H5” 2.82 0.19 Hβ Hγ 2.88 0.20 methyl group Hβ 2.91 0.20 Hγ H5” 2.99 0.21 Hα Hγ 2.99 0.21 H1’ H4’ 3.02 0.21 methyl group Hγ’ 3.14 0.22 methyl group H5’ 3.14 0.22 Hγ’ H4’ 3.16 0.22 Hγ’ H5’ 3.22 0.22 methyl group Hγ 3.23 0.22 H3’ H2 3.23 0.22 Hγ H5’ 3.27 0.23 Hβ H5” 3.30 0.23 Hγ H4’ 3.50 0.24 H8 H5” 3.52 0.25 H4’ H8 3.54 0.34 methyl group H4’ 3.56 0.25 Hβ H5’ 3.60 0.25 methyl group H5” 3.61 0.25 methyl group H3’ 3.74 0.27 H2’ H8 3.79 0.26 Hα Hγ’ 3.79 0.26 methyl group H1’ 4.18 0.32 H2 H2’ 4.29 0.39 methyl group Hα 4.32 0.31 Hβ H2 4.32 0.30 H2 methyl group 5.16 0.38 H2 H1’ 5.22 0.50 H8 Hβ 5.34 0.38 H8 H3’ 5.82 0.41 https://doi.org/10.1371/journal.pcbi.1007904.t001 Table 1. Interproton distances in SAM measured with NMR in 25˚C. https://doi.org/10.1371/journal.pcbi.1007904.t001 representative conformations of SAM from MD simulation. SAM in water As a measure of the fit we used weighted RMSD of the interatomic distances—the weights were derived from the error for a given distance measured with the NMR (ei) and were taken as proportional to its square inverse wi ¼ e2 i for the i-th distance. Given that a single conformation cannot reproduce the NMR distances well, we utilized combinations of representative structures (centroids) for selected clusters. We tested every combination of clusters (up to 4 clusters) with their popula- tions varying from 0% to 100%. Then we calculated the RMSD between distances obtained representative conformations of SAM from MD simulation. As a measure of the fit we used weighted RMSD of the interatomic distances—the weights were derived from the error for a given distance measured with the NMR (ei) and were taken as proportional to its square inverse wi ¼ e2 i for the i-th distance. Given that a single conformation cannot reproduce the NMR distances well, we utilized combinations of representative structures (centroids) for selected clusters. We tested every combination of clusters (up to 4 clusters) with their popula- tions varying from 0% to 100%. Then we calculated the RMSD between distances obtained representative conformations of SAM from MD simulation. As a measure of the fit we used weighted RMSD of the interatomic distances—the weights were derived from the error for a given distance measured with the NMR (ei) and were taken as proportional to its square inverse wi ¼ e2 i for the i-th distance. Given that a single conformation cannot reproduce the NMR distances well, we utilized combinations of representative structures (centroids) for selected clusters. We tested every combination of clusters (up to 4 clusters) with their popula- tions varying from 0% to 100%. Then we calculated the RMSD between distances obtained PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 6 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 3. Conformations of SAM in water based on MD-derived structures fitted to NMR data. In 80% of the calculated cluster population SAM is in syn conformation about the glycosidic angle. The remaining 20% is in the anti1 conformation. The extended methionine moiety is present in 70% of the conformer populations and its conformation resembles SAM bound to unknotted methyltransferases. SAM in water The conformations are shown from two angles and are superimposed on C5’, C4’, O4’, C1’ and N9. https://doi.org/10.1371/journal.pcbi.1007904.g003 https://doi.org/10.1371/journal.pcbi.1007904.g003 upon r−6 averaging of clusters contributions and NMR data. Clustering of the MD simulation with RMSD cutoff of 1.25Å resulted in 90 clusters representing the conformational ensemble of SAM in water. We achieved the best fit to the NMR distances with 4 conformations with RMSD of 0.49Å (Fig 3). The resulting cluster contributions do not agree well with populations estimated based on MD simulations. This shows, that even though the applied force field allows sampling of relevant conformations of SAM, the correct reproduction of their probabil- ity distribution is much more difficult. The best fitted conformations are mostly syn about the glycosidic angle and account for 80% of the calculated cluster population. From anti conformations only anti1 is present (20%) The best fitted conformations are mostly syn about the glycosidic angle and account for 80% of the calculated cluster population. From anti conformations only anti1 is present (20%) —none of the best fitted conformers has anti2 conformation. The extended methionine moiety is present in 70% of the conformer populations and bent in 30%. We can further divide bent conformations based on the propensity of the carboxyl group of the methionine moiety to face the ribose hydroxyl groups. In 20% of the calculated cluster population methionine moiety bends away from the ribose hydroxyls (gray in Fig 3), and in the remaining 10% they are close together (pink in Fig 3). It is worth noting, that such conformations of the methionine moiety are similar to those present in SAM bound to various proteins. The extended conformation is common for the majority of unknotted MTs, the conformation with methionine bent away from the hydroxyls is frequently present in unknotted histone-lysine N-methyltransferases and the conformation where the two groups are facing each other is favored in knotted MTs. Overall, the prevailing conformation of SAM (with a population of 50%) is the extended syn conformation. All of the above shows that the conformation of SAM is unrestricted in water. The ligand samples both syn and anti conformers, as well as the extended and bent forms. SAM in water In 20% methionine moiety bends away from the hydroxyl groups of the ribose (colored with gray), similarly as in unknotted histone-lysine N-methyltransferases, whereas in rest 10% the two groups are close together (colored with pink) as in knotted methyltransferases. The prevailing conformation of SAM (colored with teal) is characterized by syn conformation about the glycosidic angle and extended methionine moiety. The conformations are shown from two angles and are superimposed on C5’, C4’, O4’, C1’ and N9. SAM in water based on MD-derived structures fitted to NMR data. In 80% of the calculated cluster population SAM is in Fig 3. Conformations of SAM in water based on MD-derived structures fitted to NMR data. In 80% of the calculated cluster population SAM is in syn conformation about the glycosidic angle. The remaining 20% is in the anti1 conformation. The extended methionine moiety is present in 70% of the conformer populations and its conformation resembles SAM bound to unknotted methyltransferases. In 20% methionine moiety bends away from the hydroxyl groups of the ribose (colored with gray), similarly as in unknotted histone-lysine N-methyltransferases, whereas in rest 10% the two groups are close together (colored with pink) as in knotted methyltransferases. The prevailing conformation of SAM (colored with teal) is characterized by syn conformation about the glycosidic angle and extended methionine moiety. The conformations are shown from two angles and are superimposed on C5’, C4’, O4’, C1’ and N9. Fig 3. Conformations of SAM in water based on MD-derived structures fitted to NMR data. In 80% of the calculate rmations of SAM in water based on MD-derived structures f Fig 3. Conformations of SAM in water based on MD-derived structures fitted to NMR data. In 80% of the calculated cluster population SAM is in syn conformation about the glycosidic angle. The remaining 20% is in the anti1 conformation. The extended methionine moiety is present in 70% of the conformer populations and its conformation resembles SAM bound to unknotted methyltransferases. In 20% methionine moiety bends away from the hydroxyl groups of the ribose (colored with gray), similarly as in unknotted histone-lysine N-methyltransferases, whereas in rest 10% the two groups are close together (colored with pink) as in knotted methyltransferases. The prevailing conformation of SAM (colored with teal) is characterized by syn conformation about the glycosidic angle and extended methionine moiety. https://doi.org/10.1371/journal.pcbi.1007904.g003 Protein-bound conformations In protein complexes, the conformational variety of SAM should be greatly limited in compar- ison to water condition. We address that issue by conducting a survey of all SAM-binding pro- teins from the RCSB Protein Data Bank (PDB) with emphasis on the type of restrictions they Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 7 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites put on the ligand. Moreover, we divide the proteins based on their function and topology, with the focus on the knotted and unknotted methyltransferases, in order to verify if the range of SAM conformations depend on the protein type. Additionally, to explore conformational free- dom of SAM, we conducted full-atom explicit solvent MD simulations of whole complexes (protein-SAM-tRNA) of two analogous MTs—knotted TrmD and unknotted Trm5. The sim- ulations were extensive—we performed 9 independent simulations of TrmD complex (jointly 3.6 μs) and 10 of Trm5 complex (jointly 4 μs). Representative conformations of SAM in methyltransferases. We extracted all confor- mations of SAM associated with knotted and unknotted methyltransferases (MTs) from struc- tures deposited in PDB. We obtained the information about the presence of the knot from KnotProt 2.0 database [9]. Using RMSD clustering we got representative conformations of SAM bound to each group and analyzed the differences between them. Conformations of SAM in knotted MTs can be represented by two clusters (Fig 4A). The knotted active site has high structural conservation and it provides a compact cavity for SAM’s adenosine moiety. Therefore, the conformational freedom of bound ligand is limited to the flexibility of the methionine moiety. Based on its relative position to adenosine we can divide SAM conformations into bent and extended. For TrmD and Nep1 proteins the whole com- plexes (protein-cofactor-substrate) are available, in which the ligand is in bent conformation, suggesting that this form is biologically active [43, 44]. In case of TrmD, it was shown that SAM in extended conformation would cause a steric clash with the target nucleobase [11]. Therefore, since the bent conformation is more frequently observed, it is considered character- istic for this group. However, both bent and extended conformations are present in knotted MTs. Interestingly, we found that methyltransferase RlmN with unknotted active site binds SAM in similarly bent fashion (Fig 4C). Protein-bound conformations RlmN is a part of radical SAM protein family that bind iron-sulfur cluster that directly interacts with SAM through its carboxyl and amino moie- ties, which stabilizes the ligand in the bent conformation [45]. This shows, that the bent con- formation is not exclusive for the knotted active site and it can also be found in different proteins. The second conformation present in knotted MTs has extended methionine moiety. Inter- estingly, it differs from the one bound to the unknotted proteins that is also extended (S1 Fig). The conformation retains the SD-O4’-N9 angle characteristic for SAMs from the knotted MTs Fig 4. Representative conformations of SAM from PDB structures, (A) SAM from knotted methyltransferases, (B) SAM from unknotted methyltransferases, (C) superposition of the bent conformations of SAM from knotted MTs (green) and unknotted MTs (blue). The conformations are superimposed on C5’, C4’, O4’, C1’ and N9. https://doi org/10 1371/journal pcbi 1007904 g004 Fig 4. Representative conformations of SAM from PDB structures, (A) SAM from knotted methyltransferases, (B) SAM from unknotted methyltransferases, (C) superposition of the bent conformations of SAM from knotted MTs (green) and unknotted MTs (blue). The conformations are superimposed on C5’, C4’, O4’, C1’ and N9. Fig 4. Representative conformations of SAM from PDB structures, (A) SAM from knotted methyltransferases, (B) SAM from unknotted methyltransferases, (C) superposition of the bent conformations of SAM from knotted MTs (green) and unknotted MTs (blue). The conformations are superimposed on C5’, C4’, O4’, C1’ and N9. htt //d i /10 1371/j l bi 1007904 004 https://doi.org/10.1371/journal.pcbi.1007904.g004 8 / 25 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites but has clearly extended methionine moiety. There are 4 (out of 20) knotted structures depos- ited in PDB with SAM bound in this form—2 of them are proteins responsible for methylation of adenosine(1067)-2’-O in 23S rRNA (PDB ids: 3gyq, 3nk7). These methyltransferases are essential for the bacterial resistance to antibiotics—to thiostrepton (TSR—thiostrepton-resis- tance methyltransferase) and nosiheptide (NHR—nosiheptide-resistance methyltransferase) [46, 47]. In both complexes, the extended conformation is stabilized by the interaction between the carboxyl and the amino group of SAM with 3 amino acids (two arginines and glutamic acid). Mutation of those residues significantly decreases or abolishes NHR activity, which suggests the conformation is biologically active [47]. Protein-bound conformations Another structure with extended methio- nine moiety of SAM belongs to RsmE protein (PDB id: 5o96), which was deposited in PDB with eight chains [48]. Conformations of SAM vary between the chains from being bent to extended. Both forms are stabilized by protein-ligand interactions and without the informa- tion of the substrate (RNA) binding mode, it is difficult to indicate the biologically active one. Similar observations can also be drawn from other RsmE structures (PDB id: 2cx8, 5vm8). Moreover, we also found that the extended methionine moiety is present in knotted pro- teins as an alternative conformation. In TrmD protein the bent SAM is biologically active [11], however, in one TrmD structure (PDB id: 5wyr) ligand in one of the active sites is extended (sinefungin, inhibitor closely related to SAM). Currently, it is unknown whether this other conformation in this protein has any function. All of the above shows, that even though the bent conformation is the most common one in the structures of knotted MTs, it is not the only occurring conformation. Despite limited data regarding biological activity of specific SAM conformations, it is highly probable that knotted binding site allows for diversity in ligand’s methionine moiety and conformations other than bent can be active. The ensemble of SAM conformations in unknotted methyltransferases is represented by ten conformations (clusters) (Fig 4B). The conformations differ mostly in the position of the methionine and adenine moiety. Two clusters have syn conformation about the glycosidic angle, however, it should be considered as uncommon in complexes of methyltransferases since it represents about 1% of the structures. One cluster shows the bent conformation similar to the one from the knotted proteins. The most frequently observed SAM conformation in unknotted MTs has extended methionine moiety and anti1 conformation. The clusters from knotted MTs can be fitted to the NMR data representing an unbound SAM with 1.26Å RMSD and from the unknotted with 1.15Å. These results compared to the 0.49Å for the MD-derived clusters show that SAM in the bound form is more restricted and cannot access all the conformations available for the free form. Additionally, the clusters from knotted and unknotted MTs achieve similar fit even though they significantly differ in size (two clusters from knotted and ten clusters from unknotted MTs). Angle distributions. Among the knotted MTs, SAM bent conformation is the most fre- quent one, which suggests its greatest significance in biological terms. Protein-bound conformations By contrast, in unknot- ted MTs the extended SAM conformation is dominant. We sought features that could objectively differentiate these two conformations. Also, we wanted to evaluate whether SAM conformational variety differs, when compared between MTs and all proteins (all proteins include MTs and non-MT proteins). Therefore, we prepared angle distributions for chosen dihedral and plain angles in SAM molecules for SAM conformations extracted from PDB structures. We made these distributions for several sets, including unknotted proteins, knotted proteins, unknotted MTs, and knotted MTs. We compared those with angle distributions for unbound SAM conformations from MD simulations and with angle values calculated from NMR data. Additionally, we covered distributions for SAM conformations from MD simula- tions of a pair of analogous MTs—knotted TrmD and unknotted Trm5. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 9 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 5. Angle SD-O4’-N9 distribution. SAM conformations from proteins (red), from simulation in water (dashed black), from simulation in MT protein environment (green), and from NMR experiment (blue). SAM conformations from knotted (A) and unknotted proteins (B). We found out that angle SD-O4’-N9 is the best measure of SAM angulation. It could be used to differentiate SAM molecules bound to unknotted and knotted proteins. This angle clearly shows that for most of the unknotted, SAM adopts extended conformation, while in the knotted—bent conformation. We scaled values from NMR data to be comparable with angle distributions from PDB and MD. Fig 5. Angle SD-O4’-N9 distribution. SAM conformations from proteins (red), from simulation in water (dashed black), from simulation in MT protein environment (green), and from NMR experiment (blue). SAM conformations from knotted (A) and unknotted proteins (B). We found out that angle SD-O4’-N9 is the best measure of SAM angulation. It could be used to differentiate SAM molecules bound to unknotted and knotted proteins. This angle clearly shows that for most of the unknotted, SAM adopts extended conformation, while in the knotted—bent conformation. We scaled values from NMR data to be comparable with angle distributions from PDB and MD. https://doi.org/10.1371/journal.pcbi.1007904.g005 We found out that the best indicator differentiating SAM’s extended and bent conforma- tions is the value of angle SD-O4’-N9 (Fig 5). Differences in its distributions clearly show that the bent conformation is predominant for knotted proteins, while the extended conformation for the unknotted ones. Protein-bound conformations SAM conformations from knotted (A) and unknotted proteins (B), knotted (C) and unknotted MTs (D). This angle shows spatial arrangement of SAM adenine in relation to ribose moiety. Stick representations depict specific SAM conformations: syn (green), anti1 (teal), and anti2 (purple). We scaled values from NMR data to be comparable with angle distributions from PDB and MD. Fig 6. Distribution of angle values of dihedral O4’-C1’-N9-C8. SAM conformations from proteins or MTs (red), from simulation in water (dashed black), from simulation in MT protein environment (green), and from NMR experiment (blue). SAM conformations from knotted (A) and unknotted proteins (B), knotted (C) and unknotted MTs (D). This angle shows spatial arrangement of SAM adenine in relation to ribose moiety. Stick representations depict specific SAM conformations: syn (green), anti1 (teal), and anti2 (purple). We scaled values from NMR data to be comparable with angle distributions from PDB and MD. Fig 6. Distribution of angle values of dihedral O4’-C1’-N9-C8. SAM conformations from proteins or MTs (red), from simulation in water (dashed black), from simulation in MT protein environment (green), and from NMR experiment (blue). SAM conformations from knotted (A) and unknotted proteins (B), knotted (C) and unknotted MTs (D). This angle shows spatial arrangement of SAM adenine in relation to ribose moiety. Stick representations depict specific SAM conformations: syn (green), anti1 (teal), and anti2 (purple). We scaled values from NMR data to be comparable with angle distributions from PDB and MD. https://doi.org/10.1371/journal.pcbi.1007904.g006 https://doi.org/10.1371/journal.pcbi.1007904.g006 with Trm5 adopts both syn and anti (in this case anti2) conformations, similarly as in the struc- tures from PDB. Taking both angle SD-O4’-N9 and dihedral O4’-C1’-N9-C8 into account, SAM conforma- tions in knotted MTs can be well distinguished from those in other proteins (S2 Fig). Addi- tionally, combination of both angles allows to differentiate SAM between bound to knotted MTs and other knotted proteins. Based on PDB data, the first group adopts SD-O4’-N9 values between 80-125˚ and O4’-C1’-N9-C8 around -20˚ to 60˚. SAM conformational restriction in knotted methyltransferases. Since syn conforma- tion of SAM does not occur in knotted MTs, we wanted to know whether any particular part of the protein interferes with such conformation. We investigated in detail eight structures of knotted MTs. Protein-bound conformations For this angle, there are nearly no differences between MTs and the set of all proteins. Only knotted MTs require SD-O4’-N9 angle value that is outside the range vis- ited by SAM in solution. Thus, binding in knotted MTs occurs via induced fit mechanism, where the ligand has to adapt to the binding site as opposed to conformational selection mech- anism observed in unknotted MTs. Also, in the case of MD simulations of protein-SAM-tRNA complexes, the angle distributions allow to distinguish between SAM conformations from knotted and unknotted proteins. The other interesting angle is the dihedral O4’-C1’-N9-C8 (Fig 6). It can be used to differen- tiate syn, anti1, and anti2. Angles with values between –100˚ and –150˚ represent syn confor- mations. In proteins, syn SAMs are observed far less often than in water, where they seem to dominate. Interestingly, SAMs bound to knotted proteins do not adopt this conformation at all. Dihedrals with values between around –30˚ and 30˚ correspond to anti1, while those of 50˚ to 100˚—to anti2. Distributions for knotted proteins and knotted MTs differ with one peak around 75˚ to 100˚. This is the outcome of the presence of knotted SAM synthases in the first distribution. Also, SAM in unknotted MTs adopt anti2 conformation more often than anti1, in contrast to the exclusiveness of anti1 in knotted MTs. This is the result of the shape of knotted MTs SAM binding site, particularly the adenine-binding loop, which we discuss in detail below. SAM in simulation with TrmD adopts mainly anti1 conformation, as expected from a knotted MT. Interestingly, there is a small population of dihedral values between -50˚ and – 100˚. This is an intermediate state, indicating an attempt to change conformation from anti1 to syn. During a visual inspection of the simulation trajectory, we observed that the full transi- tion is impossible due to ligand’s steric clashes with the part of the knot. After being shortly in an unfavorable intermediate state, SAM returned to anti1 conformation. SAM in simulation PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 10 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 6. Distribution of angle values of dihedral O4’-C1’-N9-C8. SAM conformations from proteins or MTs (red), from simulation in water (dashed black), from simulation in MT protein environment (green), and from NMR experiment (blue). PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 Protein-bound conformations Because of its clear impact on SAM conformation, we looked into adenine-binding loop in knotted MTs. First, we investigated the structure of the loop. We superimposed the proteins by SAM ade- nines’ heavy atoms for eight protein-SAM complexes from aforementioned PDB structures (S3 Fig). We divided the proteins into two groups based on the length of the loop. The more numerous one, with five proteins, includes loops with the same length. Depending on how we define beginning and end of loop, this group is built with 10 to 12 amino acids. The second group also has equal length, in this case of 14 to 16 amino acids. We calculated RMSD for backbone of both these groups. Within the set of shorter loops, RMSD values range from 1.06 to 3.14 Å. For longer loops these values fall between 1.78 to 2.95 Å. Then, we superimposed loops in both groups by their main chains and once again calculated RMSD. This time for the shorter loops it varied from 0.72 to 1.96 Å, and for longer from 1.67 to 1.96 Å. These results show that adenine-binding loop’s structure and position in relation to SAM are very highly conserved features in knotted methyltransferases. Next we evaluated whether these loops’ geometry is unique or common among proteins. We looked for similarities between all proteins deposited in PDB and representative structures of two loops—one short (from PDB id: 4fak), and one long (from PDB id: 1x7p). For the latter loop we found 1629 similar fragments, for the former—17457. These results indicate that geometry of the adenine-binding loop is not a unique one. Therefore, the loops’ specific fea- tures, when it comes to SAM binding, should be sought elsewhere. We also inspected Rama- chandran plots for these eight loops. Nearly all amino acids have allowed conformations (S4 Fig). In order to evaluate sequence similarity among SAM binding sites of knotted MTs, we con- ducted sequence alignment for 20 selected, most possibly distinct knotted MTs (S5 Fig). When it comes to the sequence, the best preserved regions are the knot and the adenine-binding loop. The best preserved amino acid is a glycine inside the knot. It creates hydrogen bonds with SAM ribose fragment. The other highly conserved amino acids include Leu and Ile in the loop. Both take part in adenine binding. Protein-bound conformations We found that for these proteins, adenine’s arrangement typical for syn confor- mation is blocked mainly by amino acids in the adenine-binding loop from one side and by the knot from the other side (Fig 7). Also, possibly due to the compactness of the knotted binding site, methionine moiety is prone to bent towards adenine. Therefore, adenine cannot PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 11 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 7. Structure of knotted binding site with highlighted regions that prevent SAM (stick representation) from binding in syn conformation. The most important parts of the binding site, that impose anti conformation, are: adenine-binding loop (green), and part of the knot that sterically blocks adenine arrangement in syn conformation (red). Panel A shows superposition of 8 representative knotted MTs’ binding sites, superimposed on SAM ribose and adenine heavy atoms. Panel B depicts molecular surfaces of adenine binding loop and part of the knot, and shows the compactness of the binding site (PDB id: 2egv). https://doi.org/10.1371/journal.pcbi.1007904.g007 Fig 7. Structure of knotted binding site with highlighted regions that prevent SAM (stick representation) from binding in syn conformation. The most important parts of the binding site, that impose anti conformation, are: adenine-binding loop (green), and part of the knot that sterically blocks adenine arrangement in syn conformation (red). Panel A shows superposition of 8 representative knotted MTs’ binding sites, superimposed on SAM ribose and adenine heavy atoms. Panel B depicts molecular surfaces of adenine binding loop and part of the knot, and shows the compactness of the binding site (PDB id: 2egv). Fig 7. Structure of knotted binding site with highlighted regions that prevent SAM (stick representation) from binding in syn conformation. The most important parts of the binding site, that impose anti conformation, are: adenine-binding loop (green), and part of the knot that sterically blocks adenine arrangement in syn conformation (red). Panel A shows superposition of 8 representative knotted MTs’ binding sites, superimposed on SAM ribose and adenine heavy atoms. Panel B depicts molecular surfaces of adenine binding loop and part of the knot, and shows the compactness of the binding site (PDB id: 2egv). https://doi.org/10.1371/journal.pcbi.1007904.g007 https://doi.org/10.1371/journal.pcbi.1007904.g007 https://doi.org/10.1371/journal.pcbi.1007904.g007 https://doi.org/10.1371/journal.pcbi.1007904.g007 adopt syn conformation due to steric clashes. The adenine-binding loop is situated just after the knotted region, and is also a characteristic feature of knotted proteins’ binding site. Protein-bound conformations Despite the lack of unique structural features, the adenosine-binding loop is one of the most important parts of SAM binding site in knotted MTs. The lack of this loop in unknotted MTs suggests that the loop’s presence could be determined by the knot. To conclude, the loop’s structure is not unique, but its presence is. 12 / 25 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites SAM binding: Protein environment. Knowing that the knot and the adenine-binding loop appear to be crucial for SAM binding in knotted MTs, we investigated further SAM-pro- tein interactions among various protein groups (S6 Fig). SAM binding: Protein environment. Knowing that the knot and the adenine-binding loop appear to be crucial for SAM binding in knotted MTs, we investigated further SAM-pro- tein interactions among various protein groups (S6 Fig). We selected 12 representative structures of possibly most distinct knotted MTs that form dimers. While in PDB for some enzymes there are no structures with bound SAM, we also took into account those with S-adenosylhomocysteine (SAH), as its binding mode is nearly the same. SAM and SAH compounds were analyzed for their interactions with amino acids at the binding site, with the focus on three parts of ligands: adenine, ribose (Fig 8), and methionine or homocysteine (for convenience this part is referred to as “methionine chain”). Ribose part of SAM or SAH interacts mainly with Gly (11 out of 12 cases) and Leu (9/12). In 7 out of 12 structures both amino acids are involved. The adenine part usually forms interactions with Leu (8/12) and Ile (8/12). It is important to mention that these amino acids interact with both parts of the ligand using their main chains. Methionine chain has diverse but rather infrequent interactions. In some crystal structures, this part of the ligand is also truncated, so it is difficult to quantify and describe interactions in this SAM or SAH region, but generally they seem to correspond mainly to hydrogen bonds and salt bridges. We investigated 3 available structures of knotted MTs that occur in monomeric form. Here, we also accepted structures with SAH. Ribose interacts with Leu and Gly, nitrogen base with Leu and Lys. Methionine chain forms interactions mainly with Asp, and in 2 cases with Thr. https://doi.org/10.1371/journal.pcbi.1007904.g008 Protein-bound conformations For comparison, we looked into 20 chosen conformations of SAM or SAH in representative unknotted MT structures. In this set ribose interacts mainly with Glu (12/20) and Asp (7/20), and usually with 2 amino acids of the same type at one time: Glu (11/20), Asp (7/20). These amino acids were expected, as they are typical for canonical Rossmann β2-Asp/Glu motif [49]. Nitrogen base forms interactions with Asp (11/20), Ala (8/20), Leu (7/20), and Phe (6/20). Interactions of methionine chain are rather diverse. In 15 cases salt bridges are found. This part of the ligand interacts most commonly with Asp (13/20). SAM molecules in unknotted MTs exhibit considerable differences when compared to knotted MTs. Here, we see more interactions with the methionine chain, less with adenine, and interactions of ribose with acidic amino acids’ side chains. We found 11 PDB structures of knotted SAM synthases with cocrystallized SAM. This is the only knotted group with bound SAM apart from MTs that we encountered. As such, it is Fig 8. Most frequent interactions of specific amino acids with SAM or SAH ribose (A), and adenine (B). Here, we compared interactions in complexes of SAM or SAH with 20 unknotted MTs (blue), 12 knotted MT dimers (red), 3 knotted MT monomers (yellow), and 11 knotted SAM synthases (green). Fig 8. Most frequent interactions of specific amino acids with SAM or SAH ribose (A), and adenine (B). Here, we compared interactions in complexes of SAM or SAH with 20 unknotted MTs (blue), 12 knotted MT dimers (red), 3 knotted MT monomers (yellow), and 11 knotted SAM synthases (green). https://doi.org/10.1371/journal.pcbi.1007904.g008 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 13 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites important to know whether this group behaves similar to knotted MTs in terms of SAM-pro- tein interactions. In 10 out of 11 structures there is an interaction between ribose part of SAM and Asp side chain. In 9 out of 11 proteins ribose interacts with 2 Asp side chains. In all cases we observed π-π interactions of adenine, 9 of them with Phe, 2 with Tyr. In 9 structures we found also interactions between SAM nitrogen base and Arg main chain. The methionine moiety of SAM creates mostly salt bridges. This part of the ligand interacts primarily with side chains of Asp (11/11), Lys (9/11), Glu (8/11), and Gln (8/11). Protein-bound conformations Clearly, these interactions are dif- ferent from those of knotted MTs. They could be described as somehow similar to unknotted proteins interactions but should be treated as a separate group. The reason for the difference between knotted synthases and MTs lies probably in the size of the knot. In these synthases, the knotted region spans nearly the whole protein, and has almost no impact on the structure of the binding site. By contrast, in knotted MTs the knot is deep, and takes considerable part in forming the SAM binding site. Also, we observed another type of SAM conformation in unknotted histone-lysine N- methyltransferases. These conformations are considerably different than in other unknotted proteins. Their methionine moiety is characteristically contorted in a direction different than in most SAMs. We encountered similar conformations in MD simulation in water, although in those the methionine chain has an arrangement more similar to SAM bound with other unknotted proteins. This clearly shows that in some cases unknotted proteins are able to bind SAM in an unorthodox way, yet still considerably different than knotted MTs. SAM binding differs in knotted and unknotted MTs. The knotted conformations interact mainly with amino acids main chains, while the unknotted ones with side chains. The unknot- ted conformations seem to use their methionine chain more often, and to form multiple salt bridges. On the other hand, the knotted MTs create more interactions with SAM adenine (Fig 9). Knotted SAM synthases are a third group, but binding-wise more similar to unknotted MTs. It suggests that the deep trefoil knot and knot-dependent binding site structure are responsible for unique binding mode of SAM in knotted MTs. Our results explain findings of Chuang et. al., who showed that unknotting the TrmL MT via circular permutation impedes SAH binding [50]. In addition to differences in interactions between SAM in groups of knotted and unknotted MTs, we also analyzed conformational freedom of the ligand in both types of binding sites. Data obtained from MD simulations of SAM bound to MT-protein complexes show how dis- tinctively the ligand is maintained, further supporting our findings. Especially, SAM in knotted binding site differs from the one in unknotted site based on its configurational entropy (S4 Table). Moreover, the comparison of ligand’s flexibility in both sites clearly shows that the unknotted one offers more conformational freedom (Fig 10). Protein-bound conformations In particular, the mobility of SAM in Trm5 is not focused on a single part of the ligand, it is evenly distributed on the whole molecule. On the contrary, in knotted MT adenine and ribose moieties of SAM are the most stable parts of the ligand and methionine moiety is the one most mobile. The simulations also show, that methionine’s flexibility depends on the presence of the substrate (TrmD binds two ligands but only one tRNA [51]). The ligand associated with the tRNA (SAM A in Fig 10) is more stable than its counterpart from the other binding site (SAM B). Besides the amino acids involved in direct interactions with the ligand, there are other res- idues forming the binding site, but only few of them are invariant for various MTs [8, 52]. This is because the group of SAM-dependent MTs is composed of proteins differing both sequentially (even within each class) and structurally (S8 Fig, [7, 52]), which is the reason for the distinct binding motifs of SAM. However, the conservation of glycine appears to be a universal feature for SAM-dependent methyltransferases [7]. The unknotted proteins with the Rossmann Fold are known to possess a glycine-rich loop in the vicinity of the active site PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 14 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 9. Interactions of SAM at knotted (PDB id: 4fak) (A) and unknotted (PDB id: 3dmf) (B) MT binding site. This figure shows differences in binding modes of SAM in representative proteins. In unknotted MTs, SAM heavily utilizes its methionine moiety, its adenine has contact with a limited number of amino acids, and its ribose interacts with acidic amino acids. By contrast, SAM in knotted MTs forms interactions mainly using adenine moiety, which is tightly bound to adenine-binding loop. Ribose forms hydrogen bonds with Gly and Leu. In knotted MTs, methionine chain of SAM has much less contacts with amino acids. Therefore, it forms less interactions and is more loose. Fig 9. Interactions of SAM at knotted (PDB id: 4fak) (A) and unknotted (PDB id: 3dmf) (B) MT binding site. This figure shows differences in binding modes of SAM in representative proteins. In unknotted MTs, SAM heavily utilizes its methionine moiety, its adenine has contact with a limited number of amino acids, and its ribose interacts with acidic amino acids. Protein-bound conformations For example, in TrmD proteins the glutamic acid (position 110 in Fig 11) is highly conserved, possibly because of its role in maintaining the bent conformation of SAM. In the case of TrmL and RlmH it was suggested that glycine facilitate knotting [27]. All of the above show, that even universally conserved motifs can be resolved differently due to distinct protein settings. Protein-bound conformations Fig 11. Logo of the glycine motifs in knotted and unknotted MTs. Left panel represents 20 knotted proteins (Class IV; residue numbering is based on RlmH protein from Staphylococcus aureus, PDB id: 4fak, as in Fig 9), right panel 160 proteins with Rossmann Fold (Class I; residue numbering is based on RsmC protein from Thermus thermophilus, PDB id: 3dmf). Both motifs are based on the structural alignments of protein sequences with no more than 30% of sequence similarity. The motifs were visualized using WebLogo [55]. https://doi.org/10.1371/journal.pcbi.1007904.g011 [53, 54]. The glycines (G241, G243, G245 in case of RsmC protein from Thermus thermophi- lus, Fig 11) are separated by a single amino acid and the motif is preceded by highly con- served acid (either aspartic or glutamic). Similarly, knotted MTs have their glycines (G108, G112 in case of RlmH protein from Staphylococcus aureus) also positioned in the loop which is within the knot. However, here the residues determine the start and the end of the loop and are separated by at least a few amino acids (the length of the loop vary between different knotted MTs). Various types of amino acids are found in the glycine loop, depending on the specificity of the protein. For example, in TrmD proteins the glutamic acid (position 110 in Fig 11) is highly conserved, possibly because of its role in maintaining the bent conformation of SAM. In the case of TrmL and RlmH it was suggested that glycine facilitate knotting [27]. All of the above show, that even universally conserved motifs can be resolved differently due to distinct protein settings. [53, 54]. The glycines (G241, G243, G245 in case of RsmC protein from Thermus thermophi- lus, Fig 11) are separated by a single amino acid and the motif is preceded by highly con- served acid (either aspartic or glutamic). Similarly, knotted MTs have their glycines (G108, G112 in case of RlmH protein from Staphylococcus aureus) also positioned in the loop which is within the knot. However, here the residues determine the start and the end of the loop and are separated by at least a few amino acids (the length of the loop vary between different knotted MTs). Various types of amino acids are found in the glycine loop, depending on the specificity of the protein. Protein-bound conformations By contrast, SAM in knotted MTs forms interactions mainly using adenine moiety, which is tightly bound to adenine-binding loop. Ribose forms hydrogen bonds with Gly and Leu. In knotted MTs, methionine chain of SAM has much less contacts with amino acids. Therefore, it forms less interactions and is more loose. https://doi.org/10.1371/journal.pcbi.1007904.g009 Fig 10. Root Mean Square Fluctuations of SAM bound to the protein complex with substrate (tRNA) based on knotted (TrmD) and unknotted (Trm5) MTs. Structure of the ligand is colored from blue (low flexibility) to red (high flexibility). Homodimeric complex of TrmD interacts with 1 tRNA molecule, which is bound to one of the active sites and the ligand depicted here as SAM A is part of this site. SAM B is bound to the other binding site. Fig 10. Root Mean Square Fluctuations of SAM bound to the protein complex with substrate (tRNA) based on knotted (TrmD) and unknotted (Trm5) MTs. Structure of the ligand is colored from blue (low flexibility) to red (high flexibility). Homodimeric complex of TrmD interacts with 1 tRNA molecule, which is bound to one of the active sites and the ligand depicted here as SAM A is part of this site. SAM B is bound to the other binding site https://doi.org/10.1371/journal.pcbi.1007904.g010 15 / 25 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 11. Logo of the glycine motifs in knotted and unknotted MTs. Left panel represents 20 knotted proteins (Class IV; residue numbering is based on RlmH protein from Staphylococcus aureus, PDB id: 4fak, as in Fig 9), right panel 160 proteins with Rossmann Fold (Class I; residue numbering is based on RsmC protein from Thermus thermophilus, PDB id: 3dmf). Both motifs are based on the structural alignments of protein sequences with no more than 30% of sequence similarity. The motifs were visualized using WebLogo [55]. Fig 11. Logo of the glycine motifs in knotted and unknotted MTs. Left panel represents 20 knotted proteins (Class IV; residue numbering is based on RlmH protein from Staphylococcus aureus, PDB id: 4fak, as in Fig 9), right panel 160 proteins with Rossmann Fold (Class I; residue numbering is based on RsmC protein from Thermus thermophilus, PDB id: 3dmf). Both motifs are based on the structural alignments of protein sequences with no more than 30% of sequence similarity. The motifs were visualized using WebLogo [55]. Conclusions We conducted a comprehensive analysis of an ubiquitous ligand S-adenosylmethionine (SAM) conformational space and factors that affect its vastness. The study was carried out from two perspectives: free form of SAM in water and a protein-bound form. We performed the analysis based on the detailed NMR study and extensive computational approach including all-atom molecular dynamics simulations in explicit solvent and database search. The analysis indicates that large conformational freedom of unbound SAM is significantly restricted upon binding to protein targets, and furthermore that some bound conformations are unlikely to occur in solution. SAM samples various conformations mainly in terms of its glycosidic angle (syn or anti) and overall angulation (extended or bent). Syn conformation is common in water, barely pres- ent in proteins, and absent in knotted proteins. There is a limited rotation of the glycosidic angle in knotted methyltransferases (only anti1) and more freedom in unknotted MTs. In knotted MTs, SAM usually adopts bent conformation, however, in 20% of the structures, it has extended methionine moiety. Interestingly, both bent and extended conformations can be bio- logically active in the knotted methyltransferases (e.g. bent in TrmD protein and extended in NHR). This suggests that the knot is imposing restrictions not to the methionine moiety of SAM as was previously assumed, but to the adenine. SAM binding mode in knotted proteins 16 / 25 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites involves tight adenine binding, and loose methionine moiety. By contrast, the unknotted pro- teins utilize methionine chain more often, and form fewer interactions with adenine. We created a “map” of distinct SAM interactions with focus on differences between knotted and unknotted MTs, which may act as a basis for the design of novel, selective TrmD inhibi- tors. We show that even conserved glycine-rich motif, common for methyltransferases, is dif- ferently incorporated in these proteins. It turns out that in the knotted MTs it is the knot and adenine-binding loop that are essential for the unique SAM binding mode. Entanglement in proteins is a relatively new, challenging topic. Since the proteins are mostly unknotted, it may appear that nature have developed mechanisms to avoid entangle- ments altogether, although it is not entirely clear why. Moreover—does the presence (or the absence) of a knot in a protein provide any clues to its function or origin? NMR spectroscopy 20 mM (5.08 mg/500 μl) solution of (2S)-2-Amino-4-[[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)- 3,4-dihydroxy- -oxolan-2-yl]methyl-methylsulfonio]butanoate (S-adenosylmethionine, SAM) was prepared in a phosphate buffer (pH = 6.50, 500 mM in D2O). Phosphate buffer consisted of 100 mM sodium phosphate (pH = 6.50), 10 mM MgCl2, 2 mM dithiothreitol (DTT), 0.05 mM ethylenediaminetetraacetic acid (EDTA) and 100 mM NaCl in D2O. Appropriate pH of the phosphate buffer was obtained by mixing NaH2PO4 and Na2HPO4 in a suitable ratio: 26.82 mg Na2HPO4 2 H2O (177.99 g/mol) and 48.20 mg NaH2PO4 2 H2O (137.99 g/mol) for 1 ml D2O. The mixture was transferred into standard 5 mm NMR tube. The measurements were performed on 700 MHz Agilent DirectDrive2 spectrometer equipped with a room- temperature HCN probe, temperature controlled at 25˚C. Each spectrum was obtained with water suppression using presaturation. 2D rotating frame nuclear Overhauser effect spectra (ROESY) were recorded in a phosphate buffer, only with EDTA, with a spin lock time of 300 ms. A spectral width of 15.9 ppm was used in both dimensions. 256 indirect evolution time increments were recorded after 32 steady-state scans. For each FIDs 3348 complex data points were acquired for accumulated 16 scans. A relaxation delay between scans was 3 s. Conclusions Do different entan- glements play any role in the binding process or the catalysis? Our study clearly shows the difference between binding mechanism based on knotted and unknotted methyltransferases. We anticipate that such differences are also present in other types of proteins with distinct topologies. Analysis of the chemical structure of SAM Chemical shifts of SAM nuclei were obtained from analysis of 1H NMR, 2D HSQC, 2D HMBC, 2D DQF-COSY, 2D ROESY, 2D Z-TOCSY spectra (Figs 2 and 12 and Table 2) and compared with previously reported results [39, 56]. The obtained 1D and 2D data set was Fou- rier transformed and processed using nmrPipe [57] and Mnova NMR software. MD simulation Molecular Dynamics (MD) simulations of a free ligand in solvent was done using GROMACS 5.0.2 [59] package and AMBER99 force field [60] with improved parameters for the ligand [42]. The simulation was done for 1μs in constant temperature (298K) and pressure (1 atm) with 150 mM of NaCl present. The system was equilibrated for 100 ps each in NVT and NPT ensembles. The simulations of the whole complexes of TrmD and Trm5 (proteins, SAM, and tRNA) were also done in GROMACS 5.0.2, but with CHARMM36 force field. The simulations were atomistic with explicit water present. The crystal structure of TrmD used as the starting con- figuration was PDB id: 4yvi, for Trm5 2zzm. Simulations were conducted using the same methodology as described before [11]. The appropriate amino acid protonation states in pH 8 were obtained using PDB2PQR server [61]. The charge of the systems were neutralized with addition of NaCl ions. The cutoff for electrostatic and van der Waals interactions were set at length of 12 Å. The entropy of SAM in the binding site was estimated using Principal Component Analysis and g_anaeig program from the GROMACS package (quasi-harmonic approximation). Calculation of the distance between atoms in the chemical structure of SAM 2.34 27.69 γ t 3.47 41.22 γ0 t 3.68 41.22 https://doi.org/10.1371/journal.pcbi.1007904.t002 https://doi.org/10.1371/journal.pcbi.1007904.t002 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 18 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites cross relaxation distance between the H-1’ and H-2’ protons was used (2.90 Å with the 0.2 Å uncertainty), according to literature [40]: r ¼ Iref I 1 6 rref ð1Þ r ¼ Iref I 1 6 rref ð1Þ Assuming independent variables, error propagation was calculated (form. 2).fifififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififi Assuming independent variables, error propagation was calculated (form. 2).fififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififi Dr ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Iref I 1 6 Drref !2 þ 1 6 ðIrefÞ 5 6 I 1 6 rrefDIref !2 þ 1 6 ðIrefÞ 1 6rref I 7 6 DI !2 v u u t ð2Þ ð2Þ where: Δr—uncertainty of measurement (error of calculated distance between atoms); rref—reference distance between the protons in SAM’s structure (H-1’ to H-2’), according to literature [40], amounting to 2.9 Å; Δrref—error of reference rref, equal to 0.2 Å ([40]); Δrref—error of reference rref, equal to 0.2 Å ([40]); f ΔI—peak intensity error; f ΔI—peak intensity error; Iref—intensity of the reference correlation peak (H-1’ to H-2’); ΔIref—reference peak intensity error. Calculation of the distance between atoms in the chemical structure of SAM For analysis of SAM conformations the obtained 2D ROESY data set was Fourier transformed, processed using nmrPipe and imported into Sparky [58]. Intensity of the correlation peaks helped to calculate the interproton distances. The distances between the atoms (r) were calcu- lated on the basis of formula 1, where I is the intensity of the cross correlation peak, while the Iref is the intensity of referencing correlation peak. As an internal reference rref of the rate of Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 17 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Fig 12. 1H NMR spectra of SAM in 25˚C. https://doi.org/10.1371/journal.pcbi.1007904.g012 Fig 12. 1H NMR spectra of SAM in 25˚C. https://doi org/10 1371/journal pcbi 1007904 g012 Fig 12. 1H NMR spectra of SAM in 25˚C. https://doi.org/10.1371/journal.pcbi.1007904.g012 Table 2. 1H and 13C chemical shifts in 25˚C. Atom’s number Multiplet structure Chemical shift in 1H NMR spectrum [ppm] Chemical shift in 13C NMR spectrum [ppm] 2 s 8.27 155.70 8 s 8.30 143.73 1’ d 6.11 91.81 2’ t 4.96 75.16 3’ t 4.60 75.36 4’ mult. 4.57 80.92 5’ d 3.92-3.94 46.87 5” d 4.03-4.05 46.87 methyl group s 2.98 26.13 α t 3.78 55.35 β quart. 2.34 27.69 γ t 3.47 41.22 γ0 t 3.68 41.22 https://doi.org/10.1371/journal.pcbi.1007904.t002 utational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 18 / 25 Fig 12. 1H NMR spectra of SAM in 25˚C. Fig 12. 1H NMR spectra of SAM in 25˚C. Table 2. 1H and 13C chemical shifts in 25˚C. Table 2. 1H and 13C chemical shifts in 25˚C. Atom’s number Multiplet structure Chemical shift in 1H NMR spectrum [ppm] Chemical shift in 13C NMR spectrum [ppm] 2 s 8.27 155.70 8 s 8.30 143.73 1’ d 6.11 91.81 2’ t 4.96 75.16 3’ t 4.60 75.36 4’ mult. 4.57 80.92 5’ d 3.92-3.94 46.87 5” d 4.03-4.05 46.87 methyl group s 2.98 26.13 α t 3.78 55.35 β quart. Analysis of SAM-protein complexes Angle distributions of SAM conformations were calculated using GROMACS 5.0.2 [59] pack- age. We prepared distributions for the MD simulation and for all sets of SAM conformations extracted from protein-ligand complexes: unknotted proteins, knotted proteins, unknotted MTs, and knotted MTs. We obtained those complexes from PDB, and evaluated whether the protein is knotted or not using KnotProt 2.0 database [9]. Analysis of knotted MTs was carried out mainly with Schroedinger Maestro 2017-1. This includes RMSD calculations, and preparation of Ramachandran plots. The eight knotted MTs we used to investigate syn arrangement interruption and adenine-binding loop structure were (PDB ids): 1uak, 1x7p, 2egv, 2v3k, 3nk7, 4fak, 4yvg, and 5h5f. We chose these structures to be as sequentially different as possible. For adenine-binding loop geometry investigation we used MASTER (Method of Acceler- ated Search for Tertiary Ensemble Representatives) [63]. Screening of PDB structures was conducted with RMSD cutoff equal to 2 Å, and by fitting fragments through main chain superposition. Sequence alignment of knotted MTs was conducted using PROMALS3D [64]. The cluster- ing of the sequences of SAM-dependent methyltransferases were done using CLANS [65] (with default parameters), which performs all vs. all sequence BLAST matches. From UniProt database we extracted 10 934 proteins that were classified as methyltransferases based on EC number (2.1.1) and had at least one Pfam identifier. Based on Pfam we divided this set into 5 classes: Rossmann Fold, TIM, tetrapyrrole, SPOUT and SET domain MTs. Structural alignments of protein sequences used for the generation of the glycine motifs were obtained with MUSTANG 3.2.3. [66]. All available SAM-bound structures of knotted and unknotted (with Rossmann Fold) methyltransferases were aligned. In order to obtain unbiased results, we used the sequences extracted from UniProt database to find the proteins that represent each group with 30% of sequence similarity (with CD-hit [67]). The representa- tive sequences were aligned to the structure-based alignment with MAFFT [68]. Using JalView [69] with annotations showing binding site residues (within 5 Åof SAM; in-house script), we extracted the alignment of glycine-rich motifs (without indels). The motifs were visualized using WebLogo [55]. PDB structures that we used to investigate SAM binding are described in Supplementary Materials (S1–S3 Tables). We chose one structure from each of the most distinct families of unknotted MTs, and knotted MT dimers. We used all available structures of knotted MT monomers, and knotted SAM synthases. Fitting clusters to NMR distances The clustering was done with RMSD cutoff set to 1.25Å on 27 heavy atoms using g_cluster module. Clustering of PDB-derived SAMs was based on the ligands from all of the available ligand-bound structures from Protein Data Bank [62] (20 from the knotted methyltransferases and 212 from the unknotted). Finding the best fit to the NMR data was done on sets containing 90 clusters (based on free ligand MD), 10 clusters (based on SAMs bound to unknotted MTs from PDB) and 2 clusters (based on SAMs bound to knotted MTs from PDB). Each set of conformations was considered separately and divided into the combinations of 4 structures. We tested every combination of clusters with their populations varying from 0% to 100%. Each interproton distance was r−6 averaged over the set of given conformations—the average was weighted based on the clusters PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 19 / 25 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites populations. populations. dcl ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Pn i vix6 i Pn i vi 6 s ð3Þ ð3Þ vi—weight of i-th cluster; xi—interproton distance of i-th cluster Every interproton distance was averaged based on given clusters (dcl) and then weighted RMSD between dcl and NMR distance (dNMR) was calculated.fififififififififififififififififififififififififififififififififififififi RMSD ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Pn i wiðdcl dNMRÞ 2 Pn i wi s ð4Þ ð4Þ wi—weight of i-th distance; wi ¼ e2 i , (ei is error for i-th distance from NMR experiment) wi—weight of i-th distance; wi ¼ e2 i , (ei is error for i-th distance from NMR experiment) PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 S5 Fig. Sequence alignment of 20 most distinct knotted methyltransferases. (TIF) S6 Fig. SAM conformations superimposed on ribose heavy atoms and adenine N9. Green: unknotted protein (PDB ID: 4dmg); purple: knotted MT (PDB ID: 4yvg); orange: knotted SAM synthase (PDB ID: 4ndn); teal: unknotted histone MT (PDB ID: 1n6c). A: side view; B: view from the top. (TIF) S6 Fig. SAM conformations superimposed on ribose heavy atoms and adenine N9. Green: unknotted protein (PDB ID: 4dmg); purple: knotted MT (PDB ID: 4yvg); orange: knotted SAM synthase (PDB ID: 4ndn); teal: unknotted histone MT (PDB ID: 1n6c). A: side view; B: view from the top. (TIF) Analysis of SAM-protein complexes Figs 7 and 10 were prepared in Schroedinger Maestro 2017-1. 20 / 25 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Supporting information S1 Fig. SAM conformations from MTs, superimposed on ribose and adenine heavy atoms. Panel A shows extended conformations from unknotted MTs (green), bent SAMs from knot- ted MTs (purple), and rare conformations from knotted MTs with extended methionine moi- ety (grey). Panel B depicts one structure from each of these groups. (TIF) S2 Fig. Chart showing both angle SD-O4’-N9 and dihedral O4’-C1’-N9-C8 of SAM confor- mations from unknotted (blue) and knotted (red) proteins from PDB. The right panel: dis- tributions of angle SD-O4’-N9. (TIF) S3 Fig. Adenine-binding loops of selected knotted MTs. Superimposed on SAM adenine moiety’s heavy atoms. (TIF) S4 Fig. Ramachandran plots of adenine-binding loops from eight representative knotted MTs. (TIF) S5 Fig. Sequence alignment of 20 most distinct knotted methyltransferases. (TIF) S6 Fig. SAM conformations superimposed on ribose heavy atoms and adenine N9. Green: unknotted protein (PDB ID: 4dmg); purple: knotted MT (PDB ID: 4yvg); orange: knotted SAM synthase (PDB ID: 4ndn); teal: unknotted histone MT (PDB ID: 1n6c). A: side view; B: view from the top. (TIF) S7 Fig. Schematic structure of SAM in two epimeric forms: (+)-SAM and (-)-SAM. (TIF) S8 Fig. Clustering of the sequences of SAM-dependent methyltransferases divided into 5 classes. Rossmann Fold (Class I; blue), TIM beta/alpha barrel (Class II; cyan), tetrapyrrole MTs (Class III; purple), SPOUT (Class IV; red), SET domain (Class V; green). Black color refers to unannotated methyltransferases. The proteins with similarity threshold (P) lower than 10−25 are joined by lines, which are darker the greater the similarity. The proteins belong- ing to each class are separated from other classes and are forming smaller groups, which shows that sequential differences in SAM-dependent MTs are present between as well as within each class. (TIF) S1 Table. Knotted MT dimers. (PDF) S2 Table. Knotted MT monomers. (PDF) S3 Table. Unknotted MTs. (PDF) S4 Table. Quasi harmonic approximation of the entropy contribution to free energy (TS) at T = 310 K (kJ/mol) calculated for SAM in binding sites of knotted (TrmD) and unknot- ted (Trm5) analogous methyltransferases. The calculation is based on the Principal S2 Fig. Chart showing both angle SD-O4’-N9 and dihedral O4’-C1’-N9-C8 of SAM confor- mations from unknotted (blue) and knotted (red) proteins from PDB. The right panel: dis- tributions of angle SD-O4’-N9. (TIF) S2 Fig. Chart showing both angle SD-O4’-N9 and dihedral O4’-C1’-N9-C8 of SAM confor- mations from unknotted (blue) and knotted (red) proteins from PDB. The right panel: dis- tributions of angle SD-O4’-N9. (TIF) S3 Fig. Adenine-binding loops of selected knotted MTs. Superimposed on SAM adenine moiety’s heavy atoms. (TIF) S4 Fig. Ramachandran plots of adenine-binding loops from eight representative knotted MTs. (TIF) S4 Fig. Ramachandran plots of adenine-binding loops from eight representative knotted MTs. (TIF) S5 Fig. Sequence alignment of 20 most distinct knotted methyltransferases. (TIF) S7 Fig. Schematic structure of SAM in two epimeric forms: (+)-SAM and (-)-SAM. (TIF) S8 Fig. Clustering of the sequences of SAM-dependent methyltransferases divided into 5 classes. Rossmann Fold (Class I; blue), TIM beta/alpha barrel (Class II; cyan), tetrapyrrole MTs (Class III; purple), SPOUT (Class IV; red), SET domain (Class V; green). Black color refers to unannotated methyltransferases. The proteins with similarity threshold (P) lower than 10−25 are joined by lines, which are darker the greater the similarity. The proteins belong- ing to each class are separated from other classes and are forming smaller groups, which shows that sequential differences in SAM-dependent MTs are present between as well as within each class. (TIF) Author Contributions Conceptualization: Krzysztof Kazimierczuk, Piotr Setny, Joanna I. Sulkowska. Conceptualization: Krzysztof Kazimierczuk, Piotr Setny, Joanna I. Sulkowska. Formal analysis: Agata P. Perlinska, Adam Stasiulewicz, Ewa K. Nawrocka. Funding acquisition: Agata P. Perlinska, Joanna I. Sulkowska. Investigation: Agata P. Perlinska, Adam Stasiulewicz, Ewa K. Nawrocka, Krzysztof Kazimierc- zuk, Piotr Setny, Joanna I. Sulkowska. Methodology: Krzysztof Kazimierczuk, Piotr Setny, Joanna I. Sulkowska. Supervision: Joanna I. Sulkowska. Validation: Piotr Setny, Joanna I. Sulkowska. Visualization: Agata P. Perlinska, Adam Stasiulewicz, Ewa K. Nawrocka. Writing – original draft: Agata P. Perlinska, Adam Stasiulewicz, Ewa K. Nawrocka, Krzysztof Kazimierczuk. Writing – review & editing: Agata P. Perlinska, Adam Stasiulewicz, Piotr Setny, Joanna I. Sulkowska. Component Analysis of MD simulations of protein-SAM-tRNA complexes. Entropy of SAM A from TrmD and SAM from Trm5 are statistically different (p-value = 0.0052; Student’s t- test). (PDF) Component Analysis of MD simulations of protein-SAM-tRNA complexes. Entropy of SAM A from TrmD and SAM from Trm5 are statistically different (p-value = 0.0052; Student’s t- test). (PDF) S3 Table. Unknotted MTs. (PDF) S4 Table. Quasi harmonic approximation of the entropy contribution to free energy (TS) at T = 310 K (kJ/mol) calculated for SAM in binding sites of knotted (TrmD) and unknot- ted (Trm5) analogous methyltransferases. The calculation is based on the Principal S4 Table. Quasi harmonic approximation of the entropy contribution to free energy (TS) at T = 310 K (kJ/mol) calculated for SAM in binding sites of knotted (TrmD) and unknot- ted (Trm5) analogous methyltransferases. The calculation is based on the Principal S4 Table. Quasi harmonic approximation of the entropy contribution to free energy (TS) at T = 310 K (kJ/mol) calculated for SAM in binding sites of knotted (TrmD) and unknot- ted (Trm5) analogous methyltransferases. The calculation is based on the Principal S4 Table. Quasi harmonic approximation of the entropy contribution to free energy (TS) at T = 310 K (kJ/mol) calculated for SAM in binding sites of knotted (TrmD) and unknot- ted (Trm5) analogous methyltransferases. The calculation is based on the Principal 21 / 25 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1007904 May 26, 2020 PLOS COMPUTATIONAL BIOLOGY S-adenosylmethionine conformational freedom in protein binding sites Component Analysis of MD simulations of protein-SAM-tRNA complexes. Entropy of SAM A from TrmD and SAM from Trm5 are statistically different (p-value = 0.0052; Student’s t- test). (PDF) References 1. Cantoni GL. Biological methylation: selected aspects. Annual review of biochemistry. 1975; 44(1):435– 451. https://doi.org/10.1146/annurev.bi.44.070175.002251 PMID: 1094914 2. Vidgren J, Svensson LA, Liljas A. Crystal structure of catechol O-methyltransferase. Nature. 1994; 368 (6469):354. https://doi.org/10.1038/368354a0 PMID: 8127373 3. 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https://openalex.org/W2104197488	https://bmcproc.biomedcentral.com/counter/pdf/10.1186/1753-6561-4-S2-P45	English	null	Targeted gene silencing for cancer treatment	BMC proceedings	2,010	cc-by	530	* Correspondence: ligia.cgs@gmail.com 1Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal Full list of author information is available at the end of the article POSTER PRESENTATION Open Access Open Access da Silva et al. BMC Proceedings 2010, 4(Suppl 2):P45 http://www.biomedcentral.com/1753-6561/4/S2/P45 da Silva et al. BMC Proceedings 2010, 4(Suppl 2):P45 http://www.biomedcentral.com/1753-6561/4/S2/P45 © 2010 da Silva et al; licensee BioMed Central Ltd. Author details 1 Cancer arises in the twenty-first century as one of the leading causes for mortality in the western civilization. In the last decades, several genes were identified as important players in the transformation of a normal cell into a tumor cell. Therefore, modulation of those genes is a promising strategy for cancer treatment. Gene downregulation can be mediated by small-interfering RNA (siRNA), 21-23 nucleotides long double strand of RNA, which has the potential to inhibit the expression of a target gene through specific cleavage of perfectly complementary mRNA. However, the clinical use of these molecules has been impaired by their unfavourable pharmacokinetics profile and low intracellular accumulation. Author details 1Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal. 2Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal. 3Faculty of Medical Sciences, New University of Lisbon, Lisbon, Portugal. 4Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal. Published: 24 September 2010 doi: Cite this article as: da Silva et al.: Targeted gene silencing for cancer treatment. BMC Proceedings 2010 4(Suppl 2):P45. doi: Cite this article as: da Silva et al.: Targeted gene silencing for cancer treatment. BMC Proceedings 2010 4(Suppl 2):P45. In order to address this issue, we have developed a novel targeted sterically stabilized lipid-based nanoparti- cle characterized by high siRNA encapsulation effi- ciency, efficient protection of siRNA, average size around 200 nm, and charge close to neutrality. Overall, these are nanoparticles that present adequate features for systemic administration. Our results have shown that the targeted nanoparti- cles were specifically internalized by human cancer cells (MDA-MB-435 and MDA-MB-231) and endothelial cells (HMEC-1). In experiments performed with green fluorescent protein (GFP)-overexpressing human cancer cell lines, specific downregulation of GFP, both at the protein and mRNA levels, was further observed with the targeted nanoparticle but not with the non-targeted counterpart. As the developed nanoparticle is adequate for the encapsulation and delivery of any siRNA sequence, studies with a siRNA against a therapeutic molecular target are now ongoing. Author details 1 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit d Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit d © 2010 da Silva et al; licensee BioMed Central Ltd.
https://openalex.org/W2957050886	https://diposit.ub.edu/dspace/bitstream/2445/171215/1/BaystonR.pdf	English	null	An external ventricular drainage catheter impregnated with rifampicin, trimethoprim and triclosan, with extended activity against MDR Gram-negative bacteria: an in vitro and in vivo study	The journal of antimicrobial chemotherapy/Journal of antimicrobial chemotherapy	2,019	cc-by	6,703	Received 4 March 2019; returned 3 May 2019; revised 3 June 2019; accepted 11 June 2019 Background: External ventricular drainage (EVD) carries a high risk of ventriculitis, increasingly caused by MDR Gram-negative bacteria such as Escherichia coli and Acinetobacter baumannii. Existing antimicrobial EVD cathe- ters are not effective against these, and we have developed a catheter with activity against MDR bacteria and demonstrated the safety of the new formulation for use in the brain. Objectives: Our aim was to determine the ability of a newly formulated impregnated EVD catheters to with- stand challenge with MDR Gram-negative bacteria and to obtain information about its safety for use in the CNS. Methods: Catheters impregnated with three antimicrobials (rifampicin, trimethoprim and triclosan) were chal- lenged in ﬂow conditions at four weekly timepoints with high doses of MDR bacteria, including MRSA and Acinetobacter, and monitored for bacterial colonization. Catheter segments were also inserted intracerebrally into Wistar rats, which were monitored for clinical and behavioural change, and weight loss. Brains were removed after either 1 week or 4 weeks, and examined for evidence of inﬂammation and toxicity. Results: Control catheters colonized quickly after the ﬁrst challenge, while no colonization occurred in the impregnated catheters even after the 4 week challenge. Animals receiving the antimicrobial segments behaved normally and gained weight as expected. Neurohistochemistry revealed only surgical trauma and no evidence of neurotoxicity. Conclusions: The antimicrobial catheter appears to withstand bacterial challenge for at least 4 weeks, suggest- ing that it might offer protection against infection with MDR Gram-negative bacteria in patients undergoing EVD. It also appears to be safe for use in the CNS. Roger Bayston1, Waheed Ashraf1, Ivan Pelegrin1,2, Katherine Fowkes3, Alison S. Bienemann4, William G. B. Singleton4,5 and Ian S. Scott3 Downloaded from https://academic.oup.com/jac/article/74/10/2959/5532147 by guest on 13 October 2020 1School of Medicine, University of Nottingham, Nottingham, UK; 2Infectious Diseases Department, Hospital Universitari de Bellvitge- IDIBELL, Barcelona, Spain; 3Department of Neuropathology, Nottingham University Hospitals NHS Trust, Nottingham, UK; 4Institute of Clinical Neurosciences, Faculty of Health Sciences, University of Bristol, Bristol, UK; 5Department of Paediatric Neurosurgery, Bristol Royal Hospital for Children, Bristol, UK Corresponding author. E-mail: roger.bayston@nottingham.ac.uk J Antimicrob Chemother 2019; 74: 2959–2964 doi:10.1093/jac/dkz293 Advance Access publication 13 July 2019 V C The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. 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. 2959 Introduction to ‘bundles’ of interventions. While the former shows some beneﬁt, but a tendency to provoke resistance, bundles usually bring about a reduction in infection rates, but depend on sustained compli- ance, rates rising again after some time.4 Some changes have brought about clear beneﬁt, such as tunnelling of the catheter and restricting CSF aspiration to that clinically necessary. However, fol- lowing from the successful use of antimicrobial catheters in shunt- ing, similar catheters have been introduced for EVD.1,3,5–7 Our catheters containing rifampicin and clindamycin (Bactiseal, Integra Life Sciences, Plainsboro, NJ, USA) have been successful in reducing staphylococcal infection,8 but they have no activity against Gram-negative bacteria. Though staphylococci are External ventricular drainage (EVD) of CSF is widely used as a tem- porary measure in the management of raised intracranial pressure associated with a variety of conditions including trauma, haemor- rhage, hydrocephalus, infection management and tumours. Patients requiring EVD are often in intensive care. Ventriculitis is a major complication of EVD. The reported incidence varies greatly, from 7.8% to 27%,1–3 but is usually 5%–10% of episodes. The vari- ation in incidence can be due to differences in patient populations, EVD management regimens and particularly diagnostic criteria. Many regimens have been proposed to reduce the rate of ven- triculitis associated with EVD, ranging from antibiotic prophylaxis V C The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. 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. 2959 2959 Bayston et al. traditionally the main pathogens, recently there have been reports of increasing proportions of Gram-negative bacteria causing ven- triculitis9–11 with an increase in multiresistance to antibiotics.10–12 Acinetobacter baumannii, Klebsiella pneumoniae and Enterobacter spp. are the most commonly found Gram-negative bacteria. Using similar platform technology, we have developed an EVD catheter that retains activity against staphylococci while also having activity against most Gram-negative EVD pathogens, including MDR strains. The antimicrobials in the new catheter, rifampicin, tri- methoprim and triclosan, were chosen partly for their antimicro- bial spectrum and partly because of their chemical compatibility with the impregnation process. In vitro challenge The brains containing the catheter segments were removed and sent for neuropathological examination. Catheters were removed from the brain post-mortem, after brain explantation and prior to neuropathological examination. Antimicrobial and control EVD catheters were inserted aseptically into a modular challenge apparatus with capacity for 12 catheters to be tested simultaneously.13,14 The apparatus determines the ability of impregnated catheter tubing to withstand multiple bacterial challenges in ﬂow conditions. The catheters were maintained at 37C while being perfused constantly with 20%, or 2% for Acinetobacter, tryptone soy broth (TSB; Oxoid Ltd, Basingstoke, UK) at a rate of 20mL/h. At four timepoints (Days 0, 7, 14 and 21), the catheters were inoculated with 1 mL of 105 cfu/mL suspension of the test bacteria. For each inoculation, ﬂow was stopped and the catheters clamped, and, after injection of the inoculum into the test catheter, they remained clamped for 1 h to encourage bacterial attachment. A control catheter was set up for each test strain at each challenge, and changed Antimicrobial and control EVD catheters were inserted aseptically into a modular challenge apparatus with capacity for 12 catheters to be tested simultaneously.13,14 The apparatus determines the ability of impregnated catheter tubing to withstand multiple bacterial challenges in ﬂow conditions. The catheters were maintained at 37C while being perfused constantly with 20%, or 2% for Acinetobacter, tryptone soy broth (TSB; Oxoid Ltd, Basingstoke, UK) at a rate of 20mL/h. At four timepoints (Days 0, 7, 14 and 21), the catheters were inoculated with 1 mL of 105 cfu/mL suspension of the test bacteria. For each inoculation, ﬂow was stopped and the catheters clamped, and, after injection of the inoculum into the test catheter, they remained clamped for 1 h to encourage bacterial attachment. A control catheter was set up for each test strain at each challenge, and changed Neuroimplantation Segments of the processed catheters were cut to 4 mm in length, packaged and sterilized as above. All animal work was performed in accordance with the UK Animal Scientiﬁc Procedures Act 1986 and was covered by both pro- ject and personal licences that were issued by the Home Ofﬁce. Animal licences were reviewed and approved by the University of Bristol Ethics Committee (project licence PA95E951). All efforts were made to minimize animal use and suffering. Juvenile male Wistar rats (Harlan, UK) weighing 250+5 g were group-housed in Techniplast 1500U cages with irradiated lignocel bedding and sawdust (International Product Supplies Ltd, UK). The study room was illuminated by ﬂuorescent light set to give a cycle of 12h of light and 12h of dark, and was air-conditioned. The ambient temperature was held between 17C and 22C. Subjects were randomized into one of four groups (each containing four subjects) based on duration of treatment before sacriﬁce (7 and 28days). Animals were individually anaesthetized with 2% inhaled isoﬂurane in oxygen in an anaesthetic chamber then placed in a stereotactic frame (David Kopf Instruments, Tujunga, CA, USA). Anaesthesia was maintained with inhaled 2% isoﬂurane/oxygen. The scalp fur was clipped and skin cleaned using alcoholic chlorhexidine. A midline in- cision from glabella to occiput exposing the skull was made, and a 5 mm burr hole was made 2 mm anterior and 2 mm lateral to the bregma on the right. The dura was opened and the catheter segment stereotactically placed in the frontal lobe on a stylet to a depth of 4 mm. The stylet was removed, leaving the segment in place. Bone wax was applied where needed for bone haemostasis, and the wound was closed with absorbable sutures (4/0 Vicryl Rapide V R). Intramuscular analgesia was used post- operatively (buprenorphine, 30lg/kg) and the animals were returned to their housing when recovered. Processing of catheters The method used was based on that already published.13 Brieﬂy, medical grade silicone EVD catheter tubing (Vesta Inc., Franklin, WI, USA) ID 1.5 mm, OD 3.0 mm, was cut to 35cm lengths. A solution of rifampicin (0.2%), trimethoprim (1%) and triclosan (1%) in chloroform was prepared and the catheter tubing was immersed in the solution at room temperature (22C) for 1 h. The tubing was then removed and held in a current of air for 18h for the solvent to evaporate. It was then brieﬂy rinsed in ethanol to remove any surface drug accretions, dried and packaged, and sterilized by autoclaving at 121C for 15min. After sterilizing, three of the catheters were selected for quality assessment. They were immersed in chloroform for 1 h at room temperature to extract antimicrobials, then the extracts were analysed by HPLC-MS to conﬁrm drug content. Introduction As well as reporting the in vitro ac- tivity of the catheter against these bacteria in rigorous clinically predictive tests, we have investigated the safety of the new cath- eter formulation for implantation into the brain using a rat model. when it became colonized. Each day, a sample of TSB was collected from each catheter, and viable cell counting was performed to determine bacter- ial numbers, by spreading 200 lL on a blood agar plate and incubating at 37C for 48h. All were tested in triplicate. Scanning electron microscopy was performed on segments of both control and processed catheters at the end of the challenge cycle. Segments of catheter 1.0 cm long were ﬁxed in cold acetone overnight and then cut longitudinally and dehydrated with tetramethylsilane (Sigma–Aldrich), ﬁxed to specimen stubs and sputter- coated with gold for 300 s before examination using a Jeol JSM 6060 micro- scope (Jeol Ltd, Tokyo, Japan). Test strains All 17 bacteria tested were clinical isolates. They were: MRSA NB881, Escherichia coli NB2203 and NB2365, Enterobacter cloacae NB1454C and K. pneumoniae NB914 and F3990; a series of MDR strains: A. baumannii NB893, F1865, F2653 and F3859, E. coli F3986 (ESBL producing) and E. coli F3802 (NDM-1 producing); and a series of isolates from clinical cases where the rifampicin/clindamycin (Bactiseal) catheter had failed due to intrinsic resistance to either rifampicin or clindamycin or both: methicillin-resistant Staphylococcus epidermidis NB951, NB928, NB935 and F2364, and Staphylococcus aureus (MRSA) F1836. Their characteristics are shown in Table S1 (available as Supplementary data at JAC Online). The MICs of ri- fampicin and trimethoprim were determined using Etest (bioMe´rieux, Basingstoke, UK). The MIC of triclosan was determined by microtitre plate serial dilution. All animals were examined daily for clinical signs of toxicity or changes in behaviour, and body weight was recorded. All numerical data were ana- lysed using GraphPad Prism V R. According to the experimental protocol, any animals that displayed signs of neurological deterioration and/or weight loss greater that 10% of peak body mass would be terminated by Schedule 1 killing. Groups 1 and 3 had control catheter segments implanted, and Groups 2 and 4 had impregnated catheter segments implanted. At the predeter- mined timepoints, animals were euthanized by anaesthetic overdose and then perfusion-ﬁxed with 4% paraformaldehyde (Fisher Scientiﬁc, Loughborough, UK) in PBS (Oxoid), pH 7.4. JAC Antimicrobial external ventricular drain catheter (a) (b) (c) (d) Figure 1. (a and b) Scanning electron micrographs (%10000) of the lumens of catheters after four weekly bacteria challenges followed by 6–7 days of perfusion. (a) Control catheter after challenge with K. pneumoniae NB914, at Day 7 of challenge 4. (b) Control catheter after challenge with MRSA F1836, at Day 7 of challenge 4. (c) Magniﬁcation of %500. Lumen of the processed catheter after challenge with MRSA F1836, at Day 7 of challenge 4. The lower magniﬁcation was used to enable a more extensive ﬁeld. (d) Magniﬁcation of %10000. Lumen surface of (c) with no visible bacteria remaining. No bacteria were recovered after prolonged perfusion and agar culture. (a) (c) (b) (d) (b) (d) (c) Figure 1. (a and b) Scanning electron micrographs (%10000) of the lumens of catheters after four weekly bacteria challenges followed by 6–7 days of perfusion. (a) Control catheter after challenge with K. pneumoniae NB914, at Day 7 of challenge 4. (b) Control catheter after challenge with MRSA F1836, at Day 7 of challenge 4. (c) Magniﬁcation of %500. Lumen of the processed catheter after challenge with MRSA F1836, at Day 7 of challenge 4. The lower magniﬁcation was used to enable a more extensive ﬁeld. (d) Magniﬁcation of %10000. Lumen surface of (c) with no visible bacteria remaining. No bacteria were recovered after prolonged perfusion and agar culture. ﬁbrillary acidic protein (GFAP) (Roche) (at a dilution of 1/5000), b-amyloid precursor protein (b-APP) (Roche) (1/20000) and neuroﬁlament protein (NFP) (Roche) (1/50). Immunohistochemistry was performed on a Ventana Benchmark Ultra (Roche Diagnostics) automated stainer. The slides were dewaxed using cell conditioner 1 (Roche) at 95C for 36min and the pri- mary antibodies were applied as follows: 24min (GFAP), 32min (NFP) and 36min (b-APP). Haematoxylin counterstain was applied for 12min. The slides were then mounted prior to viewing (Thermo Scientiﬁc ClearVue). Negative controls were performed by omitting the primary antibody. Positive controls were mounted on all slides having ﬁrst demonstrated cross-reactivity between human and mouse tissues. catheters inoculated with the test bacteria became colonized after four successive weekly challenges. All control catheters colonized rapidly after inoculation and reached counts of 108 cfu/mL within 6–7 days of inoculation, while counts in all the impregnated cathe- ters declined to zero within 3 days, remaining at zero until the next challenge. Neuroimplantation Analysis of the impregnated catheters showed rifampicin 1.58 mg/g, trimethoprim 18.2 mg/g and triclosan 18.4 mg/g, all within the expected range. Both the 1 week and the 4 week groups of animals remained in good health, gaining weight as normal with no sign of neurotox- icity and showing normal grooming. All rats fed normally through- out. No difference was observed between the 1 week and the 4 week groups, or between controls and impregnated catheter groups. Weight gain for the 4 week cohort is shown in Figure S1. Test strain characteristics Table S1 shows the 17 test strains and their susceptibilities to the three catheter drugs. JAC From experiment, inoculation of 200 lL of efﬂuent from the catheters onto blood agar plates incubated for 48h was sufﬁ- cient to determine absence of viable bacteria. This has been con- ﬁrmed by scanning electron microscopic examination of impregnated catheters after 4 weeks of perfusion and bacterial challenge (Figure 1a–c). No re-growth occurred in the days after counts reached zero, nor was any resistance seen. Examples are shown in Tables1 and 2 as all results were essentially similar. Neuropathology After noting the placement site, the catheter segments were removed from the brains, which were then processed and embedded in parafﬁn wax using standard laboratory protocols (PathCentre Tissue Processor: 3 day schedule). The neuropathologist carrying out the assessment (I. S. S.) was blind to group allocations. Sections were stained using haematoxylin and eosin (Leica ST5020 autostainer), and with antibodies raised against glial 2960 JAC In vitro challenge There was no subjective difference in the extent of inﬂammation between the control and impregnated groups at this stage unless there had been signiﬁcant catheter trauma. After 4weeks, the inﬂammatory inﬁltrate in the wall of the tract had subsided and the extent of the gliotic response was much reduced (GFAP; Figure2c). The specimens all showed re- sidual minor axonal injury within the tract, but the surgical inﬂam- mation appeared to have subsided. The inﬂammatory response was subjectively similar in both the control and impregnated groups after 4weeks, and the surgical inﬂammation appeared to have resolved in comparison with the 1week specimens. Examples were identiﬁed, from all experimental groups, in which there was signiﬁcant trau- matic axonal injury associated with catheter implantation (Figure2d). These cases showed tract inﬂammation with diffuse cor- tical hypoxic injury illustrated by a ‘geographical’ pattern of cortical staining with b-APP and the presence of cortical ‘red’ neurons. These effects are, most probably, secondary to traumatic vascular injury. In these cases, the tract inﬂammation was directly proportional to the extent of surgical trauma and not the contents of the catheter. numbers of MDR bacteria for at least 4 weeks. EVD catheters are not commonly used for more than 3 weeks. Activity against Gram- negative bacteria would be a signiﬁcant advantage for an EVD catheter, extending its protective spectrum beyond its proven anti- staphylococcal activity. Though failures due to intrinsically resist- ant strains of staphylococci in Bactiseal EVD catheters are rare,15 we have shown that the new formulation is active against those strains too. In addition, the results suggest that it will be equally ef- fective against highly antibiotic-resistant strains that are now being increasingly encountered. Most of the test bacteria were resistant to rifampicin, as expected. In vitro challenge However, there is some evidence that, even when no con- ventional susceptibility is detected, rifampicin might have sublethal effects on the bacterial cell,11 and this has been conﬁrmed clinical- ly.16 We were also keen to employ the Dual Drug Principle,17,18 which indicates that this combination can be expected to reduce the likelihood of mutational resistance to any of the three drugs, and we previously have demonstrated this in vitro.19 The combination of antimicrobials, and particularly the triclosan component, has not been used in a neurosurgical setting before, though triclosan is used safely in other clinical applications such as biodegradable sutures20 and urinary catheter irrigation,21,22 and has been used safely in ureteral stents23 and central venous In vitro challenge Sections through the catheter tracts after 1 week and 4 weeks were compared histologically with both control catheters and impregnated catheters. The results are illustrated in Figure 2(a–d). After 1 week, the tracts in both control and impregnated groups In all cases, control catheters challenged with 105 cfu/mL of the test bacteria colonized within a few days of inoculation, showing high bacterial counts (108 cfu/mL). None of the impregnated 2961 Bayston et al. Table 2. Results of four weekly challenges with A. baumannii NB893, showing on each challenge rising bacterial counts in the control catheter to the point where they became obstructed by bioﬁlm by Day 7 Table 1. Results of four weekly challenges with MRSA F1836, showing on each challenge rising bacterial counts in the control catheter and no growth from the antimicrobial catheters Day 1 Day 2 Day 3 Day 6 Day 7 Day 0, challenge 1 control 3.6%104 3.9%105 7.5%106 4.8%107 4.8%108 test 1 0 0 0 0 0 test 2 0 0 0 0 0 test 3 0 0 0 0 0 Day 0, challenge 2 control 2.8%104 7.5%106 1.5%107 4.5%107 1.1%108 test 1 0 0 0 0 0 test 2 0 0 0 0 0 test 3 0 0 0 0 0 Day 0, challenge 3 control 3.9%105 3.6%106 2.5%107 2.1%107 1.0%108 test 1 0 0 0 0 0 test 2 0 0 0 0 0 test 3 0 0 0 0 0 Day 0, challenge 4 control 2.5%105 6.5%106 6.6%107 2.5%108 1.1%108 test 1 0 0 0 0 0 test 2 0 0 0 0 0 test 3 0 0 0 0 0 A value of 0 corresponds to a value that was below the lower limit of detection. The negative cultures remained in the antimicrobial catheters throughout the four weekly challenges and each post-challenge perfusion (7 days). There was no growth from the antimicrobial catheters at 24 h after the ﬁrst challenge and though cultures were positive on monitoring on Day 1 after challenges 3 and 4, and Day 2 after challenge 2, they were negative by Day 3 and cultures remained negative thereafter in the antimicrobial catheters throughout the four weekly challenges and each post-chal- lenge perfusion (7 days). showed tract inﬂammation associated with a ﬁerce gliotic response (GFAP; Figure2a). Stains for b-APP revealed minor axonal damage along the margins of the tract (Figure2b). ted with a ﬁerce gliotic response revealed minor axonal damage ure2b). There was no subjective mation between the control and unless there had been signiﬁcant he inﬂammatory inﬁltrate in the the extent of the gliotic response c). The specimens all showed re- he tract, but the surgical inﬂam- The inﬂammatory response was rol and impregnated groups after ation appeared to have resolved imens. Examples were identiﬁed, which there was signiﬁcant trau- with catheter implantation ct inﬂammation with diffuse cor- ‘geographical’ pattern of cortical ce of cortical ‘red’ neurons. These ary to traumatic vascular injury. tion was directly proportional to ot the contents of the catheter. numbers of MDR bacteria fo not commonly used for more negative bacteria would be catheter, extending its prote staphylococcal activity. Thou ant strains of staphylococci we have shown that the ne strains too. In addition, the re fective against highly antib being increasingly encounter Most of the test bacte expected. However, there is s ventional susceptibility is det effects on the bacterial cell,1 ly.16 We were also keen to which indicates that this com the likelihood of mutational and we previously have demo The combination of antim nges with MRSA F1836, showing on ts in the control catheter and no ers 2 Day 3 Day 6 Day 7 05 7.5%106 4.8%107 4.8%108 0 0 0 0 0 0 0 0 0 06 1.5%107 4.5%107 1.1%108 0 0 0 0 0 0 0 0 0 06 2.5%107 2.1%107 1.0%108 0 0 0 0 0 0 0 0 0 06 6.6%107 2.5%108 1.1%108 0 0 0 0 0 0 0 0 0 that was below the lower limit of antimicrobial catheters throughout ost-challenge perfusion (7 days). Table 2. Results of four week showing on each challenge risin to the point where they became Day 1 Day 0, challenge 1 control 1.3%107 test 1 0 test 2 0 test 3 0 Day 0, challenge 2 control 2.8%107 test 1 80 test 2 275 test 3 65 Day 0, challenge 3 control 2.7%107 test 1 225 test 2 155 test 3 185 Day 0, challenge 4 control 2.7%107 test 1 160 test 2 115 test 3 105 A value of 0 corresponds to a detection. There was no growth from the ﬁrst challenge and though cultu after challenges 3 and 4, and D by Day 3 and cultures remaine catheters throughout the four lenge perfusion (7 days). tion associated with a ﬁerce gliotic response for b-APP revealed minor axonal damage e tract (Figure2b). There was no subjective of inﬂammation between the control and this stage unless there had been signiﬁcant 4weeks, the inﬂammatory inﬁltrate in the bsided and the extent of the gliotic response AP; Figure2c). The specimens all showed re- ry within the tract, but the surgical inﬂam- e subsided. The inﬂammatory response was th the control and impregnated groups after al inﬂammation appeared to have resolved week specimens. Examples were identiﬁed, roups, in which there was signiﬁcant trau- associated with catheter implantation showed tract inﬂammation with diffuse cor- rated by a ‘geographical’ pattern of cortical the presence of cortical ‘red’ neurons. These bly, secondary to traumatic vascular injury. t inﬂammation was directly proportional to uma and not the contents of the catheter. impregnated with three antimicrobials numbers of MDR bacteria for at least 4 w not commonly used for more than 3 week negative bacteria would be a signiﬁcant catheter, extending its protective spectrum staphylococcal activity. Though failures d ant strains of staphylococci in Bactiseal E we have shown that the new formulation strains too. In addition, the results suggest fective against highly antibiotic-resistan being increasingly encountered. Most of the test bacteria were resi expected. However, there is some evidence ventional susceptibility is detected, rifampi effects on the bacterial cell,11 and this has ly.16 We were also keen to employ the which indicates that this combination can the likelihood of mutational resistance to and we previously have demonstrated this The combination of antimicrobials, and component, has not been used in a neur though triclosan is used safely in other clin biodegradable sutures20 and urinary cath eekly challenges with MRSA F1836, showing on cterial counts in the control catheter and no obial catheters 1 Day 2 Day 3 Day 6 Day 7 104 3.9%105 7.5%106 4.8%107 4.8%108 0 0 0 0 0 0 0 0 0 0 0 0 104 7.5%106 1.5%107 4.5%107 1.1%108 0 0 0 0 0 0 0 0 0 0 0 0 105 3.6%106 2.5%107 2.1%107 1.0%108 0 0 0 0 0 0 0 0 0 0 0 0 105 6.5%106 6.6%107 2.5%108 1.1%108 0 0 0 0 0 0 0 0 0 0 0 0 to a value that was below the lower limit of ained in the antimicrobial catheters throughout s and each post-challenge perfusion (7 days). Table 2. Results of four weekly challenges w showing on each challenge rising bacterial cou to the point where they became obstructed by Day 1 Day 2 Day 0, challenge 1 control 1.3%107 1.8%107 2. test 1 0 0 0 test 2 0 0 0 test 3 0 0 0 Day 0, challenge 2 control 2.8%107 3.2%107 2. test 1 80 5 0 test 2 275 65 0 test 3 65 15 0 Day 0, challenge 3 control 2.7%107 1.5%108 5. test 1 225 0 0 test 2 155 0 0 test 3 185 0 0 Day 0, challenge 4 control 2.7%107 3.2%107 5. test 1 160 0 0 test 2 115 0 0 test 3 105 0 0 A value of 0 corresponds to a value that wa detection. There was no growth from the antimicrobial ﬁrst challenge and though cultures were posit after challenges 3 and 4, and Day 2 after challe by Day 3 and cultures remained negative ther catheters throughout the four weekly challe lenge perfusion (7 days). gliotic response axonal damage as no subjective the control and been signiﬁcant inﬁltrate in the gliotic response s all showed re- surgical inﬂam- ry response was ted groups after o have resolved were identiﬁed, signiﬁcant trau- r implantation with diffuse cor- attern of cortical ’ neurons. These vascular injury. y proportional to f the catheter. numbers of MDR bacteria for at least 4 not commonly used for more than 3 we negative bacteria would be a signiﬁca catheter, extending its protective spectr staphylococcal activity. Though failure ant strains of staphylococci in Bactisea we have shown that the new formula strains too. In addition, the results sugg fective against highly antibiotic-resist being increasingly encountered. Most of the test bacteria were r expected. However, there is some eviden ventional susceptibility is detected, rifam effects on the bacterial cell,11 and this h ly.16 We were also keen to employ th which indicates that this combination the likelihood of mutational resistance and we previously have demonstrated th The combination of antimicrobials, a 1836, showing on catheter and no Day 6 Day 7 8%107 4.8%108 0 0 0 5%107 1.1%108 0 0 0 1%107 1.0%108 0 0 0 5%108 1.1%108 0 0 0 he lower limit of heters throughout fusion (7 days). Table 2. Results of four weekly challenge showing on each challenge rising bacterial to the point where they became obstructed Day 1 Day 2 Day 0, challenge 1 control 1.3%107 1.8%107 test 1 0 0 test 2 0 0 test 3 0 0 Day 0, challenge 2 control 2.8%107 3.2%107 test 1 80 5 test 2 275 65 test 3 65 15 Day 0, challenge 3 control 2.7%107 1.5%108 test 1 225 0 test 2 155 0 test 3 185 0 Day 0, challenge 4 control 2.7%107 3.2%107 test 1 160 0 test 2 115 0 test 3 105 0 A value of 0 corresponds to a value that detection. There was no growth from the antimicrob ﬁrst challenge and though cultures were po after challenges 3 and 4, and Day 2 after ch by Day 3 and cultures remained negative t catheters throughout the four weekly cha lenge perfusion (7 days). References 1 Babu MA, Patel R, Marsh WR et al. Strategies to decrease the risk of ven- tricular catheter infections: a review of the evidence. Neurocrit Care 2011; 16: 194–202. Transparency declarations R. B. is holder of patents on the process described, assigned to his university, with no personal gain. All other authors: none to declare. JAC Rats in the ﬁrst series were euthanized at 1 week to determine the effect of surgical trauma, and rats in the second series were euthanized at 4 weeks to determine any in- ﬂammation or neurotoxicity that might be caused by the antimi- crobials. Importantly, both series of rats receiving the antimicrobial catheter segments gained weight normally and displayed no signs of illness throughout, and were indistinguishable in these respects from the control animals. When explanted brains were examined neuropathologically, though the relatively large segments caused considerable surgical trauma, there was no evidence of inﬂamma- tion or toxicity that could be attributed to the antimicrobials in the catheter. We were pleased to note that US and EU regulators have recently banned triclosan in all but medical products, recognizing its importance and safety in this area. cology data on triclosan from transcutaneous, transmucous mem- brane and oral ingestion studies showing that it is safe.25 A pig femoral artery graft implantation study has also showed no in- ﬂammatory response over and above that of control catheters.26 We have previously demonstrated its lack of toxicity in the rat peri- toneal cavity.19 We therefore implanted catheter segments into the brains of a series of rats. Rats in the ﬁrst series were euthanized at 1 week to determine the effect of surgical trauma, and rats in the second series were euthanized at 4 weeks to determine any in- ﬂammation or neurotoxicity that might be caused by the antimi- crobials. Importantly, both series of rats receiving the antimicrobial catheter segments gained weight normally and displayed no signs of illness throughout, and were indistinguishable in these respects from the control animals. When explanted brains were examined neuropathologically, though the relatively large segments caused considerable surgical trauma, there was no evidence of inﬂamma- tion or toxicity that could be attributed to the antimicrobials in the catheter. We were pleased to note that US and EU regulators have recently banned triclosan in all but medical products, recognizing its importance and safety in this area. Supplementary data Table S1 and Figure S1 are available as Supplementary data at JAC Online. Table S1 and Figure S1 are available as Supplementary data at JAC Online. Table S1 and Figure S1 are available as Supplementary data at JAC Online. Funding This work was supported by an MRC Conﬁdence in Concept grant (CiC2017004). JAC Antimicrobial external ventricular drain catheter Figure 2. (a) Section showing the extent of reactive gliosis in the tract wall (GFAP) for a control catheter after 1week. The surrounding tissue gliotic response, demonstrated in both the control and impregnated groups after 1week, reﬂected surgical trauma at this site, which subsided after 4weeks. (b) Stains for b-APP revealed traumatic axonal injury (deposits are indicated by arrows) within the walls of the tracts related to surgical trauma. This was present in all groups and varied in extent, but was independent of the contents of the catheter. (c) The gliotic response at 4weeks (GFAP) was reduced in both the control and impregnated catheters. The traumatic axonal injury resulting from the catheter implant persisted at 4weeks, but the extent of inﬂammation at 4weeks was proportional to the degree of traumatic injury and there was no subjective evidence that the impregnated catheter induced either inﬂammation or glio- sis in excess of that produced by the control catheter. (d) An example of a catheter (control) at 4weeks (b-APP) showing an inﬂammatory inﬁltrate extending from the catheter tip to traverse the corpus callosum (arrow), demonstrating signiﬁcant axonal pathology in the corpus callosum, which presumably resulted in traumatic vascular injury (not shown). These cases showed tract inﬂammation at 4weeks regardless of the contents of the catheter segment tip. academic.oup.com/jac/article/74/10/2959/5532147 by guest on 13 October 2020 Figure 2. (a) Section showing the extent of reactive gliosis in the tract wall (GFAP) for a control catheter after 1week. The surrounding tissue gliotic response, demonstrated in both the control and impregnated groups after 1week, reﬂected surgical trauma at this site, which subsided after 4weeks. (b) Stains for b-APP revealed traumatic axonal injury (deposits are indicated by arrows) within the walls of the tracts related to surgical trauma. This was present in all groups and varied in extent, but was independent of the contents of the catheter. (c) The gliotic response at 4weeks (GFAP) was reduced in both the control and impregnated catheters. The traumatic axonal injury resulting from the catheter implant persisted at 4weeks, but the extent of inﬂammation at 4weeks was proportional to the degree of traumatic injury and there was no subjective evidence that the impregnated catheter induced either inﬂammation or glio- sis in excess of that produced by the control catheter. Discussion Silicone EVD catheters impregnated with three antimicrobials were able to withstand successive weekly challenges with high 2962 2962 JAC JAC (d) An example of a catheter (control) at 4weeks (b-APP) showing an inﬂammatory inﬁltrate extending from the catheter tip to traverse the corpus callosum (arrow), demonstrating signiﬁcant axonal pathology in the corpus callosum, which presumably resulted in traumatic vascular injury (not shown). These cases showed tract inﬂammation at 4weeks regardless of the contents of the catheter segment tip. Figure 2. (a) Section showing the extent of reactive gliosis in the tract wall (GFAP) for a control catheter after 1week. The surrounding tissue gliotic response, demonstrated in both the control and impregnated groups after 1week, reﬂected surgical trauma at this site, which subsided after 4weeks. (b) Stains for b-APP revealed traumatic axonal injury (deposits are indicated by arrows) within the walls of the tracts related to surgical trauma. This was present in all groups and varied in extent, but was independent of the contents of the catheter. (c) The gliotic response at 4weeks (GFAP) was reduced in both the control and impregnated catheters. The traumatic axonal injury resulting from the catheter implant persisted at 4weeks, but the extent of inﬂammation at 4weeks was proportional to the degree of traumatic injury and there was no subjective evidence that the impregnated catheter induced either inﬂammation or glio- sis in excess of that produced by the control catheter. (d) An example of a catheter (control) at 4weeks (b-APP) showing an inﬂammatory inﬁltrate extending from the catheter tip to traverse the corpus callosum (arrow), demonstrating signiﬁcant axonal pathology in the corpus callosum, which presumably resulted in traumatic vascular injury (not shown). These cases showed tract inﬂammation at 4weeks regardless of the contents of the catheter segment tip. even MDR bacteria suggests that it might be beneﬁcial in reducing EVD infections. Further studies will include insertion of whole cath- eters into a large animal model and bacterial challenge in vivo. If this study is successful, human studies will follow. catheters.24 There is also a large amount of published human toxi- cology data on triclosan from transcutaneous, transmucous mem- brane and oral ingestion studies showing that it is safe.25 A pig femoral artery graft implantation study has also showed no in- ﬂammatory response over and above that of control catheters.26 We have previously demonstrated its lack of toxicity in the rat peri- toneal cavity.19 We therefore implanted catheter segments into the brains of a series of rats. Conclusions The antimicrobial EVD catheter therefore appears to be effective against EVD pathogens and safe for implantation into the brain. The relatively long duration of protection against colonization by 2 Wong GKC, Poon WS, Wai S et al. Failure of regular external ventricular drain exchange to reduce cerebrospinal ﬂuid infection: result of a randomised controlled trial. J Neurol Neurosurg Psychiatr 2002; 73: 759–61. 2963 Bayston et al. 3 Wright K, Young P, Brickman C et al. Rates and determinants of ventriculostomy-related infections during a hospital transition to use of antibiotic-coated external ventricular drains. Neurosurg Focus 2013; 34: E12. 3 Wright K, Young P, Brickman C et al. Rates and determinants of ventriculostomy-related infections during a hospital transition to use of antibiotic-coated external ventricular drains. Neurosurg Focus 2013; 34: E12. 14 Bayston R, Lambert E. Duration of protective activity of cerebrospinal ﬂuid shunt catheters impregnated with antimicrobial agents to prevent bacterial catheter-related infection. J Neurosurg 1997; 87: 247–51. 15 Bayston R, Ashraf W. Antibiotic resistant infections with antibiotic- impregnated Bactiseal catheters for ventriculoperitoneal shunts. Br J Neurosurg 2011; 25: 780. 4 Dasic D, Hanna SJ, Bojanic S et al. External ventricular drain infection: the effect of a strict protocol on infection rates and a review of the literature. Br J Neurosurg 2006; 20: 296–300. 16 Bassetti M, Repetto E, Righi E et al. Colistin and rifampicin in the treatment of multidrug-resistant Acinetobacter baumannii infections. J Antimicrob Chemother 2008; 61: 417–20. 5 Zabramski JM, Whiting D, Darouiche RO et al. Efﬁcacy of antimicrobial- impregnated external ventricular drain catheters: a prospective, randomized, controlled trial. J Neurosurg 2003; 98: 725–30. 17 Ehrlich P. Pathology in therapeutics: scientiﬁc principles, methods and results. Lancet 1913; 445–51. 6 Gutierrez-Gonzalez R, Boto GR. Do antibiotic-impregnated catheters pre- vent infection in CSF diversion procedures? Review of the literature. J Infect 2010; 61: 9–20. 18 Zhao X, Drlica K. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from ﬂuoroquinolone studies. Clin Infect Dis 2001; 33 Suppl 3: S147–56. 7 Harrop JS, Sharan AD, Ratliff J et al. Impact of a standardized protocol and antibiotic-impregnated catheters on ventriculostomy infection rates in cere- brovascular patients. Neurosurgery 2010; 67: 187–91. 19 Bayston R, Fisher LE, Weber K. An antimicrobial modiﬁed silicone periton- eal catheter with activity against both Gram-positive and Gram-negative bacteria. Biomaterials 2009; 30: 3167–73. 8 Thomas R, Lee S, Patole S et al. Conclusions Antibiotic-impregnated catheters for the prevention of CSF shunt infections: a systematic review and meta-analysis. Br J Neurosurg 2012; 26: 175–84. 20 Barbolt TA. Chemistry and safety of triclosan, and its use as an antimicro- bial coating on coated Vicryl Plus antibacterial suture (coated polyglactin 910 suture with triclosan). Surg Infect (Larchmt) 2002; 3 Suppl 1: S45–53. 9 Hogg GM, Barr JG, Webb CH. In-vitro activity of the combination of colistin and rifampicin against multidrug-resistant strains of Acinetobacter bauman- nii. J Antimicrob Chemother 1998; 41: 494–5. 21 Holroyd S. A new solution for indwelling catheter encrustation and block- age. J Community Nurs 2017; 31: 48–52. 22 Pannek J, Vestweber AM. Clinical utility of an antimicrobial blocking solu- tion in patients with an indwelling catheter. Aktuelle Urol 2011; 42: 51–4. 10 Kim B-N, Peleg AY, Lodise TP et al. Management of meningitis due to antibiotic-resistant Acinetobacter species. Lancet Infect Dis 2009; 9: 245–55. 23 Cadieux PA, Chew BH, Nott L et al. Use of triclosan-eluting ureteral stents in patients with long-term stents. J Endourol 2009; 23: 1187–94. 11 Lee HJ, Bergen PJ, Bulitta JB et al. Synergistic activity of colistin and rifam- pin combination against multidrug-resistant Acinetobacter baumannii in an in vitro pharmacokinetic/pharmacodynamic model. Antimicrob Agents Chemother 2013; 57: 3738–45. 24 Silva Paes Leme AF, Ferreira AS, Alves FA et al. An effective and biocom- patible antibioﬁlm coating for central venous catheter. Can J Microbiol 2015; 61: 357–65. 12 Hsueh PR, Chen WH, Luh KT. Relationships between antimicrobial use and antimicrobial resistance in Gram-negative bacteria causing nosocomial infections from 1991–2003 at a university hospital in Taiwan. Int J Antimicrob Agents 2005; 6: 463–72. 25 Jones RG, Jampani HB, Newman JL et al. Triclosan: a review of effective- ness and safety in health care settings. Am J Infect Control 2000; 28: 184–96. 26 Hernandez-Richter T, Schardey HM, Lo¨hlein F et al. The prevention and treatment of vascular graft infection with a Triclosan (Irgasan)-bonded Dacron graft: an experimental study in the pig. Eur J Vasc Endovasc Surg 2000; 20: 413–8. 13 Bayston R, Grove N, Siegel J et al. Prevention of hydrocephalus shunt catheter colonisation in vitro by impregnation with antimicrobials. J Neurol Neurosurg Psychiatr 1989; 52: 605–9. 2964
https://openalex.org/W4315432351	https://zenodo.org/records/7519929/files/58-64.pdf	Quechua	null	XITOY TURIZIMI VA UNING XITOY IQTISODIYOTIGA TA'SIRI	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,319	YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech XITOY TURIZIMI VA UNING XITOY IQTISODIYOTIGA TA’SIRI TDIU Xalqaro turizm fakulteti talabasi: Akbarjonov Qobiljon TDIU Xorijiy tillar kafedrasi o‘qituvchisi: Khikmatov Dilshodjon Annotatsiya: Barchamizga ma`lumki Xitoy Xalq Respublikasi ham iqtisodiyot jihatidan, ham turizm sohasida yetakchi o`rinlarni egallagan. Shu bilan birgalikda Xitoy turizimi iqtisodiyotiga ham katta foyda keltiradi desam mubolag`a bo`lmaydi. Turizm sohasi o`ziga ichki va tashqi turizm, ta`lim turizimi, xarqaro festivallari, ovqatlanish madaniyatini ham o`z ichiga oladi. Bu sohalardan eng mashxuri tashqi turizmdir, Mazkur maqola orqali turizm sohasining Xitoy iqtisodiyotidagi tasirini atroflicha yoritishga xarakat qilindi. Annotatsiya: Barchamizga ma`lumki Xitoy Xalq Respublikasi ham iqtisodiyot jihatidan, ham turizm sohasida yetakchi o`rinlarni egallagan. Shu bilan birgalikda Xitoy turizimi iqtisodiyotiga ham katta foyda keltiradi desam mubolag`a bo`lmaydi. Turizm sohasi o`ziga ichki va tashqi turizm, ta`lim turizimi, xarqaro festivallari, ovqatlanish madaniyatini ham o`z ichiga oladi. Bu sohalardan eng mashxuri tashqi turizmdir, Mazkur maqola orqali turizm sohasining Xitoy iqtisodiyotidagi tasirini atroflicha yoritishga xarakat qilindi. Kalit so‘zlar: Ochiq eshiklar siyiosati, o‘rtaxol qatlam, Xitoyning o‘ziga xos siyosati, Xitoyning o‘ziga xos iqtisodiyoti Soʻnggi bir necha oʻn yilliklar davomida Xitoyda islohot va ochiqlik boshlanganidan beri turizm tezligi oshdi. Yangi boy oʻrta sinfning paydo boʻlishi va Xitoy hukumati tomonidan harakatlanishga qoʻyilgan cheklovlarning yumshatilishi ham ushbu sayohat oqimini kuchaytirmoqda. Xitoy dunyodagi eng yirik sayyohlik bozorlaridan biriga aylandi. Euromonitor International maʼlumotlariga koʻra, yaqin atrofdagi Osiyo mamlakatlaridagi iqtisodiy oʻsish va yuqori daromadlar Xitoyga 2030-yilga borib dunyoning birinchi raqamli sayyohlik yoʻnalishi boʻlishiga yordam beradi.1 Dunyoning boshqa mamlakatlariga qaraganda ko'proq tashrif buyuruvchilarni qabul qilish bilan bir qatorda, 2030 yilga borib 260 million turistik sayohat bilan AQSh va Germaniyani ortda qoldirib, eng ko'p sayohatchilar soniga ega bo'ladi. Xitoyga tashrif buyuruvchilarning ko‘payishi asosan iqtisodiy o‘sish va yaqin atrofdagi Osiyo mamlakatlaridagi yuqori 1 uz.wikipedia.org 58 YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech daromadlar bilan bog‘liq. Osiyolik tashrif buyuruvchilar uchun ham mamlakatga kirish imkoniyati yaxshilanmoqda, ular vizalarni avvalgidan ko'ra osonroq olishlari mumkin. Hozirda Osiyoga tashrif buyurayotgan sayyohlarning 80 foizi qit'a ichidan keladi, deydi Geerts.2 Tashqi turizmni rivojlanishiga asosiy sabalardan biri bu Xitoyning diqqatga sazovor joylaridir. Bilamizki xitoy xalqi 5-6 ming yillik tarixga egadir.3 Shuning uchun ham tarixiy obidalar, tog`lar tepasidagi ibodatxonlar, diqqatga sazovor joylari ko`pdir. Bizga malum va mashhur bo`lgan Buyuk Xitoy devorini oladigan bo`lsak; tog` yonbag`rida joylashgan, uzunligi 530 kmdan oshiq bo`lgan bu g`ozallikni ko`rish uchun yiligan taxminan 10 million sayyoh tashrif buyurishadi.4 Sayyohlar asosan iyun oyini tashrif buyurish uchun qulay vaqt deb hisoblaganlari uchun bu oyda Xitoy devori sayyohlar bilan gavjum hisoblanadi. 2 https://www.theguardian.com/travel/2018/nov/06/china-will-be-the-worlds-most-popular-holiday-destination-by- 2030 3 https://uz.wikipedia.org/wiki/Xitoy#Tarixi 4 O`zbekiton milliy enskilopediyasi 5 Quora.com 6 O`zbekiston milliy ensiklopediyasi www.theguardian.com/travel/2018/nov/06/china-will-be-the-worlds-most-popular-holiday-destination-by- YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech XITOY TURIZIMI VA UNING XITOY IQTISODIYOTIGA TA’SIRI Devor yiliga 776,16 million dollar davlat budjetiga foyda keltiradi.5 Bundan tashqari Pekin Saroyi muzeyi, Potala saroyi, Osmon ibodatxonasi, Shaolin ibodatxonasi, Lushan tog`lari va boshqalar. Quyidagi o`z tarixiga ega bo`lgan maskanlarning olib keladigan daromadi ham davlat xazinasida o`z o`rniga ega.6 4 O`zbekiton milliy enskilopediyasi 5 Q 2 https://www.theguardian.com/travel/2018/nov/06/china-will-be-the-worlds-most-popular-holiday-destination-by- 2030 3 https://uz.wikipedia.org/wiki/Xitoy#Tarixi p y y 8 https://www.studyinchina.com.my/web/page/more-than-300000-international-students/ 7 https://www.officeholidays.com/holiday-information/china YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague Czech Xitoy Xalq Respublikasining ichki turizimiga nazar tashlaydigan bo`lsak boshqa davlatlarnikida anchagina farqlanib turadi. Xar bir davlatni o`z milliy bayramlari bo`lgani kabi xitoy xalqida ham o`zining xalq, milliy bayramlari mavjud. Birgina manashu kabi bayramlarda xalqiga xordiq chiqarish maqsadida dam olish kunlarini berilishi ham Xitoyda ichki turizmni rivojlanganiga yaqol misol bo`la oladi. Agar xodim shanba yoki yakshanba kunlarida bo`ladigan dam olish kunida ishlasa, ularga asosiy ish haqqining 200 foiz miqdorida kompensatsiya to`lanadi. Yoki bo`lmasam bayram kunlarida beriladigan dam olish kunida ishlashsa asosiy ish haqqining 300 foiz miqdorida kompentatsiya to`lanadi. Xitoy qonunchiligiga ko`ra, maoshli ta`til majburiydir. 1 yildan 10 yilgacha ishlagan xodimga 5 kun beriladi. 10 59 YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech yildan 20 yilgacha ishlaganlar uchun ishlaganlarga 10 kunlik dam olish, 20 yil va undan ortiq ishlashgan xodimlarga 15 kungacha dam olish beriladi.7 Bu vaqt oralig`ida xitoy xalqi o`z diyorlaridagi jannatmakon go`shlarda xordiq chiqarishlari uchun zamin yaratilgan. “Buni iqtisodiyotga ta`siri qanday ta`siri bor”- degan savol tug`ilishi mumkin. Deylik bitta oila ta`til vaqtida barcha oilalar singari madaniy xordiq chiqarish maqsadida bir provinsiyadan boshqa bir provinsiyaga borishdi. Bitta oilaning mehmonxonada turishi, yeguligi, ovqatlanishi, sayohat qilishi, transport xizmatidan foydalanishi va boshqa-boshqalar uchun davlatga mo`maygina pul to`laydi. Boshqacha qilib oladigan bo`lsak maxsus tour xizmatlaridan foydalangan holda sayohat qilib kelishlari mumkin. Bundan ham davlat ham bir oila manfaat ko`radi. Endi o`ylab ko`ring Xitoy aholisi 1,5 milliardga yaqin, qanchadan qancha oilalar hozirgi kunda sayohatlarni amalga oshirib kelishmoqda. 7 https://www.officeholidays.com/holiday-information/china 8 https://www studyinchina com my/web/page/more-than-300000-international-students/ YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech 2) Navbatda turish. Eshitib meni eng hayratda qoldirgan narsalarimdan biri bu odamlarning ongli ravishda navbatga turishi, hech kim aytmasa ham, hech kim nazorat qilmasa ham navbatni shakllantira olishi va unga barcha birdek amal qilishidir. 2) Navbatda turish. Eshitib meni eng hayratda qoldirgan narsalarimdan biri bu odamlarning ongli ravishda navbatga turishi, hech kim aytmasa ham, hech kim nazorat qilmasa ham navbatni shakllantira olishi va unga barcha birdek amal qilishidir. 3) Ta'lim tizimiga bo'lgan e'tibor va ta'limda korrupsiyaning yo'qligi. Men xitoylarning hozirgi muvaffaqiyati asosida hozirgi xitoy ta'limi yotadi deb o'ylayman. Chunki Xitoy ko`chalarida maktab o`quvchilarini deyarli ko`rmaysiz. Chindan ham ko'chada, bozorda birorta maktab o'quvchilari deyarli yo`q. Chunki ular tong saharda maktabga ketadi, kech bo'lganda qaytadi. Agar maktabda yaxshi o'qimasa o'qishga kira olmaydi, o'qishga kira olmasa kelajak hayoti ham yo'q, qora ishchi bo'lishga majbur. O'qishga kirish esa oson emas, faqat va faqat bilimlilargina o'qishga kira oladi. Oliy ta'limda korrupsiya mutlaqo yo'q. Talabalarning o'qishi, yashashi, tadqiqot qilishi uchun barcha sharoit yaratilgan, professor- o'qituvchilarning barchasi hurmatda, oyligi katta, allambalo mashinalarda yuradi, qog'ozbozlik yo'q emas, bor, biroq, bu ilmiy tadqiqotlar uchun to'siq bo'ladigan, vaqtni o'g'irlaydigan darajada emas. 3) Ta'lim tizimiga bo'lgan e'tibor va ta'limda korrupsiyaning yo'qligi. Men xitoylarning hozirgi muvaffaqiyati asosida hozirgi xitoy ta'limi yotadi deb o'ylayman. Chunki Xitoy ko`chalarida maktab o`quvchilarini deyarli ko`rmaysiz. Chindan ham ko'chada, bozorda birorta maktab o'quvchilari deyarli yo`q. Chunki ular tong saharda maktabga ketadi, kech bo'lganda qaytadi. Agar maktabda yaxshi o'qimasa o'qishga kira olmaydi, o'qishga kira olmasa kelajak hayoti ham yo'q, qora ishchi bo'lishga majbur. O'qishga kirish esa oson emas, faqat va faqat bilimlilargina o'qishga kira oladi. Oliy ta'limda korrupsiya mutlaqo yo'q. Talabalarning o'qishi, yashashi, tadqiqot qilishi uchun barcha sharoit yaratilgan, professor- o'qituvchilarning barchasi hurmatda, oyligi katta, allambalo mashinalarda yuradi, qog'ozbozlik yo'q emas, bor, biroq, bu ilmiy tadqiqotlar uchun to'siq bo'ladigan, vaqtni o'g'irlaydigan darajada emas. 4) Ishga kirishning shaffofligi. Universitetda har yili bir necha marta talabalar uchun ish beruvchilar mehnat yarmarkasi tashkil etadi va suhbatlar o'tkazadi. Bu xo'jako'rsinga emas, aksincha, barcha ish beruvchilar kuchli kadrlarni izlaydi, ularni o'ziga jalb qilishga harakat qiladi. Boisi har bir soha bo'yicha raqobatchi bor, biror no'noq kadr tufayli mijozlarni bir soniyada yo'qotib qo'yish mumkin, shu bois ishga olishda faqat kuchli kadrlarga e'tibor beriladi, nepotizm balki juda yuqori elita qatlamida bordir, lekin oddiy odamlar qatlamida deyarli bu illatni uchratish amri- mahol. 5) Online savdo. Xitoyning bu jihati har qanday davlat uchun namuna bo'la oladi. Internet orqali istagan narsangizni sotib olishingiz mumkin, yetkazib berish xizmatlari ham soatdek ishlaydi. YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech Xitoyda ta`lim ham ancha rivojlangani sababli har yili xorijiy talabalarning soni tobora ortib bormoqda. Hozirda Xitoy Xalq Respublikasida 300 mingdan ortiq xalqaro talabalar tahsil olib kelishmoqda.8 Ko`pgina talabalrning maqsadi va niyati ham shu bo`lsa kerak. Hozir men sizlarga talabalarning bu o`lkaga ketishlarini asosiy sabablaridan bir nechtasini sanab o`taman: 1) O'z tiliga nisbatan juda katta hurmat. Xitoyliklar xitoy tilini haqiqatda sevadi. Ko'chalardagi yozuvlarning 99%i xitoy tilida yozilgan, mahsulotlar yorlig'ining barchasi xitoycha, hatto chet eldan keladigan import mahsulotlar idishiga xitoy tilida yozilgan alohida yorliq yopishtirilishi shart. Istalgan davlat saytiga kirsangiz avval xitoycha sahifa ochiladi. Barcha davlat va nodavlat tashkilotlar xitoy tilida yuritadi. Hamma hujjatlar xitoy tilida. Telefon sotib olsangiz ichi xitoycha, kompyuter, muzlatkich, xullas, hamma-hammasi xitoycha. Xitoyda shunday holatki, xitoy tilini bilmaydigan odamga bu yerda yashash juda qiyin. Xitoy tilini o'rganish uchun esa, aynan chet elliklar uchun, juda ko'plab kitoblar, qo'llanmalar, saytlar, mobil ilovalar, video darslar yaratilgan. 60 9 Texnoman.uz YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech Uydan chiqmasdan turib ham bemalol xaridlar qilishingiz, mahsulot haqida sotuvchi bilan gaplashishingiz mumkin va bu mutlaqo qulay va osonlashtirilgan. Agar biror mahsulot yoki xizmatdan ko'nglingiz to'lmasa 61 YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech pulingizning qaytarilishi kafolatlanadi. Sababi har bir mahsulot yoki xizmat haqida mijozlar fikr bildirib baho qo'yadigan joy mavjud va uni sotuvchilar nazorat qila olmaydi. Agar mahsulot haqida bir-ikki xaridor salbiy fikr bildirsa, tamom, o'sha mahsulot sotuvi keskin tushib ketadi. Shu bois online savdoda ham sotuvchilar mahsulot va xizmat sifatiga qattiq e'tibor beradilar. Online savdoning yana bir qulayligi bozorga chiqib savdo qilishdan ko'ra arzon va chegirmalarning ko'pligidir. Masalan, bitta mahsulotni do'kondan 10 yuanga oladigan bo'lsangiz aynan shu narsaning o'zini online savdo orqali bemalol 6-7 yuanga ham sotib olaverasiz. p g p 11 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849519/ YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague Czech 6) Transport. Bu jihat bo'yicha ham Xitoyga yetadigan davlat yo'q bo'lsa kerak. Bu mavzuda keyinroq batafsil to`xtalib o`tamiz, hozircha qisqacha shuni aytishim mumkinki, Xitoyda shaharlararo transport shu darajada rivojlanganki, bir shaharda yashab bemalol boshqa shaharda ishlashingiz mumkin. Bir necha o'nlab aviakompaniyalar bor, poyezdlar, avtobuslar ham xuddi shunday. Internet orqali chiptalarni bemalol bir harakat bilan xarid qila olasiz, monopoliya yo'qligi bois kompaniyalar mijozlarga turli-tuman chegirmalar taklif etadi, ayniqsa bayramlarda biznikidan farqli ravishda narxlar tushadi. Shahar ichidagi metro tizimi ham alohida mavzu. 10 dan ortiq liniyalar bor, har bir liniyaning uzunligi 50-60 km dan kam emas. Uyingiz oldidagi metro bekatidan chiqib shaharning istalgan qismiga bora olasiz. 9 Yaqinda paydo bo`lgan Covid-19 virusi sababli Xitoy Respublikasi karantin sababli uzoq muddatga yopildi. Shu sababli huduga kirish ham chiqish ham bo`lmagan. Bu Xitoyda turizmni keskin pasayishiga olib keldi. Turizm mamlakat iqtisodiyotiga katta foyda keltirayotgani uchun , iqtisodiyotiga ham katta ta`sir ko`rsatmay qolmadi. 2019-yil dekabr oyi oʻrtalarida Uxan shahrida birinchi yangi koronavirus infeksiyasi aniqlanganidan beri koronavirus kasalligi (COVID-19) butun Xitoy va butun dunyo boʻylab tez tarqaldi. 2020 yil iyun oxiriga kelib, 80 000 dan ortiq holatlar tasdiqlandi, 3000 dan ortiq o'lim. Xitoy hukumati COVID-19 pandemiyasini eng tez tarqalish tezligi, eng keng infektsiya hududi va PR China 9 Texnoman.uz 62 YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech tashkil topganidan beri eng qiyin nazorat va oldini olish bilan eng jiddiy sog'liqni saqlash favqulodda holat deb tan oldi. Xuddi shu davrda tasdiqlangan holatlar butun dunyo bo'ylab 10,0 milliondan oshib, 500,000 ga yaqin o'limga sabab bo'ldi COVID-19 ning yuqishi va tarqalish tezligi SARS, H1N1 va MERS kabi oldingi yuqumli kasalliklardan oshib, Xitoy va butun dunyo iqtisodiyotiga misli koʻrilmagan iqtisodiy zarar yetkazadi. Natijada, Xitoy yalpi ichki mahsuloti 2020- yilning birinchi choragida (1-chorak) 6,8 foizga keskin kamaydi, bu 2019-yilning birinchi choragidagi ijobiy YaIM o‘sish sur’ati 6,4 foizni tashkil etdi. XVJ shuningdek, Xitoyning YaIM o‘sishi prognozini 6 foizdan 1,0 foizga tushirdi10. Ko'pgina tadqiqotlar yuqumli kasalliklarning iqtisodiy ta'sirini baholadi . Bir qator tadqiqotlar shuni ko'rsatdiki, COVID-19 pandemiyasi qisqa muddatda Xitoyning makroiqtisodiyoti va tarmoq ishlab chiqarishiga katta zarar etkazishi mumkin. Iqtisodiy faoliyatning to'xtatilishi korxonalar ishlab chiqarishni sezilarli darajada qisqartirishi mumkin bo'lsa-da, qat'iy masofa iste'molchilarni kamroq sarflashiga olib keladi dinamik hisoblanuvchi umumiy muvozanat (CGE) modelidan foydalangan va Xitoyning YaIM 2020 yilda aholi o‘limi va pandemiya davomiyligiga qarab 0,4–6,2% ga kamayishini aniqlagan. Kirish-chiqish modelidan foydalangan va Xitoy yalpi ichki mahsuloti 0,40-0,72% ga tushishini ko'rsatdi. 10 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849519/ YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague Czech Bundan tashqari, pandemiya ishlab chiqarish tarmoqlarining, ayniqsa turizm, mehmonxona, restoran va chakana savdo kabi bevosita ta'sir ko'rsatadigan tarmoqlarga jiddiy ta'sir ko'rsatishi mumkin11 l i l id hi ki 1) i l blik i j d b YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech tashkil topganidan beri eng qiyin nazorat va oldini olish bilan eng jiddiy sog'liqni saqlash favqulodda holat deb tan oldi. Xuddi shu davrda tasdiqlangan holatlar butun dunyo bo'ylab 10,0 milliondan oshib, 500,000 ga yaqin o'limga sabab bo'ldi COVID-19 ning yuqishi va tarqalish tezligi SARS, H1N1 va MERS kabi oldingi yuqumli kasalliklardan oshib, Xitoy va butun dunyo iqtisodiyotiga misli koʻrilmagan iqtisodiy zarar yetkazadi. Natijada, Xitoy yalpi ichki mahsuloti 2020- yilning birinchi choragida (1-chorak) 6,8 foizga keskin kamaydi, bu 2019-yilning birinchi choragidagi ijobiy YaIM o‘sish sur’ati 6,4 foizni tashkil etdi. XVJ shuningdek, Xitoyning YaIM o‘sishi prognozini 6 foizdan 1,0 foizga tushirdi10. Ko'pgina tadqiqotlar yuqumli kasalliklarning iqtisodiy ta'sirini baholadi . Bir qator tadqiqotlar shuni ko'rsatdiki, COVID-19 pandemiyasi qisqa muddatda Xitoyning makroiqtisodiyoti va tarmoq ishlab chiqarishiga katta zarar etkazishi mumkin. Iqtisodiy faoliyatning to'xtatilishi korxonalar ishlab chiqarishni sezilarli darajada qisqartirishi mumkin bo'lsa-da, qat'iy masofa iste'molchilarni kamroq sarflashiga olib keladi dinamik hisoblanuvchi umumiy muvozanat (CGE) modelidan foydalangan va Xitoyning YaIM 2020 yilda aholi o‘limi va pandemiya davomiyligiga qarab 0,4–6,2% ga kamayishini aniqlagan. Kirish-chiqish modelidan foydalangan va Xitoy yalpi ichki mahsuloti 0,40-0,72% ga tushishini ko'rsatdi. Bundan tashqari, pandemiya ishlab chiqarish tarmoqlarining, ayniqsa turizm, mehmonxona, restoran va chakana savdo kabi bevosita ta'sir ko'rsatadigan tarmoqlarga jiddiy ta'sir ko'rsatishi mumkin11 YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague Czech Bu maqolani xulosasida aytmoqchimanki: 1) Xitoy Xalq Respublikasi juda boy xalq hisoblansada shu bilan birgalikda juda mehnatkash xalq ham hisoblanadi. 2) Turizm sohasi jadal rivojlanib kelayotgan shaxar. 3) Iqtisodiyot jihatdan ham yetakchi davlatlar bilan bemalol raqobat qila oladi desam mubolag`a bo`lmaydi. 4) Ta`lim tizimiga esa til ojiz. 63 YOUTH, SCIENCE, EDUCATION: TOPICAL ISSUES, ACHIEVEMENTS AND INNOVATIONS 2022 Prague, Czech Foydalanilgan adabiyotlar ro‘yxati: Foydalanilgan adabiyotlar ro‘yxati: 1. Xasanova, S. (2021). Rus tilshunosligida frazeologizmlarning tadqiqi: rus tilshunosligida frazeologizmlarning tadqiqi. Журнал иностранных языков и лингвистики, 2(7). 1. Xasanova, S. (2021). Rus tilshunosligida frazeologizmlarning tadqiqi: rus tilshunosligida frazeologizmlarning tadqiqi. Журнал иностранных языков и лингвистики, 2(7). 2. Shaxzoda, X. (2020). Lexical Properties Of Pu Songling Novels. The American journal of social science and education innovations. 2. Shaxzoda, X. (2020). Lexical Properties Of Pu Songling Novels. The American journal of social science and education innovations. 3. Xasanova, S. (2022). XITOY VA O ‘ZBEK TILLARIDA FE’L FRAZEOLOGIZMLARINING ANTONOMIYA HODISASI. Oriental renaissance: Innovative, educational, natural and social sciences, 2(Special Issue 29), 188-197. 4. Khasanova, S. A. Q. (2022). Analysis of verb phraseological units in chinese. Asian Journal of Multidimensional Research, 11(5), 280-283. 64
https://openalex.org/W3024504553	https://arpi.unipi.it/bitstream/11568/1047258/1/pathogens-09-00377-v2.pdf	English	null	Leptospira Survey in Wild Boar (Sus scrofa) Hunted in Tuscany, Central Italy	Pathogens	2,020	cc-by	10,508	Received: 20 April 2020; Accepted: 12 May 2020; Published: 14 May 2020 Abstract: Leptospirosis is a re-emerging, worldwide zoonosis, and wild boar (Sus scrofa) are involved in its epidemiology as the reservoir. The aim of this study was to investigate the prevalence of Leptospira with serological, bacteriological, and molecular assays in wild boar hunted in Tuscany (Italy) during two hunting seasons. In total, 287 specimens of sera, kidneys, and liver were collected to perform microscopic agglutination tests (MATs), isolation, and RealTime PCR to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene), and saprophytic (23S rRNA gene) Leptospira. Within sera, 39 (13.59%) were positive to the MAT, and Australis was the most represented serogroup (4.88%), followed by Pomona (4.18%), and Tarassovi (3.14%). Moreover, four Leptospira cultures were positive, and once isolates were identiﬁed, one was identiﬁed as L. borgpetersenii serovar Tarassovi, and three as L. interrogans serovar Bratislava. Pathogenic Leptospira DNA were detected in 32 wild boar kidneys (11.15%). The characterization through the ampliﬁcation of the rrs2 gene highlighted their belonging to L. interrogans (23 kidneys), L. borgpetersenii (four), and L. kirschneri (one), while nine kidneys (3.14%) were positive for intermediate Leptospira, all belonging to L. fainei. The results of this study conﬁrmed the importance of wild boar in the epidemiology of leptospirosis among wildlife in Central Italy. Keywords: leptospirosis; zoonosis; infectious disease; multilocus sequence typing (MLST); wildlife; Leptospira fainei; MAT; intermediate Leptospira Article Giovanni Cilia , Fabrizio Bertelloni * , Marta Angelini, Domenico Cerri and Filippo Fratini Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy; giovanni.cilia@vet.unipi.it (G.C.); m.angelini5@studenti.unipi.it (M.A.); domenico.cerri@unipi.it (D.C.); ﬁlippo.fratini@unipi.it (F.F.) * Correspondence: fabrizio.bertelloni@unipi.it; Tel.: +39-050-2216-969 Pathogens 2020, 9, 377; doi:10.3390/pathogens9050377 www.mdpi.com/journal/pathogens pathogens pathogens pathogens pathogens pathogens Keywords: leptospirosis; zoonosis; infectious disease; multilocus sequence typing (MLST); wildlife; Leptospira fainei; MAT; intermediate Leptospira 1. Introduction Wild boar (Sus scrofa) is a large ungulate mammal with worldwide distribution. It can live in several types of habitat, including urban and suburban areas [1,2]. Due to their high adaptability, wild boar populations have rapidly increased in number during recent years, in Europe, and especially in Italy [1,3]. In Italy, wild boar is largely spread in all areas, from the Alps to the southern part of the Italian peninsula, including the islands. There is a high density, particularly in speciﬁc regions, such as Tuscany [3–5]. The abundant presence of wild boar in the Tuscany region, as well in Central Italy, is suggested by the very high number of animals hunted in this area; every year the hunting of about 42,000 specimens is registered [1,3–5]. The massive presence of wild boar in particular areas, other than representing an important source of damage for agriculture [6], can be a severe risk to human and animal health, due to the identiﬁcation of wild boar as reservoir for many etiological agents; among them typical zoonoses, such as Leptospira [7–9]. Leptospirosis is a re-emerging zoonotic disease with worldwide spread. It is caused by Leptospira spp., a Gram-negative spirochetal bacterium [10–12]. The genus Leptospira is divided into more than 260 antigenically-diﬀerent serovars, classiﬁed as pathogenic, intermediate, and saprophytic, with diﬀerent levels of pathogenicity for animals and humans [13,14]. While pathogenic Leptospira cause mild or severe infection, intermediate Leptospira could possibly be pathogenic, causing mild infection, while 2 of 16 Pathogens 2020, 9, 377 saprophytic Leptospira are present in the environment and are non-pathogenic [13,14]. Intermediate and saprophytic Leptospira could be important due to the strictly-contact and recombination events with pathogenic serovars [15–17]. Leptospirosis occurs in tropical, subtropical, and temperate zones, where it is maintained by a large variety of both wild and domestic mammals which can play the role of Leptospira maintenance host [18–21]. The reservoir organisms generally do not develop symptoms, except after a long time [11,12]. Leptospira renal-carrying/-colonization/-localization in asymptomatic animals contributes to the maintenance of infection in a particular environment by constantly shedding bacteria through their urine. Accidental contact with Leptospira-infected animal urine causes incidental infection, and produces clinical diseases in so-called “incidental hosts” [11,21]. 1. Introduction Swine, including wild boar and pig, are recognized as maintenance hosts for Pomona, Tarassovi, and Bratislava serovars [21], but can be infected by other Leptospira serovars, in relation to both geographic area where the population lives and their behavior [22–26]. The epidemiology of leptospirosis may change over time in domestic and wild animals, and some serovars seems to be prevalent and emerging [26,27]. Moreover, intermediate Leptospira DNA has been detected in the kidneys of wild boar hunted in Liguria region (Italy), suggesting a possible infection [7]. Tuscany, as well as all of Central Italy, is a geographic area that promotes the presence and the persistence of Leptospira in the ecosystem. The features of Leptospira-spreading are the presence of several wild animals involved as reservoir, domestic animals bred in extensive farms in contact with wildlife, high presence of hunting activity, and abundance of wetlands, such as marshes, ponds, and irrigation canals [9,26,28–33]. The aim of this investigation was to detect and characterize pathogenic, intermediate, and saprophytic Leptospira in wild boar hunted in Tuscany region during two hunting seasons (2018/ 2019 and 2019/2020), in order to delineate the risk for the transmission and spreading of leptospirosis to domestic animals and humans. 2. Results Serum, kidney, and liver samples were collected from a total of 287 hunted wild boar. Two hundred wild boar were sampled during 2018/2019 hunting season— 75 from Grosseto province, 58 from Pisa province, 55 from Siena province, and 12 from Livorno province (Figure 1). In addition, 87 specimens were sampled during 2019/2020 hunting seasons with 38, 37, and 12 from Pisa, Grosseto, and Lucca provinces, respectively (Figure 1). Pathogens 2020, 9, x FOR PEER REVIEW 3 of 16 A B Figure 1. Geographical distribution of the sampling area included in the study (Tuscany region, Italy). The number of sampled hunted wild boar per province is indicated in relation to hunting seasons. Figure 1. Geographical distribution of the sampling area included in the study (Tuscany region, Italy). The number of sampled hunted wild boar per province is indicated in relation to hunting seasons. (A) Hunting season 2018/2019; (B) Hunting season 2019/2020. B A B A Figure 1. Geographical distribution of the sampling area included in the study (Tuscany region, Italy). Th b f l d h t d ild b i i i di t d i l ti t h ti Figure 1. Geographical distribution of the sampling area included in the study (Tuscany region, Italy). The number of sampled hunted wild boar per province is indicated in relation to hunting seasons. (A) Hunting season 2018/2019; (B) Hunting season 2019/2020. Pathogens 2020, 9, 377 3 of 16 Pathogens 2020, 9, 377 Pathogens 2020, 9, 377 Results on distribution of positive sera and kidney for pathogenic Leptospira in relation to hunting season, province, sex, and age class are reported in Table 1. Results on distribution of positive sera and kidney for pathogenic Leptospira in relation to hunting season, province, sex, and age class are reported in Table 1. Table 1. Distribution of positive sera and kidney for pathogenic Leptospira in relation to hunting season, province, sex, and age class. 2.1. Microscopic Agglutination Test (MAT) Overall, 39 out of 287 sera (13.59%) were positive in the MAT (Table 2). Considering each hunting season, seropositivity of 16% (32 out of 200 sera) was recorded during 2018/2019, while 8.05% (7 out of 87) was recorded during 2019/2020. Considering wild boar sex, 16 out of 118 male sera (13.55%) and 23 out of 169 (13.61%) were positive in the MAT. Moreover, in relation to age class, 20 out of 142 adult specimens’ sera (14.08%), 7 out of 42 subadult specimens’ sera (16.67%) and 12 out of 100 young specimens’ sera (12.00%) were positive in serological analysis. Table 2. Numbers of positive serological reactions detected for wild boar sera in relation to diﬀerent Leptospira serogroups at low (1:100) and high titers (1:12,800). Leptospira Serogroup Titer Total (%) 100 200 400 800 1600 3200 6400 12800 Icterohaemorrhagiae 1 1 (2.56%) Canicola 1 1 2 (5.13%) Pomona 8 1 3 12 (30.8%) Grippotyphosa Tarassovi 4 1 1 1 1 1 9 (23.1%) Australis 5 5 2 1 1 14 (35.9%) Sejroe Ballum 1 1 (2.56%) Total 17 3 10 2 2 2 2 1 39 (100%) Australis resulted the most-recorded serogroup (4.88%), followed by Pomona (4.18%), and Tarassovi (3.14%). Other antibody titers were reported for serogroup Canicola (0.70%) and for serogroups Icterohaemorrhagiae and Ballum (0.45%). The highest titer detected was 1:12,800 for serogroup Tarassovi, followed by titer of 1:6400, which was reported for serogroups Tarassovi and Ballum. Results on distribution of positive sera detected by MAT in relation to hunting season, province, sex, and age class are reported in Table 1. No statistical diﬀerences (p > 0.05) were reported for the serological positivity considering hunting seasons, provinces, and wild boar sex and age class. Table 2. Numbers of positive serological reactions detected for wild boar sera in relation to diﬀerent Leptospira serogroups at low (1:100) and high titers (1:12,800). Leptospira Serogroup Titer Total (%) 100 200 400 800 1600 3200 6400 12800 Icterohaemorrhagiae 1 1 (2.56%) Canicola 1 1 2 (5.13%) Pomona 8 1 3 12 (30.8%) Grippotyphosa Tarassovi 4 1 1 1 1 1 9 (23.1%) Australis 5 5 2 1 1 14 (35.9%) Sejroe Ballum 1 1 (2.56%) Total 17 3 10 2 2 2 2 1 39 (100%) Table 2. Numbers of positive serological reactions detected for wild boar sera in relation to diﬀerent Leptospira serogroups at low (1:100) and high titers (1:12,800). 2. Results Hunting Season Province Sex Age Class Examined Wild Boar MAT-Positive Sera (%) PCR-Positive Kidneys (%) 2018/2019 Pisa Male Adult 9 2 (22.2) 1 (11.1) (n = 200) (n = 58) (n = 30) Subadult 10 2 (20.0) 4 (40.0) Young 11 3 (27.3) 0 Female Adult 14 2 (14.3) 2 (14.3) (n = 28) Subadult 5 1 (20.0) 2 (40.0) Young 9 1 (11.1) 2 (22.2) Grosseto Male Adult 10 2 (20.0) 2 (20.0) (n = 75) (n = 29) Subadult 5 1 (20.0) 0 Young 14 1 (7.1) 3 (21.4) Female Adult 22 2 (9.09) 1 (4.6) (n = 46) Subadult 5 0 0 Young 19 2 (10.5) 4 (21.1) Siena Male Adult 10 2 (20.0) 0 (n = 55) (n = 22) Subadult 4 1 (25.0) 0 Young 8 0 1 (12.5) Female Adult 21 5 (23.8) 3 (14.3) (n = 33) Subadult 2 0 0 Young 10 1 (10.0) 3 (30.0) Livorno Male Adult 2 0 0 (n = 12) (n = 4) Subadult 0 0 0 Young 2 1 (50.0) 1 (50.0) Female Adult 4 1 (25.0) 1 (25.0) (n=8) Subadult 0 0 0 Young 4 2 (50.0) 1 (25.0) 2019/2020 Pisa Male Adult 6 0 0 (n = 87) (n = 38) (n = 13) Subadult 4 0 0 Young 3 0 0 Female Adult 21 2 (9.52) 0 (n = 25) Subadult 1 1 (100) 0 Young 3 0 1 (33.3) Grosseto Male Adult 11 1 (9.09) 0 (n = 37) (n = 16) Subadult 1 0 0 Young 4 0 0 Female Adult 10 1 (10.0) 0 (n = 21) Subadult 5 1 (20.0) 0 Young 6 1 (16.7) 0 Lucca Male Adult 1 0 0 (n=12) (n = 4) Subadult 0 0 0 Young 3 0 0 Female Adult 4 0 0 (n = 8) Subadult 0 0 0 Young 4 0 0 Table 1. Distribution of positive sera and kidney for pathogenic Leptospira in relation to hunting season, province, sex, and age class. Table 1. Distribution of positive sera and kidney for pathogenic Leptospira in relation to hunting season, province, sex, and age class. 4 of 16 Pathogens 2020, 9, 377 2.1. Microscopic Agglutination Test (MAT) Australis resulted the most-recorded serogroup (4.88%), followed by Pomona (4.18%), and Tarassovi (3.14%). Other antibody titers were reported for serogroup Canicola (0.70%) and for serogroups Icterohaemorrhagiae and Ballum (0.45%). The highest titer detected was 1:12,800 for serogroup Tarassovi, followed by titer of 1:6400, which was reported for serogroups Tarassovi and Ballum. Results on distribution of positive sera detected by MAT in relation to hunting season, province, sex, and age class are reported in Table 1. No statistical diﬀerences (p > 0.05) were reported for the serological positivity considering hunting seasons, provinces, and wild boar sex and age class. Moreover, comparing all parameters, no statistical diﬀerences (p > 0.05) were showed in Pisa and Grosseto during the two diﬀerent hunting seasons. 2.3. Leptospira spp. Isolation, Characterization and Genotyping Four Leptospira cultures were positive after 30 days of incubation. The results, reported in Table 3, show that three isolates were obtained from subadult males hunted in Pisa province, while the other one was from an adult female hunted in Livorno. Through multilocus sequence typing (MLST) analysis, one isolate was identiﬁed as Leptospira borgpetersenii serogroup Tarassovi serovar Tarassovi (Sequence Type 153), while the other three were identiﬁed as L. interrogans serogroup Bratislava serovar Bratislava (ST 24), as reported in Table 3. Moreover, the ampliﬁcation of the rrs2 gene from kidney tissue highlights that the species belonged to L. borgpetersenii and L. interrogans, respectively. Table 3. Characterization of wild boar Leptospira isolates tested with anti-sera and multilocus sequence typing (MLST). Characterization of wild boar Leptospira isolates tested with anti-sera and multilocus sequence Table 3. Characterization of wild boar Leptospira isolates tested with anti-sera and multilocus sequence typing (MLST). Sample Wild Boar Isolates Characterization Sex Age Class Province Anti-Serum MAT Serogroup MLST (Sequence Type) Kidney 5 Male Subadult Pisa Tarassovi Tarassovi (ST 153) Kidney 14 Male Subadult Pisa Australis Bratislava (ST 24) Kidney 15 Male Subadult Pisa Australis Bratislava (ST 24) Kidney 22 Female Adult Livorno Australis Bratislava (ST 24) With regard to characterization of PCR-positive samples, ampliﬁcation of the rr2 gene highlighted that pathogenic Leptospira belonged to L. interrogans (23 kidneys), L. borgpetersenii (four) and L. kirschneri (one) (Table 4). Moreover, phylogenetic analysis identiﬁed the close relationship to their respective Leptospira species. (Figure 2). ble 4. Characterization of Leptospira species in wild boar pathogenic Leptospira-positive R-amplifying rrs2 gene. Table 4. Characterization of Leptospira species in wild boar pathogenic Leptospira-positive PCR-amplifying rrs2 gene. Sample Wild Boar Isolate Characterization Sex Age Class Province Leptospira Species Kidney 1 Female Young Pisa L. borgpetersenii Kidney 2 Female Subadult Pisa L. interrogans Kidney 3 Male Adult Pisa L. borgpetersenii Kidney 4 Female Young Pisa L. interrogans Kidney 6 Male Young Siena L. interrogans Kidney 7 Female Young Siena L. interrogans Kidney 12 Female Young Siena L. interrogans Kidney13 Female Adult Siena L. interrogans Kidney 20 Male Young Grosseto L. interrogans Kidney 24 Female Young Livorno L. interrogans Kidney 36 Female Adult Grosseto L. interrogans Kidney39 Female Young Grosseto L. interrogans Kidney 42 Female Young Siena L. interrogans Kidney 53 Female Young Grosseto L. interrogans Kidney 54 Male Young Grosseto L. kirschneri Kidney 55 Male Young Grosseto L. interrogans Kidney 58 Female Adult Pisa L. 2.2. Molecular Analysis Concerning pathogenic Leptospira, DNA was detected in 11.15% (32 out of 297) of wild boar kidneys. Table 1 shows PCR-positive kidneys in relation to hunting seasons, province and wild boar sex and age class. During the 2018/2019 and 2019/2020 hunting seasons, 15.5% (31 out of 200) and 1.15% (1 out of 87) of PCR positivity was reported among kidneys samples, respectively. Considering wild boar sex, 12 out of 118 male sera (10.16%) and 20 out of 169 (11.83%) scored positive. Moreover, in relation to age class, 10 out of 142 adult specimens’ kidneys (7.04%), 6 out of 42 subadult specimens’ kidneys (14.28%), and 16 out of 100 young specimens’ kidneys (16.00%) gave positive results in serological analysis. No statistical diﬀerences (p > 0.05) were highlighted comparing province, wild boar sex, or age class. Conversely, the incidence of pathogenic Leptospira-positive kidneys was statically higher (p ≤0.01) during 2018/2019 hunting season compared to the 2019/2020 ones. The detection of pathogenic Leptospira DNA was higher (p ≤0.01) during 2018/2019 hunting season in both Pisa and Grosseto provinces compared to the second hunting season. On the contrary, there were no statistical diﬀerences (p > 0.05) in the Pisa and Grosseto provinces during the two diﬀerent hunting seasons, comparing sex and age class of wild boar. The 3.14% (9 out of 287) of kidneys were positive for intermediate Leptospira. The positivity in relation to hunting seasons, province, wild boar sex, and age class are showed in Table 1. All the intermediate Leptospira-positive kidneys (4.5%; 9 out of 200) were collected in 2018/2019, highlighting Pathogens 2020, 9, 377 5 of 16 a statistical diﬀerence (p ≤0.01) in relation to 2019/2020 hunting season. Also, the results showed a statistically-higher infection rate (p ≤0.01) in male compared to female, and in Pisa province compared to other provinces. No statistical diﬀerence (p > 0.05) were noted among age classes. p p g g No saprophytic Leptospira DNA was detected in kidney samples. No positive reactions were recorded in wild boar livers across all specimens during the two year of investigation. 2.3. Leptospira spp. Isolation, Characterization and Genotyping Molecular phylogenetic analysis for the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri by the maximum likelihood method, based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 31 nucleotide sequences. There was a total of 452 positions in the ﬁnal dataset. Moreover, the ampliﬁcation of intermediate Leptospira 16s rRNA DNA of PCR-positive specim wed L. fainei in all nine kidneys (Table 5). Furthermore, the phylogenetic analysis identiﬁed e relationship to L. fainei specie. (Figure 3). Figure 2. Molecular phylogenetic analysis for the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri by the maximum likelihood method, based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis i l d 31 l tid Th t t l f 452 iti i th fi l d t t Figure 2. Molecular phylogenetic analysis for the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri by the maximum likelihood method, based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 31 nucleotide sequences. There was a total of 452 positions in the ﬁnal dataset. Figure 2. Molecular phylogenetic analysis for the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri by the maximum likelihood method, based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis i l d 31 l tid Th t t l f 452 iti i th fi l d t t Figure 2. Molecular phylogenetic analysis for the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri by the maximum likelihood method, based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 31 nucleotide sequences. There was a total of 452 positions in the ﬁnal dataset. i vo ved 3 uc eotide seque ces. e e was a tota o 5 positio s i t e i a dataset. Moreover, the amplification of intermediate Leptospira 16s rRNA DNA of PCR-positive specimens showed L. fainei in all nine kidneys (Table 5). Furthermore, the phylogenetic analysis identified the close relationship to L fainei specie (Figure 3) Moreover, the ampliﬁcation of intermediate Leptospira 16s rRNA DNA of PCR-positive specimens showed L. 2.3. Leptospira spp. Isolation, Characterization and Genotyping borgpetersenii Kidney 64 Female Adult Siena L. interrogans Kidney 65 Female Adult Siena L. interrogans Kidney 67 Female Adult Pisa L. interrogans Kidney 68 Female Subadult Pisa L. interrogans Kidney 71 Male Adult Grosseto L. interrogans Kidney 72 Female Young Grosseto L. borgpetersenii Kidney 75 Male Subadult Pisa L. interrogans Kidney 78 Male Young Livorno L. interrogans Kidney 97 Male Adult Grosseto L. interrogans Kidney 165 Female Young Grosseto L. interrogans Kidney 208 Female Young Pisa L. interrogans 6 of 16 Pathogens 2020, 9, 377 Kidne Kidne ogens 2020, 9, 377 6 of 1 Kidney 165 Female Young Grosseto L. interrogans Kidney 208 Female Young Pisa L. interrogans Figure 2. Molecular phylogenetic analysis for the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri by the maximum likelihood method, based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 31 nucleotide sequences. There was a total of 452 positions in the final dataset. Moreover, the amplification of intermediate Leptospira 16s rRNA DNA of PCR-positive imens showed L. fainei in all nine kidneys (Table 5). Furthermore, the phylogenetic analysis tified the close relationship to L. fainei specie. (Figure 3). Figure 2. Molecular phylogenetic analysis for the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri by the maximum likelihood method, based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 31 nucleotide sequences. There was a total of 452 positions in the ﬁnal dataset. Moreover, the ampliﬁcation of intermediate Leptospira 16s rRNA DNA of PCR-positive specimen wed L. fainei in all nine kidneys (Table 5). Furthermore, the phylogenetic analysis identiﬁed th e relationship to L. fainei specie. (Figure 3). Figure 2. Molecular phylogenetic analysis for the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri by the maximum likelihood method, based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 31 nucleotide sequences. There was a total of 452 positions in the final dataset. Moreover, the amplification of intermediate Leptospira 16s rRNA DNA of PCR-posi imens showed L. fainei in all nine kidneys (Table 5). Furthermore, the phylogenetic anal tified the close relationship to L. fainei specie. (Figure 3). Figure 2. 2.3. Leptospira spp. Isolation, Characterization and Genotyping fainei in all nine kidneys (Table 5). Furthermore, the phylogenetic analysis identiﬁed the close relationship to L. fainei specie. (Figure 3). q p Moreover, the amplification of intermediate Leptospira 16s rRNA DNA of PCR-positive specimens showed L. fainei in all nine kidneys (Table 5). Furthermore, the phylogenetic analysis identified the close relationship to L fainei specie (Figure 3) Moreover, the ampliﬁcation of intermediate Leptospira 16s rRNA DNA of PCR-positive specimens showed L. fainei in all nine kidneys (Table 5). Furthermore, the phylogenetic analysis identiﬁed the close relationship to L. fainei specie. (Figure 3). 7 of 16 Pathogens 2020, 9, 377 Table 5. Characterization of Leptospira species in wild boar intermediate Leptospira-positive PCR-amplifying 16s rRNA gene. athogens 2020, 9, x FOR PEER REVIEW Sample Wild Boar Isolate Characterization Table 5. Characterization of Leptospira species in wild boar intermediate Leptospira-positive PCR-amplifying 16s rRNA gene. Sample Wild Boar Isolate Characterization Sex Age Class Province Leptospira Species Kidney 23 Male Young Livorno L. fainei Kidney 27 Male Adult Pisa L. fainei Kidney 31 Female Adult Pisa L. fainei Kidney 56 Male Young Grosseto L. fainei Kidney 57 Male Adult Pisa L. fainei Kidney 63 Male Adult Siena L. fainei Kidney 69 Female Subadult Pisa L. fainei Kidney123 Male Adult Livorno L. fainei Kidney 153 Male Adult Siena L. fainei Sample Wild Boar Isolate Characterization Sex Age Class Province Leptospira Species Kidney 23 Male Young Livorno L. fainei Kidney 27 Male Adult Pisa L. fainei Kidney 31 Female Adult Pisa L. fainei Kidney 56 Male Young Grosseto L. fainei Kidney 57 Male Adult Pisa L. fainei Kidney 63 Male Adult Siena L. fainei Kidney 69 Female Subadult Pisa L. fainei Kidney123 Male Adult Livorno L. fainei Kidney 153 Male Adult Siena L. fainei Figure 3. Molecular phylogenetic analysis for 16s rRNA gene of Leptospira fainei, Leptospira inada Leptospira broomii, Leptospira wolffii, Leptospira licerasiae, and Leptospira venezuelensis by the maximum likelihood method based on the Tamura–Nei model. The branch lengths of the tree measured th number of substitutions per site. The analysis involved 22 nucleotide sequences. There was a total 438 positions in the final dataset. Figure 3. Molecular phylogenetic analysis for 16s rRNA gene of Leptospira fainei, Leptospira inadai, Leptospira broomii, Leptospira wolﬃi, Leptospira licerasiae, and Leptospira venezuelensis by the maximum likelihood method based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. 2.3. Leptospira spp. Isolation, Characterization and Genotyping The analysis involved 22 nucleotide sequences. There was a total of 438 positions in the ﬁnal dataset. Sample Wild Boar Isolate Characterization Sex Age Class Province Leptospira Species Kidney 23 Male Young Livorno L. fainei Kidney 27 Male Adult Pisa L. fainei Kidney 31 Female Adult Pisa L. fainei Kidney 56 Male Young Grosseto L. fainei Kidney 57 Male Adult Pisa L. fainei Kidney 63 Male Adult Siena L. fainei Kidney 69 Female Subadult Pisa L. fainei Kidney123 Male Adult Livorno L. fainei Kidney 153 Male Adult Siena L. fainei a p e Sex Age Class Province Leptospira Species Kidney 23 Male Young Livorno L. fainei Kidney 27 Male Adult Pisa L. fainei Kidney 31 Female Adult Pisa L. fainei Kidney 56 Male Young Grosseto L. fainei Kidney 57 Male Adult Pisa L. fainei Kidney 63 Male Adult Siena L. fainei Kidney 69 Female Subadult Pisa L. fainei Kidney123 Male Adult Livorno L. fainei Kidney 153 Male Adult Siena L. fainei Figure 3. Molecular phylogenetic analysis for 16s rRNA gene of Leptospira fainei, Leptospira inada Leptospira broomii, Leptospira wolffii, Leptospira licerasiae, and Leptospira venezuelensis by the maximum likelihood method based on the Tamura–Nei model. The branch lengths of the tree measured th number of substitutions per site. The analysis involved 22 nucleotide sequences. There was a total o Figure 3. Molecular phylogenetic analysis for 16s rRNA gene of Leptospira fainei, Leptospira inadai, Leptospira broomii, Leptospira wolﬃi, Leptospira licerasiae, and Leptospira venezuelensis by the maximum likelihood method based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 22 nucleotide sequences. There was a total of 438 positions in the ﬁnal dataset. 438 posit 3. Discussion Unfortunately, serological data about leptospirosis in wildlife, especially regarding wild boar, are available just in some regions. It also seems that Leptospira serovars/serogroups have a diﬀerent geographical distribution, suggesting a distinct circulation/epidemiology in other environments/ecosystems. Examining each region, Australis, Pomona, and Tarassovi, are the most-detected serogroups in the Tuscany region [22,26,31,33], In the Lombardy and Emilia Romagna regions it is Bratislava [37–39], in the Campania region it is Tarassovi [35], whereas in the Sardinia region it is Pomona and Grippotyphosa [36]. The distribution of Leptospira serovars in wild boar in Europe is also not homogeneous; high levels of Pomona infection was recorded in Germany, Croatia, Poland, and Spain [23,40–42]. Bratislava was the most-detected serovar in Sweden [25], Tarassovi in Portugal and Slovenia [24,43], Grippotyphosa in Czech Republic [44], and Hardjo in Poland [23]. Little information is available on Leptospira isolation in wild boar, especially in Italy [38]. The obtained Leptospira isolates, identiﬁed by MLST, conﬁrm the circulation of Tarassovi and Bratislava serogroups within wild boar in Tuscany. Bratislava isolation is commonly performed in wildlife due to the high spectrum of maintenance hosts [45–47], while Tarassovi is rarely isolated and detected through serology. Indeed, Tarassovi is strictly a swine-speciﬁc serovar; its isolation, reported in this investigation, seems to conﬁrm the hypothesis that wild boar could serve as reservoir of Tarassovi [26,43,48]. Only two of them (Bratislava, isolated from subadult from Pisa province; Table 3) reported correlated serological positivity for serogroup Australis at titer 1:100, while the other two gave negative results in the MAT. No correlation was found between the MAT and PCR-positive results. The seronegativity of Leptospira-positive kidneys has been previously reported for other animal species [49–52], including swine [47], suggesting an early or chronic infection. Conversely to serological results, very few studies were performed on pathogenic Leptospira DNA in wild boar kidneys. In spite of this, the prevalence of pathogenic Leptospira infection reported during the years of this investigation was very close to the results obtained in Northern Italy (11.02%) [38] and in the Liguria region (12.13%) [7]. Moreover, prevalences of 10.30% and 15.3% were found in two diﬀerent investigations performed in Japan [53,54], while 3.40% was reported in the USA [55]. Based on phylogenetic analysis, pathogenic Leptospira DNA in wild boar kidney belong to L. interrogans, L. borgpetersenii, and L. kirschneri. 438 posit 3. Discussion 3. Discussion Leptospirosis is a re-emerging worldwide public health risk, but is underestimated and characterized by a downward trend [34]. Climatic changes, rainfall, modifications of ecologica niches, and new potential maintenance hosts all represent important features involved in Leptospir Leptospirosis is a re-emerging worldwide public health risk, but is underestimated and characterized by a downward trend [34]. Climatic changes, rainfall, modiﬁcations of ecological niches, and new potential maintenance hosts all represent important features involved in Leptospira epidemiology. niches, and new potential maintenance hosts all represent important features involved in Leptospir epidemiology. Wild boar, among wildlife, is an important Leptospira reservoir and, for several areas, represent Wild boar, among wildlife, is an important Leptospira reservoir and, for several areas, represents an appropriate indicator for this zoonotic infectious disease. Wild boar, among wildlife, is an important Leptospira reservoir and, for several areas, represents an appropriate indicator for this zoonotic infectious disease. In this investigation we reported the results of serological analysis, isolation and molecula investigations performed on 287 hunted wild boar during two hunting seasons (2018/2019 and In this investigation we reported the results of serological analysis, isolation and molecular investigations performed on 287 hunted wild boar during two hunting seasons (2018/2019 and 2019/2020). i oa , a o g i i e, i a i po a ep o pi a e e oi a , o e e a a ea , ep e e an appropriate indicator for this zoonotic infectious disease. In this investigation we reported the results of serological analysis, isolation and molecula investigations performed on 287 hunted wild boar during two hunting seasons (2018/2019 and In this investigation we reported the results of serological analysis, isolation and molecular investigations performed on 287 hunted wild boar during two hunting seasons (2018/2019 and 2019/2020). investigations performed on 287 hunted wild boar during two hunting seasons (2018/2019 and 2019/2020). With regard to serological assay, the prevalence of Leptospira infection, recorded in both hunting seasons, was very similar to other studies carried out on wild boar in Tuscany [9,22,26,31,33] With regard to serological assay, the prevalence of Leptospira infection, recorded in both hunting seasons, was very similar to other studies carried out on wild boar in Tuscany [9,22,26,31,33]. Moreover, the seroprevalence reported in this investigation was very close to other data obtained in diﬀerent 8 of 16 Pathogens 2020, 9, 377 Italian regions [27,35–38]. 438 posit 3. Discussion With respect to the serovars in Italy that are more often detected through isolation or serology, [27,38,56–59] and the other serovars that are rarely seropositive [28,60], it might be hypothesized that L. kirschneri species found in wild boar kidneys could be related to serogroup Grippotyphosa, while L. borgpetersenii species could be related to the serogroups Tarassovi or Ballum. On the other hand, it is very diﬃcult to infer the serogroup related to L. interrogans positivity, due to the inclusion of Icterohaemorrhagiae, Canicola, Pomona, Australis, and Sejroe serogroups in this species. Probably, in relation to serological and bacteriological results obtained in this study, the identiﬁed L. interrogans could belong to serogroup Australis. The data reported in this investigation suggest that the liver does not seem be a Leptospira target organ in wild boar. Furthermore, it could exclude an early stage of infection (leptospiremia) and conﬁrm that positive animals are only chronic renal carriers, as also suggested by isolation from kidneys. If very little information is available on pathogenic Leptospira DNA in wild boar, there is even less data on intermediate Leptospira. To the best of these authors’ knowledge, it was only in the Liguria region of Italy that 0.49% of wild boar kidneys were positive for intermediate Leptospira DNA in the same year of this investigation [7]. As Liguria and Tuscany are two adjoining Italian regions, a large wild boar movement could be a feature of these regions [61–63]. Even though the species of intermediate Leptospira from Liguria were not identiﬁed, those found in this investigation belong to L. fainei species. L. fainei was isolated for the ﬁrst time from ﬁg and was detected in human sera in Australia [64,65] and a human infection with febrile status was reported in France (from a Portuguese citizen) and in two patients in Denmark [66,67]. Considering wild boar behavior and its ability to live in anthropomorphic environment, a transmission between human and wildlife could be possible. 9 of 16 Pathogens 2020, 9, 377 As these are the ﬁrst determination in European wildlife, more studies are needed to understand the epidemiology of this intermediate Leptospira that could causes severe infection in humans [65–67]. The statistical diﬀerence presented during the hunting seasons between pathogenic and intermediate Leptospira incidence in wild boar could be related to the temperature and the amount of rainfall recorded in Tuscany during these periods. 438 posit 3. Discussion As reported in literature, rainfall and temperature inﬂuence the incidence of leptospirosis in humans and animals [12,68–75]. Indeed, from 2018 to 2019, the temperatures and the rainfall were both higher than those from 2019 to 2020 [76–81], suggesting that these atmospheric phenomena could be involved in these seasonality incidence diﬀerences. 4.1. Sample Collection During two hunting seasons (the ﬁrst from November 2018 to January 2019 and the second from November 2019 to January 2020) hunted wild boar blood, kidney, and liver were sampled. Blood samples were collected by ocular puncture [82]. The boar’s age class was determined after assessing the degree of tooth eruption and the wear and tear of teeth of the lower jaw, considering three age classes: young (under 12 months old), sub-adult (between 12 and 24 months), and adult (over 24 months old). The animal’ sex was also recorded [83]. All animals were hunted in the Tuscany region during authorized hunting seasons (November–January), following the regional hunting law (Regolamento di attuazione della legge regionale 12 gennaio 1994, n. 3 D.P.G.R. 48/R/2017). No animals were speciﬁcally sacriﬁced for this study purpose. Animals did not present gross lesions related to infectious disease at postmortem examination, performed during sampling operations. 4.2. Microscopic Agglutination Test (MAT) Blood samples were centrifugated at 10,000 rpm for 10 minutes to obtain the serum. In order to detect Leptospira antibodies, sera were tested through microscopic agglutination test (MAT) [84]. Titer of 1:100 was considered as positive. For the MAT, live Leptospira antigens used were: Leptospira interrogans serovar Icterohaemorrhagiae (serogroup Icterohaemorrhagiae, strain RGA), L. interrogans serovar Canicola (serogroup Canicola, strain Alarik), L. interrogans serovar Pomona (serogroup Pomona, strain Mezzano), L. kirschneri serovar Grippotyphosa (serogroup Grippotyphosa, strain Moskva V), L. borgpetersenii serovar Tarassovi (serogroup Tarassovi, strain Mitis Johnson), L. interrogans serovar Bratislava (serogroup Australis, strain Riccio 2), L. interrogans serovar Hardjo (serogroup Sejroe, serovar Hardjoprajitno), and L. borgpetersenii serovar Ballum (serogroup Ballum, strain Mus 127). 4.3. Leptospira spp. Isolation 4.3. Leptospira spp. Isolation Each wild boar organ was cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (Difco, Detroit, MI, USA). Approximately 10 cm3 from each organ was homogenized with 5 mL of sterile water and 1 mL of homogenate was cultured in 5 mL of EMJH. Cultures were incubated at 30 ◦C ± 1 ◦C for 120 days and observed every 10 days under dark-ﬁeld microscopy to evaluate possible bacterial growth. 4.4. Molecular Analysis From each kidney and liver, DNA was extracted using Quick-DNA Plus Kits (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions. Two diﬀerent multiplex Realtime-PCR were employed; the ﬁrst, targeting Leptospira spp. (16S rRNA gene) and pathogenic Leptospira (lipL32 gene), was performed on all samples [85,86]. The second protocol was only performed on positive Leptospira spp. and negative lipL32 samples, targeting intermediate Leptospira (16S rRNA gene) and saprophytic Leptospira (23S rRNA gene) [16,86]. As a positive control for the lipL32 gene, DNA extracted from a pure culture of Leptospira interrogans 10 of 16 10 of 16 Pathogens 2020, 9, 377 serogroup Pomona strain Mezzano was used. As a positive control for the 23S rRNA gene for saprophytic Leptospira, DNA extracted from a pure culture of Leptospira biﬂexa serogroup Patoc strain Patoc I was used. As a negative control, sterilized saline water was used. A total reaction volume of 15 µL was prepared by using 2x QuantiTect Probe PCR Master Mix (Qiagen, Hilden, Germany), 2 µM of each primer, 500 nM of each probe, and 3 µL of DNA, as previously reported [7]. The RealTime-PCR assay was performed on a Rotorgene Corbett 6000 (Corbett Research, Sydney, Australia) with the following thermal conditions: a holding stage of 95 ◦C for 5 min and 45 cycles of 95 ◦C for 15 s and 60 ◦C for 30 s. Samples with Ct lipL32 < 35 were considered positive and those samples with 35 < Ct lipL32 ≥40 were repeated. 4.5. Leptospira spp. Characterization and Genotyping First, serogroups of the isolates were determined through the MAT using a panel of eight polyclonal anti-sera against the eight serovars reported in Section 4.2. The agglutination with speciﬁc antiserum was used to identify the presumptive strain’s serogroup [84]. Isolated Leptospira were genotyped using a multilocus sequence typing (MLST) scheme based on housekeeping genes [87–89]. Moreover, the Leptospira species were identiﬁed from positive pathogenic and intermediate Leptospira PCR reactions, using primer for rrs2 gene and 16S rRNA gene, respectively [86,88]. Moreover, the Leptospira species were identiﬁed from positive pathogenic and intermediate Leptospira PCR reactions, using primer for rrs2 gene and 16S rRNA gene, respectively [86,88]. The ampliﬁcation of each target gene was realized with HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany), and further sequenced (BMR Genomics, Padova, Italy) using the same ampliﬁcation primer sets and analyzed using BioEdit Software [90]. Phylogenetic analysis was performed by the maximum likelihood method based on the Tamura–Nei model using MEGA 10 software [91]. 4.6. Statistical Analysis Data were analyzed with chi-square (X2) test. The statistical test was used to evaluate the Leptospira infection ratio in relationship to sex (male or female), age class (young, sub-adult, or adult), province (Pisa, Lucca, Livorno, Grosseto, or Siena) and hunting season (2018/2019 or 2019/2020). Statistical signiﬁcance threshold was set at a p value ≤0.05 [92]. References 1. Massei, G.; Kindberg, J.; Licoppe, A.; Gaˇci´c, D.; Šprem, N.; Kamler, J.; Baubet, E.; Hohmann, U.; Monaco, A.; Ozolin, š, J.; et al. Wild boar populations up, numbers of hunters down? A review of trends and implications for Europe. Pest Manag. Sci. 2015, 71, 492–500. [CrossRef] 2. Castillo-Contreras, R.; Carvalho, J.; Serrano, E.; Mentaberre, G.; Fernández-Aguilar, X.; Colom, A.; González-Crespo, C.; Lavín, S.; López-Olvera, J.R. Urban wild boars prefer fragmented areas with food resources near natural corridors. Sci. Total Environ. 2018, 615, 282–288. [CrossRef] 3. Santilli, F.; Varuzza, P. 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Lombardini, M.; Meriggi, A.; Fozzi, A. Factors inﬂuencing wild boar damage to agricultural crops in Sardinia (Italy). Curr. Zool. 2017, 63, 507–514. [CrossRef] [PubMed] 6. Lombardini, M.; Meriggi, A.; Fozzi, A. Factors inﬂuencing wild boar damage to agricultural crops in Sardinia (Italy). Curr. Zool. 2017, 63, 507–514. [CrossRef] [PubMed] 7. Cilia, G.; Bertelloni, F.; Mignone, W.; Spina, S.; Berio, E.; Razzuoli, E.; Vencia, W.; Franco, V.; Cecchi, F.; Bogi, S.; et al. Molecular detection of Leptospira spp. in wild boar (Sus scrofa) hunted in Liguria region (Italy). Comp. Immunol. Microbiol. Infect. Dis. 2020, 68, 101410. [CrossRef] [PubMed] 8. Meng, X.J.; Lindsay, D.S.; Sriranganathan, N. Wild boars as sources for infectious diseases in livest humans. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2009, 364, 2697–2707. [CrossRef] [PubMed] 9. Bertelloni, F.; Mazzei, M.; Cilia, G.; Forzan, M.; Felicioli, A.; Sagona, S.; Bandecchi, P.; Turchi, B.; Cerri, D.; 9. Bertelloni, F.; Mazzei, M.; Cilia, G.; Forzan, M.; Felicioli, A.; Sagona, S.; Bandecchi, P.; Turchi, B.; Cerri, D.; Fratini, F. 5. Conclusions Acknowledgments: We thank Mario D’Incau and his team of National Reference Centre for Animal Leptospirosis (Istituto Zooproﬁlattico Sperimentale della Lombardia e dell’ Emilia Romagna “Bruno Ubertini”, via Bianchi 7/9, 25121 Brescia, Italy) for provision of the Leptospira live strains employed in this investigation. Acknowledgments: We thank Mario D’Incau and his team of National Reference Centre for Animal Leptospirosis (Istituto Zooproﬁlattico Sperimentale della Lombardia e dell’ Emilia Romagna “Bruno Ubertini”, via Bianchi 7/9, 25121 Brescia, Italy) for provision of the Leptospira live strains employed in this investigation. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions In conclusion, this investigation conﬁrms through the MAT, isolation, and molecular assays, the role of wild boar in the epidemiology of leptospirosis in Central Italy. Wild boar represents a good indicator of Leptospira circulating in a speciﬁc area where many diﬀerent animal species share the same environment. Furthermore, wild boar populations are able to live in a wide spectrum of habitat types, and, have recently reached sub-urban and urban areas. In Italy, little recent data on human leptospirosis are available; however, some studies investigated the prevalence of infection in risk categories (hunters, farmers, and forestry workers) showing serological positivity to Leptospira [93,94]. Moreover, on the basis of the most recent report on human leptospirosis in Italy [95], a high infection rate was recorded in adult males, and this could indicate that leptospirosis is strictly related to worker activity. Hunters, for example, are usually all male and over 30 years old. In particular, these peoples are exposed to an high risk of infection due to management and slaughtering of dead animals being performed with little health care [96]. Tarassovi and Bratislava are the two main serogroups that circulate within wild boar in Tuscany. Although Bratislava has been more detected, the isolation of Tarassovi suggests that wild boar could be the main reservoir. In addition, as for pathogenic Leptospira, the presence of intermediate species in wild boar kidney underlines the need to perform other studies aimed at understanding the newly- emerging species, L. fainei, in animals and in humans. Pathogens 2020, 9, 377 11 of 16 11 of 16 Author Contributions: Conceptualization, G.C., F.B., and F.F.; investigation, G.C., F.B., and M.A.; data curation, G.C., F.B., M.A., D.C., and F.F.; writing—original draft preparation, G.C., F.B., and F.F.; writing—review and editing, G.C., F.B., M.A., D.C., and F.F.; supervision, D.C. and F.F.; funding acquisition, F.F. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, G.C., F.B., and F.F.; investigation, G.C., F.B., and M.A.; data curation, G.C., F.B., M.A., D.C., and F.F.; writing—original draft preparation, G.C., F.B., and F.F.; writing—review and editing, G.C., F.B., M.A., D.C., and F.F.; supervision, D.C. and F.F.; funding acquisition, F.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by University of Pisa, grant number PRA_2018_56. Funding: This research was funded by University of Pisa, grant number PRA_2018_56. References Serological Survey on Bacterial and Viral Pathogens in Wild Boars Hunted in Tuscany. Ecohealth 2020, 17, 85–93. [CrossRef] [PubMed] g Fratini, F. Serological Survey on Bacterial and Viral Pathogens in Wild Boars Hunted in Tuscany. Ecohealth 2020, 17, 85–93. [CrossRef] [PubMed] 10. Picardeau, M. 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https://openalex.org/W4320495329	https://www.frontiersin.org/articles/10.3389/fgene.2023.1061781/pdf	English	null	Extensive set of African ancestry-informative markers (AIMs) to study ancestry and population health	Frontiers in genetics	2,023	cc-by	9,465	OPEN ACCESS REVIEWED BY Cheryl D. Cropp, Samford University, United States Zhongshan Cheng, St. Jude Children’s Research Hospital, United States Samantha Boudeau1,2,3, Meganathan P. Ramakodi1,2,3†, Yan Zhou4, Jeffrey C. Liu5,6, Camille Ragin2,3* and Rob J. Kulathinal1,3* 1Department of Biology, Temple University, Philadelphia, PA, United States, 2Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, United States, 3African Caribbean Cancer Consortium, Fox Chase Cancer Center, Philadelphia, PA, United States, 4Department of Biostatistics and Bioinformatics, Fox Chase Cancer Center, Philadelphia, PA, United States, 5Department of Otolaryngology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, United States, 6Department of Surgical Oncology, Fox chase Cancer center, Philadelphia, PA, United States Introduction: Human populations are often highly structured due to differences in genetic ancestry among groups, posing difﬁculties in associating genes with diseases. Ancestry-informative markers (AIMs) aid in the detection of population stratiﬁcation and provide an alternative approach to map population-speciﬁc alleles to disease. Here, we identify and characterize a novel set of African AIMs that separate populations of African ancestry from other global populations including those of European ancestry. Methods: Using data from the 1000 Genomes Project, highly informative SNP markers from ﬁve African subpopulations were selected based on estimates of informativeness (In) and compared against the European population to generate a ﬁnal set of 46,737 African ancestry-informative markers (AIMs). The AIMs identiﬁed were validated using an independent set and functionally annotated using tools like SIFT, PolyPhen. They were also investigated for representation of commonly used SNP arrays. markers (AIMs) to study ancestry and population health. Front. Genet. 14:1061781. doi: 10.3389/fgene.2023.1061781 Results: This set of African AIMs effectively separates populations of African ancestry from other global populations and further identiﬁes substructure between populations of African ancestry. When a subset of these AIMs was studied in an independent dataset, they differentiated people who self-identify as African American or Black from those who identify their ancestry as primarily European. Most of the AIMs were found to be in their intergenic and intronic regions with only 0.6% in the coding regions of the genome. Most of the commonly used SNP array investigated contained less than 10% of the AIMs. COPYRIGHT © 2023 Boudeau, Ramakodi, Zhou, Liu, Ragin and Kulathinal. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). TYPE Original Research PUBLISHED 23 February 2023 DOI 10.3389/fgene.2023.1061781 TYPE Original Research PUBLISHED 23 February 2023 DOI 10.3389/fgene.2023.1061781 TYPE Original Research PUBLISHED 23 February 2023 DOI 10.3389/fgene.2023.1061781 KEYWORDS Extensive set of African ancestry- informative markers (AIMs) to study ancestry and population health OPEN ACCESS EDITED BY Yassine Souilmi, University of Adelaide, Australia REVIEWED BY Cheryl D. Cropp, Samford University, United States Zhongshan Cheng, St. Jude Children’s Research Hospital, United States CORRESPONDENCE Rob J. Kulathinal, robkulathinal@temple.edu Camille Ragin, camille.ragin@fccc.edu †PRESENT ADDRESS Meganathan P. Ramakodi, CSIR-NEERI, Hyderabad Zonal Centre, Hyderabad, Telangana, India SPECIALTY SECTION This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Genetics RECEIVED 05 October 2022 ACCEPTED 20 January 2023 PUBLISHED 23 February 2023 CITATION Boudeau S, Ramakodi MP, Zhou Y, Liu JC, Ragin C and Kulathinal RJ (2023), Extensive set of African ancestry-informative markers (AIMs) to study ancestry and population health. Front. Genet. 14:1061781. doi: 10.3389/fgene.2023.1061781 OPEN ACCESS EDITED BY Yassine Souilmi, University of Adelaide, Australia REVIEWED BY Cheryl D. Cropp, Samford University, United States Zhongshan Cheng, St. Jude Children’s Research Hospital, United States CORRESPONDENCE Rob J. Kulathinal, robkulathinal@temple.edu Camille Ragin, camille.ragin@fccc.edu †PRESENT ADDRESS Meganathan P. Ramakodi, CSIR-NEERI, Hyderabad Zonal Centre, Hyderabad, Telangana, India SPECIALTY SECTION This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Genetics RECEIVED 05 October 2022 ACCEPTED 20 January 2023 PUBLISHED 23 February 2023 CITATION Boudeau S, Ramakodi MP, Zhou Y, Liu JC, Ragin C and Kulathinal RJ (2023), Extensive set of African ancestry-informative markers (AIMs) to study ancestry and population health. Front. Genet. 14:1061781. doi: 10.3389/fgene.2023.1061781 OPEN ACCESS EDITED BY Yassine Souilmi, University of Adelaide, Australia REVIEWED BY Cheryl D. Cropp, Samford University, United States Zhongshan Cheng, St. Jude Children’s Research Hospital, United States *CORRESPONDENCE Rob J. Kulathinal, robkulathinal@temple.edu Camille Ragin, camille.ragin@fccc.edu †PRESENT ADDRESS Meganathan P. Ramakodi, CSIR-NEERI, Hyderabad Zonal Centre, Hyderabad, Telangana, India SPECIALTY SECTION This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Genetics RECEIVED 05 October 2022 ACCEPTED 20 January 2023 PUBLISHED 23 February 2023 CITATION Boudeau S, Ramakodi MP, Zhou Y, Liu JC, Ragin C and Kulathinal RJ (2023), Extensive set of African ancestry-informative markers (AIMs) to study ancestry and population health. Front. Genet. 14:1061781. doi: 10.3389/fgene.2023.1061781 Introduction socioeconomic factors (Molina et al., 2008) and access to healthcare (Ragin et al., 2011), suggesting a genetic basis to these differences. Racial health disparities in populations of African descent have been extensively documented and in the United States these disparities have been observed in many diseases (Tsai et al., 2011). For example, African Americans have signiﬁcantly greater mortality and morbidity for asthma and are nearly ﬁve times more likely to be diagnosed with primary open- angle glaucoma compared to Americans with European ancestry (Barnes, 2010; Cole et al., 2021). Based on data from US cancer registries for all malignancies combined, African Americans have worse cancer incidence and survival rates compared with European Americans (Özdemir & Dotto, 2017). Not surprisingly, there are certain cancers for which the racial health disparity is more pronounced. In head and neck cancer, African Americans possess poorer survival rates than their European American counterparts even though they have a similar incidence rate (Daraei & Moore, 2015; Zavala et al., 2021). Breast cancer also has pronounced racial disparities with African American women having a 40% higher mortality rate, younger age at diagnosis, and higher incidence of aggressive forms of the disease. Interestingly, African American males also have a higher incidence of breast cancer compared to European American males (Stringer-Reasor et al., 2021). Compared to men of European ancestry, men of African or Afro-Caribbean ancestry have been found to have a higher risk of developing more aggressive forms of prostate cancer at a younger age (McHugh et al., 2021). Socioeconomic factors including healthcare access, geographical factors, lifestyle, and other behavioral factors are routinely used to explain racial health disparities in cancer (Khan et al., 2019). Yet, in head and neck cancer studies, much of this disparity remains after accounting for Much of the literature investigating racial health disparities has relied on self-identiﬁed race as a proxy for genetic ancestry. However, current characteristics for determining race, including skin color, geography, and language, are often too vague to capture true genetic ancestry in disease studies (Hunt and Megyesi, 2008). Ancestry-informative markers (AIMs) are SNPs with highly differing allele frequencies between different populations, and the differences in frequency tends to be an order of magnitude greater than the difference among continental subpopulations (Tian et al., 2006). More recently, AIMs are being integrated in biomedical studies to study associations between genetic ancestry and health as a more accurate measure of genetic ancestry. OPEN ACCESS 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. Discussion: While several functional annotations of both coding and non-coding African AIMs are supported by the literature and linked these high-frequency African alleles to diseases in African populations, more effort is needed to map genes to diseases in these genetically diverse subpopulations. The relative dearth of these African AIMs on current genotyping platforms (the array with the highest fraction, llumina’s Omni 5, harbors less than a quarter of AIMs), further demonstrates a greater need to better represent historically understudied populations. African ancestry, aims, population structure, 1000 genomes project, ancestry and health 01 Frontiers in Genetics frontiersin.org 10.3389/fgene.2023.1061781 Boudeau et al. Introduction AIMs panels are heavily used in admixture mapping in studies seeking to identify disease-associated loci in admixed populations such as African Americans (Chen et al., 2010). AIMs allow for the study of both global and local ancestry association with disease which can lead to the identiﬁcation of population-speciﬁc disease loci. Loci that are associated with increased disease risk in a population are likely to be found in regions of the genome with a high percentage of ancestry for that population (Zhang et al., 2014). While studies have shown that only 1,500–2,500 SNPs are necessary to detect ancestral chromosomal regions in admixed populations (Winkler et al., 2010), a comprehensive AIMs set is required for a ﬁner mapping of disease loci. h k l l f f In this work, we generate a novel panel of 46,437 African ancestry- informative markers that were identiﬁed using European and African subpopulations genotype data from Phase 3 of the 1000 Genomes FIGURE 1 Development of the African AIMs panel for this study. Genotype data from African (AFR) populations (Gambian, Gambia [GWD]; Esan, Nigeria [ESN]; Luhya, Kenya [LWK]; Mende, Sierra Leone [MSL]; Yoruba, Nigeria [YRI]) and European (EUR) populations (Utah residents with European ancestry [CEU]; Finnish, Finland [FIN], British; England & Scotland [GBR]; Iberian, Spain [IBS]; Tuscany, Italy [TSI]) from the 1000 Genomes Project database. AFR dataset combinations (AFR-S1 to AFR-S6) and the combined EUR dataset were generated based on the ancestry informativeness (ln) of each variant. Genetic variants with In≥ 0.25 were considered as AIMs. Each AFR subset was compared against the combined EUR set resulting in six different AIMs subsets. The SNPs common to all six AIMs subset were extracted to generate the set of 46,737 African AIMs used in this study. FIGURE 1 Development of the African AIMs panel for this study. Genotype data from African (AFR) populations (Gambian, Gambia [GWD]; Esan, Nigeria [ESN]; Luhya, Kenya [LWK]; Mende, Sierra Leone [MSL]; Yoruba, Nigeria [YRI]) and European (EUR) populations (Utah residents with European ancestry [CEU]; Finnish, Finland [FIN], British; England & Scotland [GBR]; Iberian, Spain [IBS]; Tuscany, Italy [TSI]) from the 1000 Genomes Project database. AFR dataset combinations (AFR-S1 to AFR-S6) and the combined EUR dataset were generated based on the ancestry informativeness (ln) of each variant. Genetic variants with In≥ 0.25 were considered as AIMs. Each AFR subset was compared against the combined EUR set resulting in six different AIMs subsets. Introduction (A) Selection of the ancestry informative markers (AIMs) common to the different sets of continental African AIMs to generate the ﬁnal set of 46,737 African AIMs. (B) Distribution of the number of African AIMs in 1 Mb chromosomal windows across chromosomes. Although some windows contain a much higher count, the y-Axis is capped at 300 SNPs to visualize most of the peaks which contain less than 300 SNPs per window. The location of AIMs from this set that appear in the GWAS database as associated with cancer phenotypes are shown on the karyotype plot (nine ontologies are indicated as separate colors). (C) Distribution of African AIMs across different genomic regions. FIGURE 2 Characterization of the African AIMs identiﬁed in this study. (A) Selection of the ancestry informative markers (AIMs) common to the different sets of continental African AIMs to generate the ﬁnal set of 46,737 African AIMs. (B) Distribution of the number of African AIMs in 1 Mb chromosomal windows across chromosomes. Although some windows contain a much higher count, the y-Axis is capped at 300 SNPs to visualize most of the peaks which contain less than 300 SNPs per window. The location of AIMs from this set that appear in the GWAS database as associated with cancer phenotypes are shown on the karyotype plot (nine ontologies are indicated as separate colors). (C) Distribution of African AIMs across different genomic regions. FIGURE 3 Distribution of FST among autosomal AIMs. FST was estimated for each African subpopulation (ESN, YRI, GWD, MSL, and LWK) against the European population using data from the 1000 Genomes Project. Additionally, estimates of FST were also calculated for the two other subpopulations in the dataset with high African ancestry, the Afro-Caribbean population from Barbados (ACB), and African Americans from the United States (ASW). FST was estimated for biallelic autosomal AIMs only and shows differences in the distribution in the FST estimates between the populations. FIGURE 3 Distribution of FST among autosomal AIMs. FST was estimated for each African subpopulation (ESN, YRI, GWD, MSL, and LWK) against the European population using data from the 1000 Genomes Project. Additionally, estimates of FST were also calculated for the two other subpopulations in the dataset with high African ancestry, the Afro-Caribbean population from Barbados (ACB), and African Americans from the United States (ASW). Frontiers in Genetics Introduction The SNPs common to all six AIMs subset were extracted to generate the set of 46,737 African AIMs used in this study. 02 Frontiers in Genetics frontiersin.org 10.3389/fgene.2023.1061781 Boudeau et al. FIGURE 2 (Continued) FIGURE 2 (Continued) FIGURE 2 (Continued) Project (1KGP) (Sudmant et al., 2015). This work compares populations of African and European ancestries to identify SNPs that will be highly informative for African ancestry as well as differentiating Africans from other continental groups. Additionally, this AIM set will provide an important reference panel to investigate genetic ancestry in understudied admixed populations such as African Americans and Afro-Caribbeans. While most African AIMs found in the literature are comprised of SNPs from just two subpopulations, namely CEU (US-European) and YRI 03 Frontiers in Genetics frontiersin.org Boudeau et al. 10.3389/fgene.2023.1061781 FIGURE 2 Characterization of the African AIMs identiﬁed in this study. (A) Selection of the ancestry informative markers (AIMs) common to the different sets of continental African AIMs to generate the ﬁnal set of 46,737 African AIMs. (B) Distribution of the number of African AIMs in 1 Mb chromosomal windows across chromosomes. Although some windows contain a much higher count, the y-Axis is capped at 300 SNPs to visualize most of the peaks which contain less than 300 SNPs per window. The location of AIMs from this set that appear in the GWAS database as associated with cancer phenotypes are shown on the karyotype plot (nine ontologies are indicated as separate colors). (C) Distribution of African AIMs across different genomic regions. FIGURE 2 Characterization of the African AIMs identiﬁed in this study. (A) Selection of the ancestry informative markers (AIMs) common to the different sets of continental African AIMs to generate the ﬁnal set of 46,737 African AIMs. (B) Distribution of the number of African AIMs in 1 Mb chromosomal windows across chromosomes. Although some windows contain a much higher count, the y-Axis is capped at 300 SNPs to visualize most of the peaks which contain less than 300 SNPs per window. The location of AIMs from this set that appear in the GWAS database as associated with cancer phenotypes are shown on the karyotype plot (nine ontologies are indicated as separate colors). (C) Distribution of African AIMs across different genomic regions. FIGURE 2 Characterization of the African AIMs identiﬁed in this study. Introduction FST was estimated for biallelic autosomal AIMs only and shows differences in the distribution in the FST estimates between the populations. (Yoruba) (Zhang et al., 2014), this panel provides a more extensive accounting of each of the 1KGP subpopulations in Europe (EUR) and Africa (AFR). The most highly differentiated SNPs from each AFR subpopulation relative to the EUR population based on informativeness estimates common to all AFR subpopulations were pooled to generate this panel. The African AIMs were validated using 04 Frontiers in Genetics frontiersin.org Boudeau et al. 10.3389/fgene.2023.1061781 FIGURE 4 (Continued). FIGURE 4 resulted in six separate AIMs subpanels which were subsequently intersected to identify 46,737 common African AIMs. an independent dataset of 1,448 individuals where approximately one half is of European ancestry and the other half is of varying degree of African ancestry. They were characterized for function and disease association with several gene-trait associations speciﬁc to African populations corroborated in the literature. We also ﬁnd that these SNPs are under-represented in major genotyping platforms that are in current use. This work identiﬁes a robust new panel of SNPs found in high frequency across continental African populations that have the potential to link population-speciﬁc mutations and disease, thus, providing much needed foundational data to examine understudied African populations. Population genomic differentiation analysis To validate population-speciﬁc properties of the identiﬁed AIMs, we estimated the pairwise ﬁxation index (FST) for the seven 1KGP subpopulations with signiﬁcant African ancestry (YRI, ESN, MSL, GWD, LWK, ACB, ASW) against the European population. Using the 1KGP data for these populations, we extracted SNPs identiﬁed as AIMs on the 22 autosomes using BCFTools (Danecek et al., 2021). Weir-FST estimates were calculated using VCFTools (Danecek et al., 2011) and then visualized via violin plots. Data source and AIMs identiﬁcation We performed a principal component analysis on the AIMs set to visualize their ability to differentiate between the subpopulations of African ancestry and the other subpopulations from the 1KGP. Using BCFTools, the 1KGP VCF ﬁles were converted to their binary version (.bcf). PLINK was used to prune and merge the chromosome ﬁles into one. Eigenvectors were generated and used for PCA analysis comparing the different populations. Ancestry-informative markers (AIMs) were identiﬁed using SNP data from the 1000 Genomes Project (1KGP) (1000 Genomes Project 2012; 1000 20151000 Genomes Project Consortium et al., 2015). The genotype data of African (AFR) populations (Gambian, Gambia [GWD]; Esan, Nigeria [ESN]; Luhya, Kenya [LWK]; Mende, Sierra Leone [MSL]; Yoruba, Nigeria [YRI]), and European (EUR) populations (Utah residents with European ancestry [CEU]; Finnish, Finland [FIN], British, England & Scotland [GBR]; Iberian, Spain [IBS]; Tuscany, Italy [TSI]) were downloaded from the 1KGP database (2013 release). The ancestry informativeness (In) of each genetic variant was estimated separately for six different combinations of AFR datasets (AFR-S1 to AFR-S6) and the combined EUR dataset (Figure 1) using the tool, infocalc (Rosenberg et al., 2003). Genetic variants with In≥0.25 were considered as AIMs. The analyses Frontiers in Genetics AIMs validation To test the ability of the identiﬁed AIMs to separate populations of African ancestry from those of European ancestry, we evaluated them on an independent dataset of 1,472 individuals. These individuals were recruited as part of a cohort of head and neck cancer study as cases and controls to be genotyped for an IRB-approved related study. A custom Illumina sequencing array was designed by adding a subset of the Frontiers in Genetics 05 frontiersin.org Boudeau et al. 10.3389/fgene.2023.1061781 URE 4 stering of African AIMs within and between populations. (A) Principal component analysis across all subpopulations in 1000 Genomes Project data h populations of high African ancestry in individual colors while the other subpopulations are colored in a single-color gradient (EUR: aquamarine; EAS: en; AMR: yellow; SAS: purple). (B) Principal component analysis of the AFR subpopulations (multicolor), plus the Afro-Caribbean (ACB), the African erican (ASW), and the EUR subpopulations. (C) PCA of 9,385 AIMs genotyped in an independent dataset with PC1 explaining 53.7% of the genetic ance in the data. FIGURE 4 Clustering of African AIMs within and between populations. (A) Principal component analysis across all subpopulations in 1000 Genomes Project data with populations of high African ancestry in individual colors while the other subpopulations are colored in a single-color gradient (EUR: aquamarine; EAS: green; AMR: yellow; SAS: purple). (B) Principal component analysis of the AFR subpopulations (multicolor), plus the Afro-Caribbean (ACB), the African American (ASW), and the EUR subpopulations. (C) PCA of 9,385 AIMs genotyped in an independent dataset with PC1 explaining 53.7% of the genetic variance in the data. AIMs from this panel to the Illumina GSA backbone (Inﬁnium Global Screening Array-24 Kit). DNA was extracted from the biospecimen collected from study participants as described in Blackman et al. (2018) and genotyped using the custom array. The AIMs included in the array are a subset of the 47 K that are not in linkage disequilibrium. After excluding retired SNPs, updated and combined SNPs, as well as SNPs whose probes were not able to be made, 11,377 probes were added to the GSA array. The number of AIMs successfully genotyped by all samples across two batches totaled 9,566 and the genotype data for AIMs from this panel to the Illumina GSA backbone (Inﬁnium Global Screening Array-24 Kit). DNA was extracted from the biospecimen collected from study participants as described in Blackman et al. (2018) and genotyped using the custom array. AIMs validation The AIMs included in the array are a subset of the 47 K that are not in linkage disequilibrium. After excluding retired SNPs, updated and combined SNPs, as well as SNPs whose probes were not able to be made, 11,377 probes were added to the GSA array. The number of AIMs successfully genotyped by all samples across two batches totaled 9,566 and the genotype data for 9,385 SNPs were used as the validation SNP subset via principal component analysis. Frontiers in Genetics Functional characterization We annotated and evaluated the functional role of these genetic variants by running our list of AIMs through ANNOVAR (Wang et al., 2010) using the hg19 human reference genome. The 06 frontiersin.org frontiersin.org Boudeau et al. 10.3389/fgene.2023.1061781 FIGURE 5 Distribution of minor allele frequencies in African AIMs. AIMs found in the coding regions of the genome and carrying non-synonymous mutations are juxtaposed next to the SIFT and Polyphen2 scores for each SNPs. Of the 338 exonic SNPs, 164 are non-synonymous mutations, 149 of which were scored by the tools. Excluding the X-linked SNPs and multiallelic sites, only 112 SNPs were non-synonymous and are plotted here. The color gradient for the MAF ranges from white (0) to red (0.6); the gradient for SIFT range from purple (0, damaging) to pink (1, benign), while the gradient for Polyphen2 ranges from pink (0, benign) to purple (1, damaging). Distribution of minor allele frequencies in African AIMs. AIMs found in the coding regions of the genome and carrying non-synonymous mutations are juxtaposed next to the SIFT and Polyphen2 scores for each SNPs. Of the 338 exonic SNPs, 164 are non-synonymous mutations, 149 of which were scored by the tools. Excluding the X-linked SNPs and multiallelic sites, only 112 SNPs were non-synonymous and are plotted here. The color gradient for the MAF ranges from white (0) to red (0.6); the gradient for SIFT range from purple (0, damaging) to pink (1, benign), while the gradient for Polyphen2 ranges from pink (0, benign) to purple (1, damaging). each array platform. These AIMs were then compared to a large list of imputed SNPs to estimate which of the AIMs could be imputed. pathogenicity of the AIMs was evaluated using multiple functional effect predictors: polymorphism phenotyping: PolyPhen2 (Adzhubei et al., 2013); sorting intolerant from tolerant: SIFT (Kumar et al., 2009); and two machine learning methods: metaSVM (support vector machine) and metaLR (logistic regression) (Dong et al., 2015). Frontiers in Genetics Overlap with current platforms Two intronic AIMs (rs13267382: chr8, LINC00536; rs9952980: chr18, SLC14A2) and two intergenic AIMs (rs10832963: chr11, SPTY2D1 - SRSF3P1; rs11814448: chr10) were also found to be associated with breast cancer in samples of European and Asian ancestry (Michailidou et al., 2017). rs7252505 found in the GPATCH1 gene (chr19) was associated with colorectal cancer risk (H. Wang et al., 2017) (Figure 2B). Two intronic AIMs (rs13267382: chr8, LINC00536; rs9952980: chr18, SLC14A2) and two intergenic AIMs (rs10832963: chr11, SPTY2D1 - SRSF3P1; rs11814448: chr10) were also found to be associated with breast cancer in samples of European and Asian ancestry (Michailidou et al., 2017). six subpanels were retrieved for further analyses. The usefulness of these AIMs for assessment of population structure was analyzed using data from a separate and ongoing project pointing towards the utility of this AIMs panel to study population admixture in African populations (Ramakodi et al., 2017). To characterize the distribution of these African AIMs in the genome and to evaluate their functional roles, we partitioned 1 Mbp windows across each chromosome. Figure 2B shows a genome-wide distribution of African AIMs with a signiﬁcantly high fraction found on the X-chromosome. Most autosomal 1 Mbp windows have fewer than 300 SNPs, although there are several windows with a much higher SNP density. Chromosome 17 and the X-chromosome possess regions with a much higher density of SNPs, with some chromosomal regions having up to 970 SNPs per 1 MB window. Windows that are AIMs-rich are generally distributed evenly across each chromosome (e.g., chromosomes 1 and 12) with no obvious clusters. However, there appears to be some clusters of AIMs-rich windows, as is the case of the X-chromosome harboring a large cluster near its centromere. Additionally, some of the shorter chromosomes (e.g., chr8, 12, 14, 15, 17, and 20) appear to have a higher fraction of African AIMs relative to the longer chromosomes (e.g., chr2, 3, 5, 6, and 7). Figure 2C shows the distribution of SNPs across different genomic regions and the potentially consequential roles they play in genome function. Most of the AIMs are found in two genomic regions, with just over half, 51.4%, found in the intergenic regions of the genome and 39.1% in intronic regions. The remaining SNPs are distributed in smaller fractions in intronic non-coding RNAs (5.8%), 3-prime UTRs (1.1%), with 0.7% found within 1 Mb downstream of genes and 0.6% found upstream of genes. Overlap with current platforms Only 338 SNPs, 0.6% of the entire AIMs set, were found to be in coding regions of the genome. Interestingly, when compared to all autosomal SNPs in the 1000 Genomes Project dataset, this AIMs set has signiﬁcantly fewer exonic (p-value < 2.2e-16, Fisher’s Exact test) and 5-prime UTR (p-value = 0.04685, Fisher’s Exact test) SNPs than expected. To investigate whether the regions enriched for AIMs correlate with diseases in the literature, we extracted studies from the GWAS catalog that identiﬁed AIMs from this set as associated with cancer and plotted the loci (colored dots) onto a karyotype plot (Figure 2B). AIMs, rs12916300 and rs12913832, mapped to HERC2 (chr15) and rs694339 in the CBLN2 (chr18) genes were associated with colorectal cancer risk in a European sample (Hofer et al., 2017) (Figure 2B). In an African American sample, AIM Overlap with current platforms In this study, genotype data derived from ﬁve continental African and ﬁve European subpopulations were analyzed to obtain a generalized AIMs panel for the African populations represented in the 1KGP data (Figure 1). This African-European continental comparison initially generated six AIMs subpanels ranging from 51,907 to 59,422 SNPs with each subpanel including all African populations except for one. The number of AIMs obtained from each dataset are shown in Figure 2A. All six AIMs subpanels were intersected and a total of 46,737 AIMs commonly found in each of the We also surveyed the representation of our AIMs set among common genotyping array platforms which may indicate how likely they are to be found in the literature as associated with known disease mutations. We downloaded the manifests for 25 commonly used genotyping arrays from Illumina and Affymetrix (ThermoFisher Scientiﬁc) and ascertained the number of SNPs from the AIMs set that appear on the sequencing arrays. Lastly, we estimated the fraction of our African AIMs represented on 07 frontiersin.org Boudeau et al. 10.3389/fgene.2023.1061781 FIGURE 6 Percentage of African AIMs from this study that are represented in commercial genotyping arrays. Twenty-ﬁve commonly used genotyping platforms were chosen from Illumina and Affymetrix (y-Axis) and the fraction of SNPs differentiating the African population from others (i.e., AIMs from this study) is listed for each array. Platforms are ranked from most representative of the AIMs panel from this study to the least. FIGURE 6 Percentage of African AIMs from this study that are represented in commercial genotyping arrays. Twenty-ﬁve commonly used genotyping platforms were chosen from Illumina and Affymetrix (y-Axis) and the fraction of SNPs differentiating the African population from others (i.e., AIMs from this study) is listed for each array. Platforms are ranked from most representative of the AIMs panel from this study to the least. FIGURE 6 Percentage of African AIMs from this study that are represented in commercial genotyping arrays. Twenty-ﬁve commonly used genotyping platforms were chosen from Illumina and Affymetrix (y-Axis) and the fraction of SNPs differentiating the African population from others (i.e., AIMs from this study) is listed for each array. Platforms are ranked from most representative of the AIMs panel from this study to the least. rs7252505 found in the GPATCH1 gene (chr19) was associated with colorectal cancer risk (H. Wang et al., 2017) (Figure 2B). Frontiers in Genetics Substructure in populations of African ancestry The importance of population substructure in association studies is becoming more apparent as they can confound observed genetic associations when ignored and hinder us from elucidating the genetic bases of disease. Consequently, understanding whether this set of 08 Frontiers in Genetics frontiersin.org 10.3389/fgene.2023.1061781 10.3389/fgene.2023.1061781 Boudeau et al. Boudeau et al. populations. The EUR populations appear to have much lower MAFs for these SNPs. The seven columns (1–7) with populations of African ancestry show further evidence of population substructure. We also tried to predict pathogenicity using prediction tools such as SIFT (sorting intolerant from tolerant) and PolyPhen2 (polymorphism phenotyping). Column 9 shows the SIFT results and column 10 shows the results from PolyPhen2 with damaging mutations labeled in purple (Figure 5). There are a few instances where some SNPs are identiﬁed as damaging by both predictors and these SNPs appear to be more concentrated in SNPs with higher MAFs in the AFR populations. Interestingly, many of the SNPs predicted to be damaging by SIFT have very low MAF in the EUR population. We also compared other pathogenicity tools, such as metaSVM and metalR (Dong et al., 2015), that provide an aggregate prediction score from multiple individual tools and these tools only identiﬁed two AIMs as damaging: rs12186491 maps to SPINK6, a serine protease inhibitor kazal-type 6 gene which has been found to regulate nasopharyngeal carcinoma metastasis through EGFR signaling, and rs6601495 encodes for Retinitis Pigmentosa 1-Like 1 Protein which is associated with diseases like occult macular dystrophy and retinitis pigmentosa (Zheng et al., 2017; Noel and MacDonald, 2020). African AIMs adequately identiﬁes population substructure within and between populations with shared African ancestry is an important goal. Figure 3 shows the distribution of the ﬁxation index of these AIMs in the different sub-populations of African ancestry as estimated against the EUR super-population. Based on Weir-FST statistics, there is a uniform distribution of FST estimates for the continental AFR subpopulations with the four West African subpopulations (ESN, YRI, GWD, MSL). The violin plots in Figure 3 show the most similarity. The ACB subpopulation shows a slightly different distribution compared to the AFR subpopulations though its plot still reﬂects a high degree of dissimilarity from the EUR population and close relationship to the AFR subpopulation. The ASW violin plot reveals the most divergent distribution and a much lower FST minimum relative to the other subpopulations. Discussion Using an independent dataset of racially self-identiﬁed individuals from the United States, we were able to show that our validation subset of 9,385 markers effectively separated individuals of European ancestry from those of African ancestry (Figure 4C). Additionally, these AIMs detected population differences within the group of individuals with African ancestry without being able to fully differentiate the sub-populations of African ancestry (continental African, African American, Caribbean of African ancestry). Undetected population structure can lead to spurious ﬁndings in genetic association studies. With the increased reliance on these studies to identify genetic markers associated with disease, identiﬁcation and correction for population stratiﬁcation are critical as both environmental and genetic factors can affect disease risk between populations or subpopulations (Enoch et al., 2006). In this work, we developed a set of African ancestry-informative SNPs that differentiates populations of African ancestry from others and identiﬁes substructure within populations of African ancestry based on estimates of informativeness. We identiﬁed 46,737 African ancestry-informative markers from ﬁve African subpopulations using Phase 31,000 Genomes Project data and our results suggest they convincingly aggregate populations based on their genetic ancestry and effectively separate populations of African ancestry from other major ancestral populations (Figure 4). Although the AIMs were identiﬁed from a comparison between AFR subpopulations and a combined EUR reference, Figure 4 shows that they suitably isolate populations of African ancestry from those of other continental groups including East Asian, South Asian, and groups with American ancestry. While there are several existing sets of ancestry-informative SNPs claiming to differentiate Substructure in populations of African ancestry The generated African AIMs were evaluated for their ability to properly differentiate populations of African ancestry from populations of European ancestry and the other continental ancestral groups such East Asia, South Asia, and the Americas. Using SNPs from the 1000 Genomes Project, principal component analysis reveals that this AIMs set separates African subpopulations from all other populations (Figure 4A). Based on these AIMs, the AFR subpopulations are effectively segregated from the EUR subpopulations in addition to other global ancestry groups that were not included in the generation of the set (Figure 4A). The PCA plots also show that the AIMs were able to detect population substructure within populations admixed with African ancestries such as the admixed American populations, ASW and ACB. Figure 4B shows that PC1 separates the AFR subpopulations from the EUR subpopulations and to a lesser extent it shows some degree of separation between the AFR subpopulations, explaining 16.94% of the total variance of the sampled genetic variation. PC2 and PC3 explained a combined additional 15.24% of the total genetic variance. The ﬁve continental African (AFR) subpopulations appear to cluster more tightly together while the populations of African ancestry in Southwest United States (ASW) and the African Caribbean in Barbados (ACB) subpopulations show some spread toward the European (EUR) population cluster. Generally, functional or pathogenicity information on the SNPs contained in this set of AIMs is scarce which may be a byproduct of being underrepresented in currently available genotyping platforms. An analysis of 25 popular genotyping array platforms from Illumina and Affymetrix reveals that most of the commercial arrays surveyed included less than 10% of the African AIMs (Figure 6). Combined, only 19,239 of the 46,737 AIMs in this set were found among the 8,708,293 unique SNPs included in these 25 commercial arrays. When compared to a current list of over 13 million variants (imputed SNPs from Neale lab, https://github.com/Nealelab/UK_Biobank_GWAS) that were imputed against a diverse panel, 43,647 of the 46,737 AIMs were found, indicating that most of the SNPs could be imputed. Frontiers in Genetics AIM allele frequencies and representation in common genotyping arrays Minor allele frequency (MAF) reﬂects how common an allele is in a population with low frequency alleles often associated with disease phenotypes making them markers of high interest. In Figure 5, columns 1–5 display the MAF for the ﬁve continental AFR populations (ESN, GWD, LWK, MSL, YRI), column 6: the Afro- Caribbean population, column 7: African Americans in the Southwest, and column 8: the European population. The ﬁve AFR subpopulations appear to have similar MAFs for the different SNPs while the frequencies are noticeably different in the ACB and ASW 09 frontiersin.org 10.3389/fgene.2023.1061781 Boudeau et al. Boudeau et al. alloimmunization in sickle cells patients (Williams et al., 2018; Daya et al., 2019). Over eight variants associated with prostate cancer risk have been identiﬁed on loci 8q24 (Han et al., 2016). African populations from others, they often come with limitations including that they are estimated from one subpopulation of African ancestry, usually YRI, and one of European ancestry, usually CEU (Keene et al., 2008; Cheng et al., 2009; Tandon et al., 2011; Zeng et al., 2016), though sometimes they include ASW (Chen et al., 2010). This AIM panel provides a more extensive and comprehensive set of pan- African SNPs that can help improve the accuracy of African ancestry estimates since it was established from multiple subpopulations from both the European and African populations. African populations from others, they often come with limitations including that they are estimated from one subpopulation of African ancestry, usually YRI, and one of European ancestry, usually CEU (Keene et al., 2008; Cheng et al., 2009; Tandon et al., 2011; Zeng et al., 2016), though sometimes they include ASW (Chen et al., 2010). This AIM panel provides a more extensive and comprehensive set of pan- African SNPs that can help improve the accuracy of African ancestry estimates since it was established from multiple subpopulations from both the European and African populations. This AIMs panel will signiﬁcantly contribute to the ease with which the ﬁeld integrates African genetic ancestry in population genetics studies but there remain some limitations. The 1000 Genomes Project was used to identify the AIMs for this set although it only includes six African sub- populations and does not completely encapsulate the rich genetic history of the African continent. Moreover, the included populations have small sample sizes which might not be fully representative of the diversity in those populations. AIM allele frequencies and representation in common genotyping arrays Lastly, Phase 3 data from the 1KGP was sampled at relatively low depth (7.4x) which can make it challenging to identify less common, rarer variants in the population (Byrska-Bishop et al., 2022). We developed our AIMs set to exclude SNPs that are speciﬁc to just one African subpopulation and are unlikely to be informative outside the context of that subpopulation. We also detected substructure between subpopulations of African ancestry when admixed populations with signiﬁcant African ancestry, namely ASW and ACB, were introduced (Figure 2). However, this AIMs panel of markers does not fully differentiate between subpopulations of African ancestry. This distinction highlights the consideration that should be given to both between and within population variation to effectively control for population stratiﬁcation in genetic studies. As advanced in Enoch et al. (2006) and originally by Lewontin (1972), while there is signiﬁcant diversity between populations, the bulk of human diversity is found within populations. The underrepresentation of these African AIMs on commonly used commercial genotyping arrays also contributes to the scarcity of information about their involvement of disease. However, many of these SNPs may be imputable using currently available African- inclusive panels. We searched a list of over 13 million imputable variants generated from UK BioBank data imputed using a panel made up of data from the Haplotype reference consortium, UK10K, as well as the 1000 Genomes Project reference panels, for the presence of our identiﬁed African AIMs. Out of these 46,737 AIMs, 43,647 were found on the list of imputable variants, which is expected considering that 1KPG data was included in the panel used to impute the SNPs. However, many populations in Africa remain poorly studied and imputing variants for those could be challenging. As discussed by Martin et al. (2018), imputation panels and resources have a European bias, and sequencing initiatives are biased for West African populations, thus, ignoring much of the total genetic diversity on the continent. This is a particular concern for the imputation of African genomes as the literature suggests a higher rate of genetic diversity and a lower rate of linkage disequilibrium in African populations (Bentley et al., 2020). Furthermore, most reference panels are currently not publicly available or use too small of a sample size to impute effectively (Gurdasani et al., 2015; Mathias et al., 2016; Taliun et al., 2021). AIM allele frequencies and representation in common genotyping arrays We evaluated the subset of AIMs that are in protein-coding regions for pathogenicity and association with disease as certain disease-causing variants have been found at highly differing frequencies across populations (Patterson et al., 2004). Using tools such as SIFT, Polyphen, metaSVM, and metaLR via ANNOVAR, we investigated which changes may lead to a loss of function in their associated proteins. These tools predict how non-synonymous mutations affect protein function (Flanagan et al., 2010). 0.64% of the AIMs are located in exonic regions of the genome and of that SNP pool, only the 48.52% fraction that are non-synonymous mutations were used for pathogenicity prediction. There is also a lack of agreement between each of the prediction tools which makes it challenging to interpret the predictions made for the SNPs identiﬁed as damaging. Further investigation is needed to contextualize these AIMs and elucidate their potential implications in disease. The development of ancestry-informative markers within the framework of population disease risk estimation presents an exciting opportunity to investigate the genetic bases of health disparities across heterogeneous populations with people from different genetic histories. Population structure can have signiﬁcant implications for genomics studies and simply controlling for them is not always enough to successfully account for population stratiﬁcation and to avoid such pitfalls as spurious associations (Enoch et al., 2006). As demonstrated here, having a highly speciﬁc African AIMs set can help detect ancestry differences between populations of African ancestry and others but it can also identify substructure within populations of African ancestry that should be further surveyed in association studies. This approach increases statistical power and can lead to the identiﬁcation of true associations between the SNP markers of interest and the disease/trait in association studies. Many studies have identiﬁed associations between genetic ancestry and health and, more recently, there is a trend toward the identiﬁcation of AIMs associated with disease though the studies of African ancestry are still few. Studies like these allow for increased granularity in the analysis of ancestry and health. Considering that self-reported race is an inconsistent and unreliable substitute for genetic ancestry, the AIMs set presented here provides a means for researchers to uncover the impact of ancestry on disease and phenotype. Frontiers in Genetics References Cheng, C.-Y., Kao, W. H. L., Patterson, N., Tandon, A., Haiman, C. A., Harris, T. B., et al. (2009). Admixture mapping of 15,280 african Americans identiﬁes obesity susceptibility loci on chromosomes 5 and X. PLoS Genet. 5 (5), e1000490. doi:10.1371/journal.pgen. 1000490 1000 Genomes Project Consortium, 2012 1000 Genomes Project Consortium (2012). An integrated map of genetic variation from 1,092 human genomes. Nature 491 (7422), 56–65. doi:10.1038/nature11632 1000 Genomes Project Consortium, 2012 1000 Genomes Project Consortium (2012). An integrated map of genetic variation from 1,092 human genomes. 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(2015). Racial disparity among the head and neck cancer population. J. Cancer Educ. 30 (3), 546–551. doi:10.1007/s13187-014-0753-4 Bentley, A. R., Callier, S. L., and Rotimi, C. N. (2020). Evaluating the promise of inclusion of African ancestry populations in genomics. Npj Genomic Med. 5 (1), 5. AIM allele frequencies and representation in common genotyping arrays These Although most of our identiﬁed AIMs are in non-coding regions, they can still play a role in the genetic basis of health disparities and how genetic ancestry can inﬂuence disease risk, although these association data are sparser. One such case of that demonstrates the functional impact of non-coding AIMs from association literature is the African ancestry variant, rs72725854, a rare variant found in an enhancer region at 8q24 which has been shown to regulate multiple lnRNA genes and the MYC oncogene to inﬂuence prostate cancer risk in men of African ancestry (Darst et al., 2020; Walavalkar et al., 2020). Additionally, the GWAS literature is ripe with SNPs that have been found to be associated with disease phenotypes such as differential survival in head and neck cancer, differences in prostate cancer risk, and differences in diagnosis stage in breast cancer (Al- Alem et al., 2014; Irizarry-Ramírez et al., 2017; Ramakodi et al., 2017). Certain chromosomes stood out as having a higher proportion of AIMs relative to their size such as the X-chromosome and chromosomes 4, 8, 12, 14, 15, and 17, all of which have a higher proportion of AIMs than the much larger chromosome 1. Interestingly, loci on some of these chromosomes have been identiﬁed in the literature as associated with diseases in populations of African ancestry including loci 8p23 and 8q24 and asthma risk and the association of rs75853687 on chromosome 5 with 10 frontiersin.org Boudeau et al. 10.3389/fgene.2023.1061781 African AIMs, allowing researchers to apply a set of markers spanning the whole genome, will hopefully provide new avenues to study disease genetics in a large, diverse, and understudied population, and helps elucidate the contribution of local ancestry to disease risk and health. supported by the Chan Zuckerberg Initiative. This work is supported in part by 5P30CA006927 and by the TUFCCC/HC Regional Comprehensive Cancer Health Disparity Partnership, Award Number U54 CA221704 from the National Cancer Institute of National Institutes of Health (NCI/NIH). Publication of this article was funded in part by the Temple University Libraries Open Access Publishing Fund. Its contents are solely the responsibility of the authors and do not necessarily represent the ofﬁcial views of the NCI/NIH. Supplementary material This work was supported by the American Cancer Society (RSG-14- 033-01-CPPB to CR) and the National Cancer Institute (CA006927). MR is partially supported by the William J. Avery Postdoctoral Research Fellowship (Fox Chase Cancer Center). SB, RJK, and CR are partially The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2023.1061781/ full#supplementary-material Data availability statement Publicly available datasets were analyzed in this study. This data can be found here: 1000 Genomes project. The list of ancestry- informative markers developed in this study is available in Supplementary Table S1. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 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. Author contributions SB performed the analyses, generated the tables and ﬁgures, wrote the ﬁrst draft of the manuscript, and managed all subsequent drafts. RK and CR contributed to the conception and design of the study. MR developed the AIMs pipeline, identiﬁed the AIMs set. YZ designed the custom array and processed the genotype data. RK, CR, SB, and MR wrote sections of the manuscript. 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W3206282166.txt	https://www.mimmun.ru/mimmun/article/download/2427/1444	en		DYNAMIC CHANGES IN CARDIOVASCULAR RISK BIOMARKERS AND CYTOKINES OF MYOCARDITIS-FREE PATIENTS WITH DECOMPENSATED HEART FAILURE AND ISCHEMIC SYSTOLIC DYSFUNCTION	Medicinskaâ immunologiâ	2,021	cc-by	3,808	Медицинская иммунология 2021, Т. 23, № 4, стр. 839-844 © 2021, СПб РО РААКИ Краткие сообщения Short communications Medical Immunology (Russia)/ Meditsinskaya Immunologiya 4, pp. 839-844 2021, Vol. 23, © 2021, SPb RAACI ДИНАМИКА БИОМАРКЕРОВ КАРДИОВАСКУЛЯРНОГО РИСКА И ЦИТОКИНОВ У БОЛЬНЫХ С ДЕКОМПЕНСИРОВАННОЙ СЕРДЕЧНОЙ НЕДОСТАТОЧНОСТЬЮ С СИСТОЛИЧЕСКОЙ ДИСФУНКЦИЕЙ ИШЕМИЧЕСКОГО ГЕНЕЗА ПРИ НАЛИЧИИ И ОТСУТСТВИИ МИОКАРДИТА Огуркова О.Н.1, Кручинкина Е.В.1, Гусакова А.М.1, Суслова Т.Е.1, Рябов В.В.1, 2 Научно-исследовательский институт кардиологии ФГБНУ «Томский национальный исследовательский медицинский центр Российской академии наук», г. Томск, Россия 2 ФГБОУ ВО «Сибирский государственный медицинский университет» Министерства здравоохранения РФ, г. Томск, Россия 1 Резюме. Развитие и прогрессирование сердечной недостаточности связано с различными патофизиологическими механизмами, особый интерес представляет изучение воспалительной реакции, как фундаментального звена в патогенезе ХСН и ее основного компонента – декомпенсации. Проведено открытое, нерандомизированное, проспективное исследование по изучению клинико-морфологических особенностей субклинического воспаления у больных с острой декомпенсацией ишемической хронической сердечной недостаточностью со сниженной фракцией выброса. В исследование были включены 25 больных с декомпенсацией ишемической ХСН с ФВ левого желудочка < 40% в возрасте от 35 до 75 лет (60,12±9,3) с подписанным информированным согласием. В данном исследовании изучалась динамика содержания в сыворотке крови С-реактивного белка (СРБ), N-концевого фрагмента белка-предшественника мозгового натрийуретического пептида (NT-proBNP), растворимого ST2 (sST2), рецептора инсулиноподобного фактора роста 1 (IGF-1R), интерлейкина-6 (IL-6), интерлейкина-10 (IL-10), фактора некроза опухоли-α (TNFa) на Multiplex Instrument FLEXMAP 3D Luminex Corporation. Все обследованные пациенты были разделены на две группы в зависимости от диагностированного миокардита: пациенты без признаков миокардита и пациенты с миокардитом. Установлено, что в группе пациентов с диагностированным миокардитом наблюдалось повышенное содержание СРБ, IGF-1R, IL-6 и IL-10, TNFa по сравнению с группой пациентов без миокардита. Средние концентрации NT-proBNP и sST2 в обеих группах не различались. При последующем визите через год наблюдалось снижение содержания СРБ, NT-proBNP, IL-6 в обеих группах. В группе больных миокардитом наблюдалось повышение содержания sST2, IGF-1R, IL-10. Таким образом, проведенное в динамике исследование выявило достоверные различия в изменении сывороточной активности про- и противовоспалительных цитокинов и биомаркеров сердечно-сосудистого риска у пациентов с декомпенсированной сердечной недостаточностью с систолической дисфункцией при диагностированном миокардите и при его отсутствии. Ключевые слова: воспаление, цитокины, биомаркеры, сердечная недостаточность, миокардит Адрес для переписки: Огуркова Оксана Николаевна Научно-исследовательский институт кардиологии 634012, Россия, г. Томск, ул. Киевская, 111а. Тел.: 8 (960) 975-45-44. E-mail: ogurkovaon@mail.ru Address for correspondence: Ogurkova Oksana N. Cardiology Research Institute 634012, Russian Federation, Tomsk, Kievskaya str., 111a. Phone: 7 (960) 975-45-44. E-mail: ogurkovaon@mail.ru Образец цитирования: О.Н. Огуркова, Е.В. Кручинкина, А.М. Гусакова, Т.Е. Суслова, В.В. Рябов «Динамика биомаркеров кардиоваскулярного риска и цитокинов у больных с декомпенсированной сердечной недостаточностью с систолической дисфункцией ишемического генеза при наличии и отсутствии миокардита» // Медицинская иммунология, 2021. Т. 23, № 4. С. 839-844. doi: 10.15789/1563-0625-DCI-2280 © Огуркова О.Н. и соавт., 2021 For citation: O.N. Ogurkova, E.V. Kruchinkina, A.M. Gusakova, T.E. Suslova, V.V. Ryabov “Dynamic changes in cardiovascular risk biomarkers and cytokines of myocarditisfree patients with decompensated heart failure and ischemic systolic dysfunction”, Medical Immunology (Russia)/ Meditsinskaya Immunologiya, 2021, Vol. 23, no. 4, pp. 839-844. doi: 10.15789/1563-0625-DCI-2280 DOI: 10.15789/1563-0625-DCI-2280 839 Огуркова О.Н. и др. Ogurkova O.N. et al. Медицинская Иммунология Medical Immunology (Russia)/Meditsinskaya Immunologiya DYNAMIC CHANGES IN CARDIOVASCULAR RISK BIOMARKERS AND CYTOKINES OF MYOCARDITIS-FREE PATIENTS WITH DECOMPENSATED HEART FAILURE AND ISCHEMIC SYSTOLIC DYSFUNCTION Ogurkova O.N.a, Kruchinkina E.V.a, Gusakova A.M.a, Suslova T.E.a, Ryabov V.V.a, b a Cardiology Research Institute, Tomsk National Research Medical Centre, Russian Academy of Sciences, Tomsk, Russian Federation b Siberian State Medical University, Tomsk, Russian Federation Abstract. The development and progression of heart failure is associated with a variety of pathophysiological mechanisms, of particular interest is the study of the inflammatory response as a fundamental link in the pathogenesis of CHF and its main component – decompensation. An open, non-randomized, prospective study was carried out to evaluate the clinical and morphological features of subclinical inflammation in patients with acute decompensation of ischemic chronic heart failure with a reduced ejection fraction. The study included 25 patients with decompensated ischemic CHF with left ventricular ejection fraction < 40% aged 35 to 75 years (60.12±9.3 y. o.). In this study the dynamics of the serum content of C-reactive protein (CRP), N-terminal fragment of the brain natriuretic peptide precursor protein (NT-proBNP), soluble ST2(sST2), insulin-like growth factor-1 receptor (IGF-1R), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNFα) was performed by multiplex immunoassay using the FLEXMAP 3D. All studied patients were divided into two groups depending on the diagnosed myocarditis: patients with no signs of myocarditis and patients with myocarditis. It was found that in the group of patients with diagnosed myocarditis there was an increased content of CRP, IGF-1R, IL-6 and IL-10, TNFα compared to the group of patients without myocarditis. The median concentrations of the NT-proBNP and sST2 in both groups did not differ. At the follow-up visit a year later, there was a decrease in the content of CRP, NT-proBNP, IL-6 in both groups. In the group of patients with myocarditis, an increase in the content of sST2, IGF-1R, IL-10 was observed. Thus, the study carried out in dynamics revealed significant differences in the degree of changes in the serum activity of pro- and anti-inflammatory cytokines and biomarkers of cardiovascular risk in patients with decompensated heart failure with systolic dysfunction with diagnosed myocarditis and in its absence. Keywords: inflammation, cytokines, biomarkers, heart failure, myocarditis Introduction Decompensation of CHF is a serious clinical and public health problem with a high level of mor bidity, mortality and rate of hospitalizations [6]. The development and progression of heart failure are associated with a variety of pathophysiological mechanisms, of particular interest is examining an inflammatory response as a fundamental link in the CHF pathogenesis and its main component – decompensation [7]. An inflammatory concept has been formulated, which is based on the persistent inflammation present at the early stages of CHF, as well as at later stages, when CHF is verified [6]. Recently, the study of inflammation markers and their role in cardioprotective mechanisms, as well as growth factors and cytokines as prognostically relevant biomarkers of developing heart failure deserved particular interest [4, 5]. It is known that imbalance of pro- and anti-inflammatory cytokines significantly contributes to development and progression of CHF, but the results of experimental and exploratory cli nical studies have not led to the emergence of a 840 single effective anti-inflammatory strategy in CHF syndrome [7]. Purpose of the study: to study the dynamics and relationship of serum cardiovascular risk biomarkers and cytokines in patients with decompensated heart failure with ischemic systolic dysfunction with/wit hout myocarditis using xMAP technology. Materials and methods An open, non-randomized, prospective study was carried out to evaluate the clinical and morphological features of subclinical inflammation in patients with acute decompensation of ischemic chronic heart failure with reduced ejection fraction. This study is registered at the ClinicalTrials.gov, ID: NCT02649517. The study included 25 patients with decompensated ischemic CHF with left ventricular ejection fraction < 40% aged 35 to 75 years (60.12±9.3 y.o.), who signed informed consent, 6 months after successfully performed angioplasty and/or coronary artery bypass grafting. The therapy performed in patients at the time of enrolling to the study corresponded to the 2021, Т. 23, № 4 2021, Vol. 23, 4 Биомаркеры и цитокины у больных с сердечной недостаточностью Biomarkers and cytokines in patients with heart failure current recommendations such as: beta-blockers, diuretics, mineralocorticoid receptor antagonists, cardiac glycosides. Exclusion criteria were: acute coronary syndrome less than 6 months before hospi talization; hemodynamically significant valvular heart disease; severe concomitant diseases that could affect the course of the underlying disease and the study results. All patients underwent invasive coronary angioplasty to exclude the progression of coronary artery atherosclerosis, as well as endomyocardial biopsy with immunohistological analysis to verify myocarditis. This article analyzes the dynamics of biomarkers in such patients by using Milliplex kit (Merck KGaA, Darmshdadt) (n = 10). The diagnosis of myocarditis was established in 5 patients according to immunohistochemical criteria: the presence of at least 14 leukocytes/mm2, with the presence of CD3 positive T-lymphocytes ≥ 7 cells/mm2. Fasting blood sampling for biomarkers from the cubital vein was carried out during hospitalization of patients and one year later at the checkpoint visit. The evaluation serum of high-sensitive C-reactive protein (hCRP) concentration was carried out in blood serum by ELISA (Biomedica); level of N-terminal fragment of brain natriuretic peptide precursor protein (NTproBNP) pg/ml was determined using Human Cardiovascular Disease Panel 1. Levels of soluble ST2 (sST2) ng/ml, insulin-like growth factor-1 receptor (IGF-1R) pg/ml were investigated by using Human Cardiovascular Disease Panel 5. The level of interleukin-6 (IL-6) pg/ml, interleukin-10 (IL-10) pg/ml, tumor necrosis factor-α (TNFα) pg/ml was measured by using Human Cytokines/Chemo kines-38 kit. All studies were performed using Multiplex Instrument FLEXMAP 3D Luminex Corporation and MILLIPLEX Analyst 5.1 software (Merck KGaA, Milliplex; Darmshdadt), the Core Facility “Medical genomics”, Tomsk NRMC. Results and discussion All patients were divided into two groups depen ding on the diagnosed myocarditis: patients with no signs of myocarditis were included in group 1 and patients with myocarditis were included in group 2. Clinical characteristics are presented in Table 1. Examining serum biomarkers from patients during hospitalization showed that in the group of patients with diagnosed myocarditis, there was an increased content of C-reactive protein, insulin-like growth factor receptor-1, interleukins-6 and 10, tumor necro sis factor -α compared to the group of patients without myocarditis. The median NT-proBNP concentration in both groups was comparable and exceeded the pathologically significant level of 125 pg/ml. The sST2 level in both groups was also comparable on admission, but did not exceed the pathologically significant level of 30 ng/ml. The data are presented in Table 2. A decreased level of C-reactive protein was noted in both groups, at the follow-up visit one year later in patients without myocarditis, the observed decrease was by 58%, while in the group with diagnosed myocarditis it was decreased only by 28%. The NT-proBNP content in both groups decreased by 40% one year later, but its concentration also remained above the pathologically significant level. The level of sST2 one year later in the group of patients without myocarditis remained unchanged; in the group of patients with myocarditis, it was increased by 76%. In the group of patients with myocarditis, the IGF-1R content increased by 5-fold, while the median IGF-1R concentration in the blood serum of patients without myocarditis remained at the level comparable to that observed at the time of hospitalization. The content of interleukin-6 in both groups of patients decreased by 48 and 43%, respectively. The content of interleukin-10 in the group of patients with myocarditis increased by 18% compared to the baseline level; in the group of patients without myocarditis, the level of IL-10 remained unchanged. In both groups, the content of TNFα decreased by 12 and 20%, respectively. Thus, the study of the dynamics of biomarkers in both groups revealed decreased concentrations of inflammation markers such as CRP, IL-6, and NT-proBNP a marker of the risk of heart failure after 12 months of observation. Moreover, in the group of patients with myocarditis, there was increased content of sST2, IGF-1R, IL-10 after 12 months of observation. Correlation analysis in the group of patients without myocarditis revealed a negative relationship between the content of NTproBNP and IGF-1R (r = -0.70, p < 0.05). In both groups of patients, a positive relationship with high correlation coefficient was found between serum level of sST2 and IGF-1R (r = 0.90) p < 0.05 on admission and at the follow-up visit one year later. Cytokines are a key element of the immune system in the development of acute and chronic inflammation. The majority of chronic inflammatory diseases, regardless of their organ location, are based on imbalanced production between pro- and antiinflammatory mediators [4]. Therefore, the basis for the treatment of any inflammatory process is attempted to normalize it by any means. It has now been established that not only lymphocytes and macrophages, but also other cells are capable of synthesizing cytokines, and in this respect the heart is a unique source of their production [9]. The concentration of hCRP is considered as the most sensitive and specific labo ratory marker of inflammation and tissue damage [8] and, as we demonstrated in our study, it correlates with the synthesis of IL-6 both with/without myo carditis, which, in turn, plays an important role in the developing inflammation. It is known that cardiomyocytes in patients with heart failure produce 841 Огуркова О.Н. и др. Ogurkova O.N. et al. Медицинская Иммунология Medical Immunology (Russia)/Meditsinskaya Immunologiya TABLE 1. BASELINE CHARACTERISTICS OF PATIENTS WHO WERE INCLUDED IN THE STUDY, Me (Q0.25-Q0.75) Parameters Group 1 (n = 5) Group 2 (n = 5) 58.0 (51.0-61.0) 61.0 (56.0-67.0) Body Mass Index, kg/m 31.6 (30.3-34.7) 28.0 (25.30-31.07) Duration of chronic heart failure, month 24.0 (12.7-77.0) 12.0 (8.0-48.0) Time between the last myocardial infarction before the development of chronic heart failure, month 26.0 (6.0-114.0) 80.0 (13.0-120.0) Age, years 2 Functional class of CHF by (NYHA) before hospitalization: II 4 2 III – 3 IV 1 – Systolic pressure, mmHg 130.0 (100.0-138.0) 120.0 (100.0-130.0) Diastolic pressure, mmHg 80.0 (60.0-80.0) 70.0 (60.0-80.0) Heart rate, beats/min. 68.0 (64.0-82.0) 72.0 (68.0-90.0) Respiratory rate, breath/min. 18.0 (17.0-20.0) 18.0 (16.0-20.0) A2 receptor Blockers/ ACE inhibitors – 1 Beta-blockers 3 – Cardiac glycosides - 2 Diuretics 4 4 Acyclovir – 2 Viferon 1 4 Medical treatment during hospitalization: TABLE 2. CYTOKINE LEVEL, Me (Q0.25-Q0.75) Baseline Parameter С-RP(h) mg/l Nt-proBNP pg/ml sST2 ng/ml IGF-1R pg/ml At 12 mounts Group 1 (n = 5) Group 2 (n = 5) Group 1 (n = 5) Group 2 (n = 5) 6.59** (1.03-9.66) 8.26 (1.34-10.20) 2.77 (1.99-4.32) p = 0.06 5.93 (1.69-10.46) 579.70 (93.13-683.71) p = 0.06 525.31 (212.34-532.80) p = 0.06 362.12 (163.79-656.05) 323.40 (268.72-625.72) 2.22 (1.83-4.38) 2.14* (1.35-3.36) 2.10* (1.83-6.54) 8.58 (5.90-10.98) 6639.96 (4213.32-7839.19) 8245.50** (7275.64-8819.10) 7758.29* (5552.64-15202.21) 39808.16 (23040.85-95407.03) IL-6 pg/ml 6.79 (4.69-7.05) p = 0.06 8.11 (5.33-9.80) p = 0.06 3.50 (3.48-6.87) 4.60 (4.02-8.84) IL-10 pg/ml 1.70 (1.28-2.41) 2.57 (0.92-3.60) 1.55* (1.23-2.10) 3.16 (3.09-6.25) TNFα pg/ml 7.62* (4.98-12.65) 20.46 (19.53-23.90) 6.71* (6.42-11.60) 16.53 (11.60-17.81) Note. , statically significant within the group 1 vs 2; *, statically significant within the group Baseline vs At 12 mounts; Group 1, there is no myocarditis; Group 2, there is myocarditis. 842 2021, Т. 23, № 4 2021, Vol. 23, 4 Биомаркеры и цитокины у больных с сердечной недостаточностью Biomarkers and cytokines in patients with heart failure TNFα, even without manifested inflammatory pro cess. In addition, TNFα induces the process of car diomyocyte programmed death, which loses its compensatory character under the pathological con ditions posing TNFα as having fundamental im portance in myocardial remodeling. [8]. In our study, an increased content of serum TNFα was noted in the group of patients with myocarditis; after 12 months of observation, its level remained unchanged, whereas content of interleukin 10 tendned to increase. It is known that the anti-inflammatory cytokine IL-10 can inhibit TNFα production and counterbalance its negative effects in heart failure [2]. However, the results of clinically examined IL-10 in heart failure and its progression are controversial. On the one hand, there is an evidence of decreased IL-10 levels in HF and left ventricular remodeling; on the other hand, there are reports showing its increased levels and elevated mortality in HF patients with a simultaneous increased IL-10 and TNFα concentrations [1]. IL-10 can function as a component of the feedback mechanism: elevated levels of TNFα in HF stimulate the secretion of IL-10, and IL-10, on the contrary, suppresses excessive activity of pro-inflammatory cytokines; however, a role of IL-10 regardless of TNFα, cannot be ruled out as the production of IL-10 is induced together with pro-inflammatory cytokines, TNFα in particular in inflammatory processes and in HF [3]. It was supported by showing positive correlations between the content of TNFα and IL-10 (r = 0.70) p < 0.05 in the group of patients with myocarditis. Among diverse existing modern biochemical markers, natriuretic peptides have been introduced into routine clinical practice, proving to act as markers of myocardial stress, myocardial dysfunction and heart failure [9]. Currently, not a single biomarker can account for all aspects of CHF syndrome and development of its decompensation [9]. According to the numerous modern studies, sST2 has recently been included in the European and American guidelines for CHF treatment. The data obtained in the group of patients with diagnosed myocarditis after 12 months of observation demonstrate not only increased serum sST2 content, but also a positive relationship with proinflammatory cytokines. It is possible that the level of sST2 expression not only confirms the inflammatory mechanism of CHF development, but also reflects the activity of systemic inflammatory response upon disease progression. In recent years, the association of IGF-1 with cardiovascular diseases as an independent risk factor has been discussed, but the results of these studies are rather contradictory. IGF-1 under the influence of growth hormone is secreted in the liver as well as also produced in cardiomyocytes, smooth muscle cells and fibroblasts; it is capable of exerting an insulin-like metabolic effect that exerts a significant role in regulating the structure and function of the myocardium and blood vessels [1]. There is evidence that decreased synthesis of IGF-1 promotes enhanced apoptosis of cardiomyocytes and progression of myocardial fibrosis that appears to be a predictor of decompensated chronic heart failure [3,9]. IGF-1 exerts its action through ubiquitously expressed receptors (IGF-1R) including cardiomyocytes, which determines their role as modulators of myocardial structure and function. Insulin-like growth factors in the bloodstream are associated with proteins that play a key role in the bioavailability of ligands, as they compete with IGF-1R for IGF-I. By binding to IGF-1, IGFPs thereby inhibit proliferation signals and, according to the feedback loop, IGF-1R becomes elevated if IGF-1R is over-activated. There is evidence that proinflammatory cytokines can be involved in blocking IGF-1 specific receptor binding due to the phosphorylation of serine residues in IRS [9]. It is possible that the significant increase in the IGF-1R content obtained in our study in the group of patients with myocarditis underlies hor monal and neurohumoral processes and serves as a biochemical marker of altered metabolic processes, whereas established positive relationships with inflam mation markers seem to be a predictor of aggravated chronic heart failure decompensation, indicating an unfavorable prognosis. Thus, a study in the dynamics of biomarkers in patients with diagnosed myocarditis demonstrated increased level of IGF-1R, soluble ST2, IL-10, a decrease in NL-6, NT-proBNP and C-reactive protein and their close inter-relationship. In the group of patients without myocarditis, there was a decrease in IL-6, NT-proBNP and C-reactive protein, the levels of other biomarkers remained unchanged. The follow-up study revealed significant differences in magnitude of changes in serum level of pro- and anti-inflammatory cytokines and biomarkers of cardiovascular risk in patients with decompensated heart failure with systolic dysfunction with/without diagnosed myocarditis. The established relationships allow us to assume that the presence of myocarditis in patients with decompensated heart failure with ischemic systolic dysfunction leads to formation of more unfavorable relationships in the feedback loop of cytokine secretion, leading to evenly greater shift in the cytokine balance towards quantitative predominance of pro-inflammatory cytokines. 843 Огуркова О.Н. и др. Ogurkova O.N. et al. Медицинская Иммунология Medical Immunology (Russia)/Meditsinskaya Immunologiya References 1. Gennadinik A.G., Nelaeva A.A. 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Interleukin-10 and pro / antiinflammatory cytokine balance in heart failure in patients with type 2 diabetes mellitus. Ukrainian Therapeutic Journal, 2012, no. 3-4, pp. 58-63. (In Russ.) 9. Zakirova A.N., Fatkullina E.Z., Zakirova N.E., Zhamalov L.M. Insulin-like growth factor-1 and myocardial remodeling in patients with arterial hypertension and metabolic syndrome. Rational Pharmacotherapy in Cardiology, 2015, Vol. 11, no. 5, pp. 489-495. (In Russ.) Авторы: Огуркова О.Н. – к.м.н., научный сотрудник отделения клинической лабораторной диагностики, Научно-исследовательский институт кардиологии ФГБНУ «Томский национальный исследовательский медицинский центр Российской академии наук», г. Томск, Россия Кручинкина Е.В. – младший научный сотрудник отделения неотложной кардиологии, Научноисследовательский институт кардиологии ФГБНУ «Томский национальный исследовательский медицинский центр Российской академии наук», г. Томск, Россия Гусакова А.М. – к.фарм.н., научный сотрудник отделения клинической лабораторной диагностики, Научно-исследовательский институт кардиологии ФГБНУ «Томский национальный исследовательский медицинский центр Российской академии наук», г. Томск, Россия Суслова Т.Е. – к.м.н., руководитель отделения клинической лабораторной диагностики, Научноисследовательский институт кардиологии ФГБНУ «Томский национальный исследовательский медицинский центр Российской академии наук», г. Томск, Россия Рябов В.В. – д.м.н., заместитель директора по научной и лечебной работе, Научно-исследовательский институт кардиологии ФГБНУ «Томский национальный исследовательский медицинский центр Российской академии наук»; заведующий кафедрой кардиологии ФПК и ППС ФГБОУ ВО «Сибирский государственный медицинский университет» Министерства здравоохранения РФ, г. Томск, Россия Поступила 15.03.2021 Отправлена на доработку 01.06.2021 Принята к печати 09.06.2021 844 Authors: Ogurkova O.N., PhD (Medicine), Research Associate, Department of Clinical Laboratory Diagnostics, Cardiology Research Institute, Tomsk National Research Medical Centre, Russian Academy of Sciences, Tomsk, Russian Federation Kruchinkina E.V., Junior Research Associate, Department of Emergency Cardiology, Cardiology Research Institute, Tomsk National Research Medical Centre, Russian Academy of Sciences, Tomsk, Russian Federation Gusakova A.M., PhD (Pharmacology), Research Associate, Department of Clinical Laboratory Diagnostics, Cardiology Research Institute, Tomsk National Research Medical Centre, Russian Academy of Sciences, Tomsk, Russian Federation Suslova T.E., PhD (Medicine), Head, Department of Clinical Laboratory Diagnostics, Cardiology Research Institute, Tomsk National Research Medical Centre, Russian Academy of Sciences, Tomsk, Russian Federation Ryabov V.V., PhD, MD (Medicine), Deputy Director for Research and Clinical Services of Cardiology Research Institute, Tomsk National Research Medical Centre, Russian Academy of Sciences; Head, Cardiology Department of the Continuous Medical Education Faculty, Siberian State Medical University, Tomsk, Russian Federation Received 15.03.2021 Revision received 01.06.2021 Accepted 09.06.2021
https://openalex.org/W4391282592	https://ojs.cbn.ac.id/index.php/pemimpin/article/download/1190/433	Indonesian	null	PELATIHAN PEMBUATAN E-LKPD BERBASIS GOOGLE FROM MODEL PROJECT BASED LEARNING BAGI MAHASISWA PGSD	Pengabdian Masyarakat Ilmu Pendidikan	2,024	cc-by-sa	4,396	Abstrak Abstrak Pelatihan Pembuatan E-Lembar Kerja Peserta Didik (E-LKPD) dengan Model Project Based Learning (PBL) berbasis Google Form bagi Mahasiswa Pendidikan Guru Sekolah Dasar (PGSD) merupakan upaya untuk meningkatkan keterampilan mahasiswa dalam menciptakan materi pembelajaran interaktif. Penelitian ini fokus pada penggunaan Google Form dan pendekatan PBL untuk menciptakan E-LKPD yang relevan dengan kurikulum. Metode pelatihan mencakup pemaparan konsep, demonstrasi penggunaan Google Form, dan proyek kolaboratif dalam menerapkan PBL. Evaluasi dilakukan terhadap kualitas E-LKPD yang dihasilkan dan kemampuan mahasiswa dalam menerapkan PBL. Hasilnya menunjukkan peningkatan kualitas E- LKPD dan pemahaman yang lebih baik mengenai PBL. Pendidikan berkembang seiring dengan kemajuan teknologi, menuntut para pendidik untuk mengadopsi metode yang inovatif dalam proses pembelajaran. Khususnya dalam Pendidikan Guru Sekolah Dasar (PGSD), integrasi teknologi menjadi aspek penting untuk menciptakan pengalaman belajar yang relevan dan menarik bagi mahasiswa. Salah satu alat teknologi yang sangat berguna dalam konteks ini adalah Google Form, yang tidak hanya menyediakan platform yang mudah diakses tetapi juga memungkinkan pembuatan materi pembelajaran yang interaktif dan adaptif. p Dalam konteks ini, pengembangan E-Lembar Kerja Peserta Didik (E- LKPD) berbasis Google Form menjadi fokus utama. E-LKPD ini bukan hanya sekadar materi statis, tetapi sebuah alat yang dapat diakses secara daring, memungkinkan interaksi, pengayaan konten, dan evaluasi yang lebih dinamis. Namun, untuk mencapai hal ini, diperlukan pemahaman yang baik tentang bagaimana mengintegrasikan teknologi ini ke dalam strategi pembelajaran yang sudah ada, dan di sinilah Model Project Based Learning (PBL) memainkan peran krusial Kata Kunci : (E-LKPD), Google Form, Project Based Learning (PBL) Kata Kunci : (E-LKPD), Google Form, Project Based Learning (PBL) PBL menawarkan pendekatan yang melibatkan pembelajaran berbasis proyek, di mana mahasiswa secara aktif terlibat dalam penyelesaian masalah nyata. Dalam konteks pelatihan bagi mahasiswa PGSD, PBL memberikan kerangka kerja yang tepat untuk mengaplikasikan pengetahuan yang mereka pelajari ke dalam konteks praktis pembuatan E- LKPD. Proses ini melibatkan pemahaman kurikulum, penerapan kreativitas, dan penggunaan teknologi Google Form sebagai alat pembuatan E-LKPD yang inovatif. PELATIHAN PEMBUATAN E-LKPD BERBASIS GOOGLE FROM MODEL PROJECT BASED LEARNING BAGI MAHASISWA PGSD 1Lolita Anna Risandy, 2Septiana Sholikhah, 3Putri Zudhah Ferryka, 4Anggi Firnanda 1,2,3,4 Universitas Widya Dharma Klaten, Jawa Tengah 1Lolita Anna Risandy, 2Septiana Sholikhah, 3Putri Zudhah Ferryka, 4Anggi Firnanda 1,2,3,4 Universitas Widya Dharma Klaten, Jawa Tengah 1 lolitaanna02@gmail.com, 2 septianasholikhah99@gmail.com, 3 zudhahputri@gmail.com, 4 anggifirnanda1906@gmail.com 1 lolitaanna02@gmail.com, 2 septianasholikhah99@gmail.com, 3 zudhahputri@gmail.com, 4 anggifirnanda1906@gmail.com il.com, 2 septianasholikhah99@gmail.com, 3 zudhahputri@gmail.com, 4 anggifirnanda1906@gmail.com Volume 4 No 1 – Januari 2024 Penyusunan Materi dan Rencana Pelatihan y Materi pelatihan dirancang berdasarkan prinsip PBL dengan penekanan pada integrasi teknologi Google Form dalam pembuatan E-LKPD. Dokumentasi dan panduan langkah demi langkah disusun untuk memandu peserta dalam mengembangkan E-LKPD yang sesuai dengan kebutuhan kurikulum di SD. Langkah Persiapan Pertama, pendekatan dalam perencanaan kegiatan ini melibatkan analisis terhadap kurikulum PGSD dan kebutuhan teknologi yang mendukung pembelajaran. Tim fasilitator melakukan penelitian literatur untuk mengevaluasi prinsip PBL dan pemanfaatan Google Form dalam menciptakan E-LKPD yang efektif. Pelaksanaan Pelatihan Kegiatan dimulai dengan sesi pengantar yang memperkenalkan konsep PBL dan potensi Google Form dalam pembelajaran interaktif. Sesi praktik langsung memungkinkan peserta untuk berkolaborasi dalam pengembangan E-LKPD. Ada juga ruang untuk diskusi reflektif dan sesi tanya jawab guna memastikan pemahaman yang komprehensif. Evaluasi dan Penilaian Evaluasi dilakukan dalam beberapa tahap. Pertama, penilaian formatif selama proses pelatihan untuk memastikan pemahaman yang berkesinambungan. Kedua, penilaian terhadap E-LKPD yang dihasilkan peserta, termasuk aspek kreativitas, relevansi kurikulum, dan kegunaan teknologi. Feedback dari peserta juga diambil sebagai masukan untuk pengembangan lebih lanjut. 2. METODE ABDIMAS Kegiatan Abdimas (Pengabdian kepada Masyarakat) yang mengusung pelatihan Pembuatan E-Lembar Kerja Peserta Didik (E-LKPD) dengan pendekatan Model Project Based Learning (PBL) berbasis Google Form bagi Mahasiswa Pendidikan Guru Sekolah Dasar (PGSD) merupakan upaya yang dilakukan untuk memperkuat keterampilan mahasiswa dalam menghadapi tuntutan zaman yang kian digital dan inovatif. Diseminasi Hasil dan Pengembangan Lanjutan Hasil pelatihan, baik berupa E-LKPD yang dibuat maupun hasil evaluasi, didiseminasi melalui seminar, publikasi, atau forum diskusi untuk berbagi pengalaman dan mendukung pengembangan berkelanjutan. Pelatihan ini juga menjadi landasan untuk pengembangan program serupa di masa depan. Melalui metode ini, diharapkan para mahasiswa PGSD dapat mengasah keterampilan teknologi, kreativitas, serta penerapan konsep pembelajaran inovatif dalam menciptakan E-LKPD yang relevan dan adaptif. Selain itu, metode ini juga memberikan kontribusi positif dalam meningkatkan pemahaman mahasiswa mengenai pemanfaatan teknologi dalam konteks pendidikan dasar. Abtsract Training on Creating Student E- Worksheets (E-LKPD) using the Google Form-based Project Based Learning (PBL) Model for Elementary School Teacher Education (PGSD) Students is an effort to improve students' skills in creating interactive learning materials. This research focuses on the use of Google Forms and the PBL approach to create E-LKPD that is relevant to the curriculum. Training methods include concept exposure, demonstration of the use of Google Forms, and collaborative projects in implementing PBL. Evaluation is carried out on the quality of the E-LKPD produced and students' ability to apply PBL. The results show an increase in the quality of E-LKPD and a better understanding of PBL. Pelatihan ini dirancang untuk memberikan pemahaman mendalam tentang cara membuat E-LKPD yang tidak hanya informatif tetapi juga interaktif dan sesuai dengan kebutuhan kurikulum di Sekolah Dasar. Melalui pendekatan kolaboratif dan sesi praktik langsung, diharapkan mahasiswa mampu mengembangkan keterampilan desain, teknologi, serta pemahaman yang lebih mendalam tentang bagaimana metode pembelajaran seperti PBL dapat diintegrasikan ke dalam proses pembuatan E-LKPD. p Dengan menggabungkan konsep teknologi informasi dan strategi pembelajaran inovatif, diharapkan pelatihan ini dapat mempersiapkan mahasiswa PGSD menjadi pendidik yang adaptif, kreatif, dan mampu menggunakan teknologi sebagai alat untuk meningkatkan pengalaman belajar peserta didik di era digital. Keywords: (E-LKPD), Google Form, Project Based Learning (PBL). Keywords: (E-LKPD), Google Form, Project Based Learning (PBL). 19 JURNAL PEMIMPIN – PENGABDIAN MASYARAKAT ILMU PENDIDIKAN E-ISSN: 2808-5132 19 \| H a l Volume 4 No 1 – Januari 2024 Volume 4 No 1 – Januari 2024 ganda, isian singkat, dan tampilan yang dapat disesuaikan memungkinkan mahasiswa untuk menciptakan E- LKPD yang bervariasi dan interaktif. Ketersediaan integrasi dengan Google Sheets sebagai database penyimpanan jawaban juga memudahkan pemantauan dan analisis respons peserta didik. ganda, isian singkat, dan tampilan yang dapat disesuaikan memungkinkan mahasiswa untuk menciptakan E- LKPD yang bervariasi dan interaktif. Ketersediaan integrasi dengan Google Sheets sebagai database penyimpanan jawaban juga memudahkan pemantauan dan analisis respons peserta didik. p y p j j g p p p Penggunaan Google Form juga memungkinkan adanya adaptasi terhadap kebutuhan kurikulum yang beragam. Mahasiswa PGSD dapat membuat E-LKPD yang sesuai dengan tujuan pembelajaran, kompetensi, dan indikator yang terdapat dalam kurikulum yang berlaku. Fleksibilitas dalam menyusun soal, materi tambahan, atau sumber daya belajar lainnya memungkinkan adanya penyesuaian terhadap kebutuhan spesifik peserta didik. Selain itu, interaktifitas Google Form memperkaya pengalaman belajar. Mahasiswa dapat mengintegrasikan multimedia, seperti gambar, video, atau tautan ke sumber belajar lainnya, sehingga E-LKPD tidak hanya statis tetapi juga menarik dan memperkaya pengalaman peserta didik dalam proses pembelajaran. Dalam konteks adaptabilitas, Google Form memungkinkan respons dinamis terhadap kebutuhan peserta didik. Mahasiswa dapat merancang E-LKPD yang memberikan umpan balik otomatis, memberikan soal b b i di k j l b h b d k j b dib ik D Selain itu, interaktifitas Google Form memperkaya pengalaman belajar. Mahasiswa dapat mengintegrasikan multimedia, seperti gambar, video, atau tautan ke sumber belajar lainnya, sehingga E-LKPD tidak hanya statis tetapi juga menarik dan memperkaya pengalaman peserta didik dalam proses pembelajaran. Selain itu, interaktifitas Google Form memperkaya pengalaman belajar. Mahasiswa dapat mengintegrasikan multimedia, seperti gambar, video, atau tautan ke sumber belajar lainnya, sehingga E-LKPD tidak hanya statis tetapi juga menarik dan memperkaya pengalaman peserta didik dalam proses pembelajaran. Dalam konteks adaptabilitas, Google Form memungkinkan respons dinamis terhadap kebutuhan peserta didik. Mahasiswa dapat merancang E-LKPD yang memberikan umpan balik otomatis, memberikan soal berbasis respons, atau menyediakan penjelasan tambahan berdasarkan jawaban yang diberikan. Dengan demikian, E-LKPD dapat menyesuaikan diri dengan tingkat pemahaman dan kecepatan belajar masing-masing peserta didik. y p j g p y p g p p p j Dalam konteks adaptabilitas, Google Form memungkinkan respons dinamis terhadap kebutuhan peserta didik. Mahasiswa dapat merancang E-LKPD yang memberikan umpan balik otomatis, memberikan soal berbasis respons, atau menyediakan penjelasan tambahan berdasarkan jawaban yang diberikan. Dengan demikian, E-LKPD dapat menyesuaikan diri dengan tingkat pemahaman dan kecepatan belajar masing-masing peserta didik. Namun, untuk mencapai efektivitas yang maksimal, penting bagi mahasiswa PGSD untuk memahami secara menyeluruh tujuan pembelajaran, karakteristik peserta didik, serta menyusun E-LKPD dengan desain yang mempertimbangkan prinsip-prinsip pedagogi yang sesuai. Selain itu, pelatihan dan bimbingan yang memadai dalam pemanfaatan fitur-fitur Google Form juga diperlukan agar mahasiswa dapat memanfaatkannya secara optimal dalam menciptakan E-LKPD yang efektif, interaktif, dan adaptif sesuai dengan kebutuhan kurikulum di Sekolah Dasar. 3. HASIL DAN PEMBAHASAN A. Efektivitas penggunaan Google Form dalam menciptakan E-Lembar Kerja Peserta Didik (E- LKPD) yang interaktif dan adaptif sesuai dengan kebutuhan kurikulum bagi mahasiswa PGSD Efektivitas penggunaan Google Form dalam menciptakan E-Lembar Kerja Peserta Didik (E-LKPD) yang interaktif dan adaptif sesuai kebutuhan kurikulum bagi mahasiswa Pendidikan Guru Sekolah Dasar (PGSD) menjadi aspek penting dalam mempersiapkan mereka sebagai calon guru yang beradaptasi dengan tuntutan teknologi saat ini. g Pertama-tama, Google Form memberikan kemudahan dalam pembuatan kuesioner dan formulir daring yang dapat diakses dengan mudah oleh pengguna. Dalam konteks pembelajaran, fitur-fitur seperti pilihan 20 \| H a l 20 \| H a l C. Proses pelatihan Pembuatan E-LKPD berbasis Google Form dengan pendekatan PBL memengaruhi pemahaman mahasiswa PGSD terhadap konsep desain pembelajaran yang inovatif. Proses pelatihan Pembuatan E-Lembar Kerja Peserta Didik (E-LKPD) berbasis Google Form dengan pendekatan Model Project Based Learning (PBL) memberikan dampak yang signifikan terhadap pemahaman mahasiswa Pendidikan Guru Sekolah Dasar (PGSD) terhadap konsep desain pembelajaran yang inovatif. p p p j y g Pertama-tama, pendekatan PBL dalam pelatihan menciptakan lingkungan belajar yang kolaboratif dan interaktif. Melalui pembelajaran berbasis proyek, mahasiswa PGSD tidak hanya diberi pengetahuan teoritis, tetapi juga diberi kesempatan untuk mengaplikasikan pengetahuan tersebut dalam konteks praktis. Dalam hal ini, penggunaan Google Form sebagai alat untuk membuat E-LKPD memberikan kesempatan bagi mahasiswa untuk memperoleh pemahaman yang lebih mendalam tentang bagaimana teknologi dapat diintegrasikan dalam pembelajaran. Selama proses pelatihan, mahasiswa PGSD terlibat dalam aktivitas merancang, mengembangkan, dan mengimplementasikan E-LKPD. Mereka belajar untuk memahami kebutuhan peserta didik mereka, memikirkan strategi pengajaran yang sesuai, dan menciptakan materi pembelajaran yang relevan, menarik, serta responsif terhadap kebutuhan kurikulum. Hal ini memberi kesempatan bagi mereka untuk menggali konsep desain pembelajaran yang inovatif. Selain itu, proses pelatihan ini memungkinkan mahasiswa untuk mengalami secara langsung bagaimana teknologi dapat menjadi alat yang powerful dalam mendukung pembelajaran. Mereka tidak hanya belajar tentang fitur-fitur teknologi Google Form, tetapi juga mengerti bagaimana teknologi tersebut dapat diaplikasikan dalam menciptakan pengalaman pembelajaran yang lebih dinamis dan interaktif bagi peserta didik. Proses refleksi yang terintegrasi dalam PBL juga memberikan ruang bagi mahasiswa PGSD untuk mengevaluasi karya mereka. Mereka dapat mengidentifikasi kelebihan, kekurangan, dan perbaikan yang mungkin diperlukan dalam E-LKPD yang telah mereka buat. Hal ini membantu mereka untuk terus meningkatkan kualitas desain pembelajaran yang inovatif melalui penggunaan teknologi. g p j y g p gg g Namun, pemahaman yang mendalam tentang konsep desain pembelajaran yang inovatif memerlukan proses yang berkelanjutan. Penting bagi institusi pendidikan untuk memberikan dukungan kontinu, melalui workshop, pembimbingan, atau diskusi, agar mahasiswa terus dapat mengembangkan pemahaman dan keterampilan dalam merancang pembelajaran yang inovatif menggunakan teknologi seperti Google Form. Dengan demikian, proses pelatihan ini tidak hanya menciptakan pemahaman yang lebih baik, tetapi juga memberi dorongan untuk terus meningkatkan kualitas desain pembelajaran yang adaptif dan inovatif di masa mendatang. B. Sejauh mana Model Project Based Learning (PBL) dapat meningkatkan keterampilan mahasiswa PGSD dalam mengintegrasikan teknologi (Google Form) dalam pembuatan materi pembelajaran. B. Sejauh mana Model Project Based Learning (PBL) dapat meningkatkan keterampilan mahasiswa PGSD dalam mengintegrasikan teknologi (Google Form) dalam pembuatan materi pembelajaran. Model Project Based Learning (PBL) telah terbukti menjadi metode pembelajaran yang efektif dalam mengembangkan keterampilan praktis dan konseptual bagi mahasiswa Pendidikan Guru Sekolah Dasar (PGSD). Ketika PBL dipadukan dengan integrasi teknologi, seperti penggunaan Google Form dalam pembuatan materi pembelajaran, hal itu membuka pintu untuk pengalaman pembelajaran yang lebih mendalam dan relevan. PBL menekankan pembelajaran melalui proyek, di mana mahasiswa terlibat aktif dalam menyusun solusi atas masalah nyata atau simulasi kasus yang mendekati konteks kehidupan sehari-hari. Dalam konteks pembelajaran untuk PGSD, PBL memberikan kesempatan bagi mahasiswa untuk merancang materi pembelajaran yang konkret dan sesuai dengan kebutuhan peserta didik di tingkat sekolah dasar. Keterampilan yang dikembangkan melalui PBL termasuk keterampilan kolaborasi, pemecahan masalah, dan kreativitas. Ketika mahasiswa PGSD menggunakan PBL untuk membuat materi pembelajaran dengan Google Form, mereka secara alamiah terlibat dalam kolaborasi, diskusi, dan pemecahan masalah untuk menciptakan E-Lembar Kerja Peserta Didik (E-LKPD) yang sesuai dengan tujuan kurikulum dan memanfaatkan teknologi yang tersedia. g y g PBL juga memungkinkan mahasiswa untuk belajar dengan cara yang lebih mandiri. Dalam konteks integrasi Google Form, mahasiswa dapat mengeksplorasi berbagai fitur yang ditawarkan platform tersebut untuk membuat E-LKPD yang interaktif dan responsif. Mereka dapat memanfaatkan pengetahuan teknologi yang mereka peroleh untuk mengkreasikan pertanyaan yang dinamis, menyesuaikan tipe-jawaban, menyusun evaluasi berbasis respons, serta menambahkan elemen multimedia yang memperkaya materi pembelajaran. p y g p y p j Selain itu, PBL memacu mahasiswa PGSD untuk memiliki keterampilan analisis yang kuat. Mereka tidak hanya membuat E-LKPD secara mekanis tetapi juga secara kritis mempertimbangkan cara terbaik untuk menyampaikan materi yang relevan, menarik, dan bermanfaat bagi peserta didik mereka. Integrasi Google Form dalam PBL memberikan peluang bagi mahasiswa untuk memahami bagaimana teknologi dapat diintegrasikan dengan baik dalam pendidikan dan bagaimana hal itu dapat meningkatkan pengalaman belajar. g g p g p g p g j Namun, keberhasilan pengintegrasian PBL dan Google Form membutuhkan pendekatan yang terencana dan bimbingan yang memadai dari dosen atau fasilitator. Pelatihan yang tepat dan bimbingan dalam mengenali 21 \| H a l Volume 4 No 1 – Januari 2024 potensi teknologi dan mengaplikasikannya dalam konteks pembelajaran menjadi kunci bagi mahasiswa PGSD untuk mengoptimalkan penggunaan Google Form dalam menciptakan materi pembelajaran yang efektif. C. Proses pelatihan Pembuatan E-LKPD berbasis Google Form dengan pendekatan PBL memengaruhi pemahaman mahasiswa PGSD terhadap konsep desain pembelajaran yang inovatif. Volume 4 No 1 – Januari 2024 dalam menyusun materi pembelajaran yang sesuai dengan kurikulum, mudah dipahami oleh peserta didik, serta memanfaatkan teknologi untuk memperkaya pengalaman belajar. Cara mengatasinya: Fasilitasi diskusi terstruktur tentang kurikulum dan pendekatan desain pembelajaran yang relevan dengan kebutuhan peserta didik di tingkat sekolah dasar. Bimbingan intensif dari dosen atau pembimbing yang berpengalaman juga akan membantu mahasiswa untuk mengarahkan pemikiran mereka ke arah yang lebih produktif. dalam menyusun materi pembelajaran yang sesuai dengan kurikulum, mudah dipahami oleh peserta didik, serta memanfaatkan teknologi untuk memperkaya pengalaman belajar. Cara mengatasinya: Fasilitasi diskusi terstruktur tentang kurikulum dan pendekatan desain pembelajaran yang relevan dengan kebutuhan peserta didik di tingkat sekolah dasar. Bimbingan intensif dari dosen atau pembimbing yang berpengalaman juga akan membantu mahasiswa untuk mengarahkan pemikiran mereka ke arah yang lebih produktif. 3. Keterbatasan Akses atau Sarana Pendukung: Tidak semua mahasiswa mungkin memiliki akses yang sama terhadap perangkat lunak atau perangkat keras yang dibutuhkan untuk mengembangkan E-LKPD yang kompleks. Cara mengatasinya: Institusi pendidikan dapat menyediakan akses yang sama bagi semua mahasiswa ke perangkat lunak dan perangkat keras yang diperlukan. Selain itu, pilihan alternatif seperti kemitraan dengan lembaga lain atau penggunaan perpustakaan universitas untuk akses perangkat mungkin dapat membantu mengatasi hambatan ini. 4. Kesulitan dalam Kolaborasi dan Penerapan PBL secara Efektif: Proyek bersama dalam PBL memerlukan kerja tim yang solid. Mahasiswa mungkin menghadapi tantangan dalam berkolaborasi dan membagi tugas dengan efektif. Cara mengatasinya: Sesi pelatihan tentang kerja tim, komunikasi yang efektif, dan manajemen proyek dapat membantu mahasiswa memahami pentingnya kolaborasi yang kuat dalam PBL. Memberikan ruang bagi mereka untuk berinteraksi dan bekerja sama dalam proyek-proyek kecil dapat meningkatkan kemampuan kerja tim mereka. 5. Kendala Waktu dan Tekanan Belajar: Mahasiswa PGSD sering kali memiliki jadwal yang padat dan tuntutan akademis yang tinggi. Hal ini dapat menghambat mereka untuk sepenuhnya fokus pada proses pengembangan E-LKPD dengan pendekatan PBL. Cara mengatasinya: Penting bagi institusi untuk memberikan dukungan dan fleksibilitas dalam jadwal, serta memberikan bantuan yang tepat agar mahasiswa dapat mengelola waktu mereka dengan efisien. Mendorong mereka untuk membuat rencana kerja yang terstruktur juga dapat membantu mengurangi tekanan belajar yang berlebihan. Mengatasi tantangan-tantangan ini membutuhkan perencanaan yang matang dan dukungan yang tepat dari institusi pendidikan. Melalui pendekatan yang holistik, seperti kombinasi pelatihan teknis, bimbingan akademis, dan lingkungan belajar yang mendukung, mahasiswa PGSD dapat mengatasi tantangan ini dan mengembangkan E-LKPD yang inovatif dengan pendekatan PBL menggunakan Google Form. D. Tantangan utama yang dihadapi mahasiswa PGSD dalam mengembangkan E-LKPD menggunakan Google Form dengan pendekatan PBL, dan bagaimana cara mengatasinya. Mahasiswa Pendidikan Guru Sekolah Dasar (PGSD) menghadapi beberapa tantangan signifikan dalam mengembangkan E-Lembar Kerja Peserta Didik (E-LKPD) menggunakan Google Form dengan pendekatan Model Project Based Learning (PBL). Beberapa tantangan utama meliputi: 1. Keterbatasan Pengetahuan Teknologi: Banyak mahasiswa PGSD mungkin memiliki keterampilan teknologi dasar, tetapi tidak cukup familiar dengan fitur-fitur khusus Google Form yang dapat meningkatkan interaktivitas dan efektivitas E-LKPD. Cara mengatasinya: Penyediaan pelatihan yang komprehensif tentang fitur-fitur Google Form yang relevan untuk pembuatan E-LKPD. Workshops, tutorial, atau sumber daya online dapat membantu mahasiswa memahami cara memanfaatkan secara maksimal alat tersebut. 1. Keterbatasan Pengetahuan Teknologi: Banyak mahasiswa PGSD mungkin memiliki keterampilan teknologi dasar, tetapi tidak cukup familiar dengan fitur-fitur khusus Google Form yang dapat meningkatkan interaktivitas dan efektivitas E-LKPD. Cara mengatasinya: Penyediaan pelatihan yang komprehensif tentang fitur-fitur Google Form yang relevan untuk pembuatan E-LKPD. Workshops, tutorial, atau sumber daya online dapat membantu mahasiswa memahami cara memanfaatkan secara maksimal alat tersebut. p g Cara mengatasinya: Penyediaan pelatihan yang komprehensif tentang fitur-fitur Google Form yang relevan untuk pembuatan E-LKPD. Workshops, tutorial, atau sumber daya online dapat membantu mahasiswa memahami cara memanfaatkan secara maksimal alat tersebut. 2 Kesulitan dalam Merancang Materi yang Relevan: Mahasiswa mungkin mengalami kesulitan 2. Kesulitan dalam Merancang Materi yang Relevan: Mahasiswa mungk 22 \| H a l 22 \| H a l E. Dampak pelatihan ini terhadap kesiapan mahasiswa PGSD dalam mengaplikasikan keterampilan teknologi dan pendekatan inovatif dalam mengajar di lingkungan sekolah dasar Pelatihan mengenai Pembuatan E-Lembar Kerja Peserta Didik (E-LKPD) berbasis Google Form dengan pendekatan Model Project Based Learning (PBL) memiliki dampak yang substansial terhadap kesiapan mahasiswa Pendidikan Guru Sekolah Dasar (PGSD) dalam mengaplikasikan keterampilan teknologi dan pendekatan inovatif dalam mengajar di lingkungan sekolah dasar. 1. Penguasaan Teknologi: Pelatihan ini memperkuat pemahaman dan keterampilan mahasiswa dalam menggunakan teknologi, khususnya Google Form, sebagai alat untuk menciptakan materi pembelajaran yang interaktif dan adaptif. Mahasiswa menjadi lebih terampil dalam memanfaatkan fitur-fitur teknologi yang relevan dengan kebutuhan pembelajaran di sekolah dasar. 1. Penguasaan Teknologi: Pelatihan ini memperkuat pemahaman dan keterampilan mahasiswa dalam menggunakan teknologi, khususnya Google Form, sebagai alat untuk menciptakan materi pembelajaran yang interaktif dan adaptif. Mahasiswa menjadi lebih terampil dalam memanfaatkan fitur-fitur teknologi yang relevan dengan kebutuhan pembelajaran di sekolah dasar. 2. Kreativitas dan Inovasi dalam Pembelajaran: Dampak utama dari pelatihan ini adalah peningkatan kreativitas dan inovasi dalam merancang materi pembelajaran. Mahasiswa belajar untuk berpikir di luar kotak, mengintegrasikan teknologi ke dalam rencana pembelajaran mereka, dan menciptakan pengalaman belajar yang lebih menarik dan relevan bagi peserta didik di tingkat sekolah dasar. 23 \| H a l 4. KESIMPULAN PBL menjadi teras yang memungkinkan mahasiswa untuk melihat pembelajaran sebagai proses dinamis, bukan sekadar transfer informasi. Mereka belajar untuk merancang proyek pembelajaran yang menantang, berkolaborasi dalam tim, dan memanfaatkan teknologi sebagai alat untuk menciptakan pengalaman belajar yang lebih bermakna. Seiring dengan itu, mahasiswa juga terlibat dalam proses refleksi yang mendalam, mempertimbangkan bagaimana pendekatan inovatif ini dapat diterapkan secara efektif di lingkungan sekolah dasar. Dampak dari pelatihan ini tidak hanya berhenti pada penguasaan teknologi atau desain pembelajaran inovatif, tetapi meluas ke kesiapan mahasiswa dalam menghadapi tantangan masa depan dalam dunia pendidikan. Mereka menjadi agen perubahan yang siap memimpin integrasi teknologi di lingkungan pendidikan, membawa inovasi dalam strategi pembelajaran, serta memperluas kemungkinan-kemungkinan untuk peserta didik di era digital ini. Dalam konteks ini, pelatihan menciptakan landasan yang kokoh bagi mahasiswa PGSD untuk menjadi pendidik yang adaptif, kreatif, dan siap untuk menjawab dinamika pendidikan yang terus berubah. Dengan pengalaman ini, mereka siap membawa perubahan dalam pembelajaran di sekolah dasar, mendorong adopsi teknologi dalam pengajaran, dan memperkaya pengalaman belajar siswa dengan metode yang lebih inovatif. Oleh karena itu, pelatihan ini tidak hanya menjadi titik awal bagi kemampuan teknologi, tetapi juga sebuah pijakan penting untuk membentuk pendidik yang mampu menghadapi dan menginspirasi masa depan pendidikan yang lebih cerah. Volume 4 No 1 – Januari 2024 4. KESIMPULAN Pembahasan tentang pelatihan Pembuatan E-Lembar Kerja Peserta Didik (E-LKPD) berbasis Google Form dengan pendekatan Model Project Based Learning (PBL) bagi mahasiswa Pendidikan Guru Sekolah Dasar (PGSD) menggambarkan transformasi signifikan dalam persiapan calon pendidik untuk menghadapi era pendidikan yang semakin terkini dan teknologi yang terus berkembang. Melalui pelatihan ini, mahasiswa PGSD bukan hanya mengasah keterampilan teknologi dengan memanfaatkan Google Form untuk menciptakan materi pembelajaran interaktif, tetapi juga mengintegrasikan aspek inovatif dan kreatif dalam proses pengajaran. Dari pemahaman yang dalam tentang desain pembelajaran yang adaptif hingga kesiapan menghadapi tantangan teknologi di lingkungan sekolah, pelatihan ini membekali mahasiswa dengan kemampuan yang jauh lebih luas daripada sekadar penguasaan teknologi. Mereka belajar untuk berpikir kritis, merancang pembelajaran yang responsif terhadap kebutuhan peserta didik, dan siap menghadapi dinamika pendidikan yang terus berubah. Selain itu, mahasiswa juga dilatih untuk menjadi agen perubahan yang membawa inovasi ke lingkungan sekolah, memimpin penggunaan teknologi, dan menggali potensi pembelajaran yang lebih efektif. Pelatihan ini bukan hanya tentang penggunaan alat teknologi, melainkan tentang mengubah paradigma pendidikan menuju pendekatan yang lebih adaptif, interaktif, dan relevan dengan kebutuhan peserta didik di era digital. Dengan demikian, pelatihan ini berperan penting dalam menciptakan calon-calon guru yang tidak hanya terampil dalam teknologi, tetapi juga siap menghadapi dan menginspirasi perkembangan pendidikan di masa depan. g , p j g p g p g p p g p p Pelatihan mengenai pembuatan E-Lembar Kerja Peserta Didik (E-LKPD) dengan pendekatan Model Project Based Learning (PBL) berbasis Google Form bagi mahasiswa Pendidikan Guru Sekolah Dasar (PGSD) membawa dampak yang mendalam terhadap paradigma pendidikan dan persiapan calon pendidik. Pelatihan ini tidak hanya tentang penggunaan teknologi dalam pembelajaran, tetapi juga mengenai memahami esensi dari pendekatan inovatif dalam menyusun materi pembelajaran yang lebih adaptif, interaktif, dan sesuai dengan kebutuhan peserta didik. Mahasiswa PGSD tidak hanya menjadi terampil dalam mengoperasikan Google Form, tetapi juga mampu menerapkan keterampilan desain pembelajaran yang relevan, responsif, dan kreatif. j di ki k h i k lih b l j b i di i PBL menjadi teras yang memungkinkan mahasiswa untuk melihat pembelajaran sebagai proses dinamis, bukan sekadar transfer informasi. Mereka belajar untuk merancang proyek pembelajaran yang menantang, berkolaborasi dalam tim, dan memanfaatkan teknologi sebagai alat untuk menciptakan pengalaman belajar yang lebih bermakna. Seiring dengan itu, mahasiswa juga terlibat dalam proses refleksi yang mendalam, mempertimbangkan bagaimana pendekatan inovatif ini dapat diterapkan secara efektif di lingkungan sekolah dasar. Kusuma, A. I., Sari, W. P., & Hikmawati, A. (2021). Peningkatan Kompetensi Guru dan Tenaga Kependidikan Melalui Pelatihan Teacherpreneur Berbasis Digital Profile Builder. Jmm (Jurnal Masyarakat Mandiri), Volume 4 No 1 – Januari 2024 3. Kesiapan Menghadapi Tantangan Teknologi di Sekolah: Mahasiswa PGSD dilatih untuk menjadi lebih siap dalam menghadapi tantangan teknologi yang mungkin mereka temui di lingkungan sekolah. Mereka menjadi lebih percaya diri dalam menghadapi perubahan teknologi dan lebih mudah menyesuaikan diri dengan alat-alat baru yang mungkin diperkenalkan di masa depan. 4. Keterampilan Manajemen Pembelajaran yang Lebih Efektif: Melalui PBL, mahasiswa PGSD tidak hanya mempelajari cara menggunakan teknologi untuk membuat materi pembelajaran, tetapi juga memahami bagaimana mengelola pembelajaran yang melibatkan teknologi tersebut. Mereka belajar tentang desain pembelajaran yang adaptif, evaluasi yang relevan, dan integrasi teknologi dalam proses pengajaran. p p g j 5. Kesiapan Beradaptasi dengan Perkembangan Pendidikan: Sebagai calon guru, mahasiswa PGSD perlu mempersiapkan diri untuk terus belajar dan beradaptasi dengan perkembangan terbaru dalam pendidikan. Pelatihan ini memberikan dasar yang kuat untuk mereka terus belajar dan memperbaharui keterampilan mereka seiring dengan perkembangan teknologi dan strategi pembelajaran yang baru. 6. Kesiapan untuk Memimpin Perubahan: Mahasiswa PGSD yang terlatih dalam penerapan teknologi dan pendekatan inovatif dapat menjadi agen perubahan di lingkungan sekolah dasar. Mereka dapat memimpin atau berkontribusi dalam pengadopsian teknologi di sekolah dan menjadi sumber inspirasi bagi rekan-rekan sejawatnya. Melalui pelatihan ini, mahasiswa PGSD tidak hanya memperoleh keterampilan teknologi yang diperlukan, tetapi juga mengembangkan sikap adaptif dan kreatif yang esensial untuk menghadapi tuntutan zaman yang kian digital dan dinamis dalam lingkungan pendidikan sekolah dasar. Dampak ini mempersiapkan mereka secara komprehensif untuk menjadi pendidik yang efektif dan adaptif di masa depan. Gambar 1. Dokumentasi Kegiatan Gambar 1. Dokumentasi Kegiatan Gambar 1. Dokumentasi Kegiatan 24 \| H a l 24 \| H a l DAFTAR PUSTAKA Kusuma, A. I., Sari, W. P., & Hikmawati, A. (2021). Peningkatan Kompetensi Guru dan Tenaga Kependidikan Melalui Pelatihan Teacherpreneur Berbasis Digital Profile Builder. Jmm (Jurnal Masyarakat Mandiri), 25 \| H a l 25 \| H a l Indah, M. (2023). Pengembangan LKPD Interaktif dengan Liveworksheet Berbasis Model Discovery Learning pada Materi Zat Aditif dan Zat Adiktif untuk Meningkatkan Keterampilan Proses Sains (Doctoral dissertation, Universitas Lampung). Volume 4 No 1 – Januari 2024 5(5), 2476-2487. 5(5), 2476-2487. MUHAMMAD IKHLAS, J. A. Y. A. (2023). PENGARUH E-LKPD DALAM LIVEWORKSHEETS BERBASIS PROBLEM BASED LEARNING TERHADAP KEMAMPUAN LITERASI SAINS SISWA KELAS VIII PADA MATERI SISTEM EKSKRESI. Dwiyanti, N. K. E. M., Rati, N. W., & Lestari, L. P. S. (2023). Dampak Model Problem Based Learning Berbantuan Liveworksheet Terhadap Hasil Belajar IPA Siswa Kelas V SD. Jurnal Ilmiah Pendidikan dan Pembelajaran, 7(2). Dwiyanti, N. K. E. M., Rati, N. W., & Lestari, L. P. S. (2023). Dampak Model Problem Based Learning Berbantuan Liveworksheet Terhadap Hasil Belajar IPA Siswa Kelas V SD. Jurnal Ilmiah Pendidikan dan Pembelajaran, 7(2). Syazali, M., Erfan, M., Amrullah, L. W. Z., & Hasanuddin, H. (2023). STRATEGIC ANALYSIS OF TOOLS/MEDIA COMBINATION FOR DISTANCE LEARNING IN SCIENCE COURSES. COMPUTATIONAL INTELLIGENCE. Yustina, Y., Mahadi, I., Daryanes, F., Alimin, E., & Nengsih, B. (2022). The Effect of Problem-based Learning through Blended Learning on Digital Literacy of Eleventh-Grade Students in SMA Negeri Plus Riau on Excretory System Material. Jurnal Pendidikan IPA Indonesia, 11(4). UMMU, K. (2023). PENGEMBANGAN e-LKPD PEMBELAJARAN IPA TIPE CONNECTED BERBASIS MULTI REPRESENTASI UNTUK MENINGKATKAN KEMAMPUAN REPRESENTASI DAN LITERASI SAINS PESERTA DIDIK (Doctoral dissertation, UNIVERSITAS LAMPUNG). Aryanitha, N. P. A. (2022). Pengembangan Lembar Kerja Peserta Didik (Lkpd) Interaktif Berbasis Mind Mapping Pada Muatan IPA Materi Otot Manusia Siswa Kelas V SD Negeri 4 Penatih (Doctoral dissertation, Universitas Pendidikan Ganesha). Aryanitha, N. P. A. (2022). Pengembangan Lembar Kerja Peserta Didik (Lkpd) Interaktif Berbasis Mind Mapping Pada Muatan IPA Materi Otot Manusia Siswa Kelas V SD Negeri 4 Penatih (Doctoral dissertation, Universitas Pendidikan Ganesha). DEVITHA OCTAVIANA, W. I. R. A. W. A. N. (2023). PENGEMBANGAN E-LKPD BERBASIS HOTS MATERI PECAHAN BERBANTU LIVEWORKSHEETS BERORIENTASI PADA PEMAHAMAN KONSEP MATEMATIKA SISWA KELAS V (Doctoral dissertation, Universitas Mataram). Indah, M. (2023). Pengembangan LKPD Interaktif dengan Liveworksheet Berbasis Model Discovery Learning pada Materi Zat Aditif dan Zat Adiktif untuk Meningkatkan Keterampilan Proses Sains (Doctoral dissertation, Universitas Lampung). 26 \| H a l 26 \| H a l
https://openalex.org/W4308745676	https://link.springer.com/content/pdf/10.1007/s11217-022-09855-3.pdf	English	null	Exploring Criticality in Chinese Philosophy: Refuting Generalisations and Supporting Critical Thinking	Studies in philosophy and education/Studies in philosophy & education	2,022	cc-by	11,709	Studies in Philosophy and Education (2023) 42:123–141 https://doi.org/10.1007/s11217-022-09855-3 Studies in Philosophy and Education (2023) 42:123–141 https://doi.org/10.1007/s11217-022-09855-3 * Ian H. Normile ian.normile@ed.ac.uk 1 University of Edinburgh, Edinburgh, UK Exploring Criticality in Chinese Philosophy: Refuting Generalisations and Supporting Critical Thinking Ian H. Normile1 Accepted: 8 September 2022 / Published online: 9 November 2022 © The Author(s) 2022 Keywords Critical thinking · Criticality · Critique · Chinese philosophy · Chinese international students · Higher education Abstract Much of the literature exploring Chinese international student engagement with critical thinking in Western universities draws on reductive essentialisations of ‘Confucianism’ in efforts to explain cross-cultural differences. In this paper I review literature problematising these tendencies. I then shift focus from inferences about how philosophy shapes culture and individual students, toward drawing on philosophy as a ‘living’ resource for under- standing and shaping the ideal of critical thinking. A cross disciplinary approach employs historical overview and philosophical interpretation within and beyond the Confucian tra- dition to exemplify three types of criticality common in Chinese philosophy. These are criticality within tradition, criticality of tradition, and critical integration of traditions. The result is a refutation of claims or inferences (intentional or implicit) that Chinese philoso- phy is not conducive to criticality. While this paper focuses on types of criticality, it also reveals a common method of criticality within Chinese philosophy, in the form of ‘creation through transmission’. This resonates with recent research calling for less confrontational and more dialogical engagement with critical processes. However, I also draw attention to examples of confrontational argumentation within Chinese philosophy, which may provide valuable resources for educators and students. Finally, I conclude careful and explicit con- sideration is needed regarding the types of criticality sought within Western universities to prevent educators and students from ‘speaking past’ one and other instead of ‘speaking with’ one and other in critical dialogue. Keywords Critical thinking · Criticality · Critique · Chinese philosophy · Chinese international students · Higher education Keywords Critical thinking · Criticality · Critique · Chinese philosophy · Chinese international students · Higher education Introduction Scholars reviewing research on Chinese international student engagement with critical thinking in Western1 universities have identified tendencies to draw on reductive essen- tialisations of ‘Confucian heritage’ to explain difficulties engaging with critical thinking (Clark and Gieve, 2006; Heng, 2018; Li, 2017; Moosavi, 2020; O’Dwyer, 2016). This paper reviews and contributes to a growing body of literature problematising such essen- tialism and generalisation. To avoid these problems, I shift focus from inferences about how philosophy manifests in contemporary culture, specific practices, or groups of stu- dents, toward drawing on philosophy—past and present—as a ‘living’ resource for under- standing and actively shaping the normative concept of critical thinking. This theoretical shift is justified by the fact that critical thinking is not a description of how people do think, but an ideal of how people ought to think in certain situations for certain purposes. Furthermore, the norms and ideals shaping dominant conceptions of critical thinking are typically rooted in, and derived from, the Western philosophic traditions (Tan, 2017). How- ever, this is not because ‘the West’ (an equally problematic term of generalisation) has any monopoly on criticality. Consequently, exploring Chinese philosophy may shed new light on an old concept. Light much needed in the increasingly international contexts of Western higher education. I begin by providing an outline of critical thinking and its relationship to traditions. The aim is to draw on commonly agreed features of criticality to facilitate exploration of its manifestations within Chinese philosophy. I then review literature problematising tenden- cies toward cultural generalisation and reductive essentialisation of Chinese philosophy within existing research. Next, I draw on a cross-disciplinary approach, employing his- torical overview and philosophical analysis to show three types of criticality within Chi- nese philosophy. These are criticality within tradition, criticality of tradition, and critical integration of traditions. This is done by exploring criticality within the Confucian tradi- tion through examples from the Analects and the critical evolution of Confucian theory through Mencius and Xunzi. I then briefly consider the influence of Buddhist metaphysics on Neo-Confucianism to exemplify critical integration of traditions within Chinese phi- losophy. Next, I shift attention beyond Confucianism, to show criticality of tradition along with further critical integration of traditions. This is done by looking at Daoist and Mohist philosophy of the ancient Warring States Period and the impact of Western philosophy in China beginning in the nineteenth century. 1 Research consulted is primarily from the UK and North America. 123456789) 1 3 3456789) 1 3 I. H. Normile 124 Criticality and Tradition Critical thinking has a long history of definition and redefinition (Ennis, 2016; Johnson and Hamby, 2015). For the purposes of this paper, I draw on basic but broadly agreed aspects of critical thinking, conceived of as a process of reasoning about what to believe and how to act, the exercising of which requires a combination of knowledge, skills, and disposi- tions (Bailin and Siegel, 2003; Ennis, 2016; Facione, 1990). Such a simplified definition cannot capture the complexity of all criticality. However, this very general approach high- lights a key aspect of critical thinking; the fact that it is necessarily criteriological (ibid). There must be some form of criteria by which reasons for thought and action are justified. The origin and nature of the criteriological framework(s) guiding criticality within and across contexts are the source of much philosophical debate. Some suggest such frame- works can only be achieved through consensus and practice (Rorty, 1991). Others argue the very possibility of commensurability between critical frameworks logically entails trans- cendent meta-criteria capable of guiding universal rationality, and thus criticality (Siegel, 2017). Biesta and Stams draw on Derrida to claim, “…there are no pure, uncontaminated, original criteria on which we can simply and straightforwardly base our judgments” (2001, p. 68). In this view we are always embedded within a context of assumptions, the ultimate boundaries of which cannot be comprehended or transcended, yet criticality can reveal new possibilities without recourse to foundational, self-selected, or transcendent criteria (2001). This is an important debate for criticality, the justification of critique within philosophy, and questions about the nature of rationality. However, it is not a debate I can settle here. In this paper, I am not claiming to provide a ‘new’ conception of critical thinking. Instead, I am pointing out the unsettled complexity of the topic and drawing on examples from Chinese philosophy to provide perspectives typically not considered in the critical thinking literature. What is most relevant for this paper is that the sources of criteria guiding criticality are commonly understood to be contextually dependent on factors such as discipline, practice, and culture (Evers, 2007; McPeck, 2016). What constitutes good reasons, which values guide judgment, and how those values are conceived may vary depending on the purposes toward which critical thinking is directed and the context in which it is practiced. Introduction I conclude by considering the implications of this work for research and practice. The prioritisation of breadth over depth in this approach is deemed necessary because the diversity of Chinese philosophy is not well represented in Anglophone critical think- ing literature (Moosavi, 2020). Furthermore, attention to breadth serves my primary aims of problematising reductive generalisations and refuting claims or inferences (intentional or implicit) that Chinese philosophy is not conducive to criticality. Importantly, in dem- onstrating the critical capacity of Chinese philosophy, I make no effort to ‘explain’ or describe Chinese culture or students, both of which are too diverse and dynamic to gener- alise. This is not to deny the interconnectivity of philosophy, culture, and individuals but simply an analytical separation facilitating focus on one aspect of an interrelated totality. My point is that if Chinese international students struggle with critical thinking, it is not 1 3 Exploring Criticality in Chinese Philosophy: Refuting… 125 the inevitable result of inherently uncritical philosophy. Quite the contrary, Chinese phi- losophy contains great potential as a ‘living’ resource capable of informing the conceptu- alisation and practice of critical thinking. In service of actualising this potential, I provide examples of criticality within Chinese philosophy. Importantly, while the focus here is on Chinese philosophy, this work has relevance for anyone interested in exploring and better understanding criticality within and across traditions more generally. Criticality and Tradition In such an analogy, one of the most fundamental assumptions is that a particular game (a tradition) ought to be played. The most likely underlying aim is to ‘win’ the game. However, other aims, such as enjoyment or display of style may also influence the nature of play. The rules of the game are equivalent to the criteria guiding criticality. Using this analogy, dogmatism is playing the game without questioning the aims or rules. This may be highly uncriti- cal if it involves playing in a proscribed manner or by a dictated strategy. However, it may also include a degree of critical (perhaps calculated is a better word) reasoning about overall aims and what moves to make in service of achieving those aims without questioning the rules of the game. Criticality within tradition, questions the rules of the game. This does not require breaking the rules, as there are means by which the rules of a game can be agreeably changed by the participants. Criticality of tradition suggests playing a new game altogether. This may result in radical transformation akin to a Kuh- nian paradigm shift or serve to strengthen existing assumptions and reaffirm the value of the current game. Critical integration between traditions arises when a new game is encountered, providing opportunities to combine criticality within and of tradition to either internally transform the rules of the existing game, or perhaps create a new game altogether. It is not, however, simply abandoning one game to play another, as that would lack integration. Finally, I must note why I use criticality and critical thinking as interchangeable syno- nyms. A case can be made for separating the two concepts (Davies, 2015). While I am open to the possibility of such a distinction having value in many contexts, I make no such distinction here because I think this separation sells short the broader aims, and spirit, of criticality in higher education. Many theorists and practitioners lament critical think- ing being taught and practiced in Western universities as an instrumentalised processes of proposition evaluation and argumentation (Barnett, 1997; Davies, 2015; Thayer-Bacon, 1998). Criticality and Tradition Con- sequently, to account for contextual variance, the preceding conception of critical think- ing can be expanded to include reflexive examination, and potential transformation, of the aims, assumptions, and criteria guiding critical thinking and action. Barnett and Davies call this a form of ‘metacritique’ (Barnett, 1997; Davies, 2015) and Lipman identifies it as the ‘self-correcting’ aspect of criticality (2012). Critical thinking, then, is not only a pro- cess of reasoning about what to believe and how to act, but also a process that questions the aims, assumptions, and criteria guiding reasoning itself. This conception of critical think- ing recognises the ‘internal’ coherence of reasoning may vary by context. However, it also 1 3 3 3 I. H. Normile 126 requires questioning the aims and assumptions that guide that reasoning. Consequently, the antithesis of criticality is ideology, the unquestioning adherence to any set of aims and assumptions as an immutable guide to reasoning, belief, and action. requires questioning the aims and assumptions that guide that reasoning. Consequently, the antithesis of criticality is ideology, the unquestioning adherence to any set of aims and assumptions as an immutable guide to reasoning, belief, and action. The primary ‘contexts’ under consideration in this paper are various traditions of Chinese philosophy. While the boundaries of traditions are difficult to delineate, they also provide enough pragmatic clarity to meaningfully explore criticality without having to resolve debates on the ‘ultimate’ nature of criticality. Traditions are defined by con- stellations of aims, assumptions, and criteria. For example, ancient Confucian traditions assume the value of learning and ritual in meeting the aims of social harmony. These, and other aims and assumptions, guide reasoning within the tradition. The fact that tra- ditions (philosophical and otherwise) are shaped by pre-existing aims and assumptions does not preclude, but in fact creates, the possibility for criticality. For example, a tradi- tion may encounter what MacIntyre calls an ‘epistemic crisis’ resulting from inadequacy in practical explanation or breakdowns of internal coherence (1990; 1988). This can derive from new experiences and ideas or contact with other traditions, which creates opportunities for criticality within tradition, of tradition, or critical integration between traditions. However, holding any assumptions as unassailable, particularly in the face of epistemic crisis or when encountering alternatives, constitutes uncritical dogmatism.f These different types of criticality and how they articulate with traditions can be understood through analogy with the playing of a game. Problematising the ‘Construct of the Chinese Learner’ Why do Chinese international students struggle to think critically while studying in West- ern universities? This is a problematic question, laden with assumptions (that they do), cultural generalisation (that all Chinese students are somehow similar), and, very often, philosophical reductivism (the nature of that similarity is a homogeneous form of ‘Confu- cianism’). Despite these problems, it is also a question many educators and students find themselves asking, because many students do struggle with critical thinking while studying abroad (Durkin, 2007; Sun et al., 2018; Wu and Hammond, 2011; Wu, 2015). Research also shows challenges with critical thinking within Chinese higher education (Jiang, 2013; Li and Wegerif, 2013; Tan, 2020; Tian and Low, 2011). This leads some scholars to claim ‘Confucian culture’ is not conducive to ‘Western’ style criticality (Atkinson, 1997; McBride et al., 2002). In an example of extreme generalisation, Dong claims, It has been commonly acknowledged that Chinese traditional culture is generally uncritical… Confucianism shaped a tradition that valued respect for parents and the elderly, the collective good, social order, and harmony. This is in contrast with ancient Greek civilization, which valued independent thought, reason, and ability to debate and argue in public (2015, p. 357). It is unclear why respect for elders and pursuit of social harmony (aims shared by many Ancient Greek philosophers) are necessarily uncritical. Furthermore, implicit in this state- ment is the idea that ‘Chinese traditional culture’ is essentially ‘Confucian’. While there is no doubt Confucian philosophy has an immense impact on Chinese culture, such an obser- vation overlooks the diversity and plurality of culture, while also obscuring the complexi- ties of Confucian philosophy as a resource for actively reshaping culture and reconceptual- ising normative concepts like critical thinking. Reductive essentialisation of Confucian philosophy is increasingly seen as problematic. Ryan and Louie contend that treating 2,500 years of ‘Confucianism’ as the same thing is like treating the various manifestations of Christianity as essentially homogenous (2007). After all, it could be (rather reductively) argued that Catholics, Quakers, and the Ku Klux Klan are all ‘Christians’. Furthermore, contemporary politics probably exert more influ- ence on culture, and certainly on the teaching and learning of critical thinking in con- temporary China, than any philosophical tradition (Zhang, 2017). Criticality and Tradition The intention here is not to diminish the centrality of thinking, or value of logic, to critical thinking, but simply to recognise the types of criticality I aim to identify within Chinese philosophy are not focused only on procedures of thought or discursive processes, but on critical changes to the way people reason and judge what to believe and how to act. 1 3 Exploring Criticality in Chinese Philosophy: Refuting… 127 I am concerned with demonstrating criticality that changes the ‘rules’ of the game or pro- poses an entirely new ‘game’ to play. This is the type of criticality many suggest is absent from Chinese philosophical traditions. My aim is to refute any such assumption. I am concerned with demonstrating criticality that changes the ‘rules’ of the game or pro- poses an entirely new ‘game’ to play. This is the type of criticality many suggest is absent from Chinese philosophical traditions. My aim is to refute any such assumption. Problematising the ‘Construct of the Chinese Learner’ Along these lines, any lack of opportunity to cultivate and practice critical thinking in Chinese education as the result of historical or contemporary political circumstances does not necessarily indicate a cultural or philosophical disinclination toward, or lack of ability to engage in, critical thinking (Bali, 2015; Tian and Low, 2011). It is also important to note research showing the challenges of engaging with critical thinking in a foreign language (Floyd, 2011). Lin- guistic barriers should not be misconstrued as conceptual impediments or lack of capacity. Furthermore, research also shows that while many Chinese students initially struggle with critical thinking while studying abroad, they are capable of developing and learning the required skills and dispositions over time (Wu, 2015). Thus, it is problematic to assume the difficulties some Chinese students face while studying abroad are the result of ‘deficit’ 1 3 128 I. H. Normile instead of merely challenges arising from difference (Heng, 2018). Indeed, it is likely most Chinese students do not lack critical thinking, but simply engage in the process differently (Evers, 2007; Mason, 2013; Shaheen, 2016). This leads some to argue that imposing a Western-centric theory of critical thinking in culturally diverse contexts could be construed as a form of ‘intellectual colonialism’ (Indelicato and Prazic, 2019; Moosavi, 2020). Indeed, as Hammersley–Fletcher and Han- ley point out, critical thinking may become ironically uncritical if it finds itself as a mecha- nism for “reproducing the interests of particular groups and constraining thought within the boundaries of Western traditions” (2016, p. 990). However, there is nothing ‘colonial’ about Western universities drawing on Western intellectual traditions and practices. Indeed, many international students may choose to study abroad precisely because they want expo- sure and experience with these traditions and practices. Nevertheless, it is important to rec- ognise the teaching and practice of critical thinking without consideration of its conceptual heritage and underlying assumptions may simply fail to be as efficient or effective in cross- cultural contexts. What is at stake is not necessarily a matter of aims but of effectiveness. However, as I discuss in the conclusion of this paper, questions about the aims of students, educators, and institutions regarding they type of criticality each seeks are important and potentially problematic if inexplicit or unaligned. Existing research on Chinese student engagement with critical thinking gives very lit- tle attention to how criticality manifests in Chinese philosophy. Problematising the ‘Construct of the Chinese Learner’ This is surprising given the degree to which Western philosophy is mined for resources (e.g., epistemological the- ory, logic, dialectic argumentation) for conceptualising and practicing critical thinking. If critical thinking is more than a ‘Western’ construct, it would seem relevant to explore the phenomenon in other traditions. Some scholars have been making headway in this area. For example, Tan articulates a ‘Confucian conception of criticality’ as a form of action oriented judgment (2017). Lam works to reconcile Confucianism and a generally Western- derived conception of critical rationalism (2017). Sigurosson, acknowledges Confucianism is rarely seen to promote criticality and typically regarded as conservative, reactionary, and ideological (2017). In aiming to rectify this, he argues that the “transformative self- critical attitude” of ancient Confucian philosophy exemplifies a form of deep criticality often neglected within Western conceptions (2017, p. 133). However, such work seems to be the exception, not the norm. Furthermore, while these more nuanced examinations of Confucian conceptions of criticality are valuable, it is less common to see considera- tion of Chinese philosophy beyond Confucianism within the Anglophone critical thinking literature. This paper builds on the work of those aiming to understand the value of Con- fucianism for critical thinking, while also expanding the project to consider other aspects of Chinese philosophy for the same purposes. The goal here is not to ‘explain’ culture or individual student behaviour, but to provide historical and philosophical resources for bet- ter understanding the concept and practice of critical thinking, which is not a description of something we are bound to by tradition, but an ideal of how we ought to think and act within and across traditions. Confucian Criticality This section explores manifestations of criticality within the Confucian tradition. The natu- ral starting place is with Confucius (c. 551–479 BCE), whose project is undoubtedly con- servative. Confucius aims to rectify an idealised past, not to create a new future. Along 1 3 Exploring Criticality in Chinese Philosophy: Refuting… 129 these lines he proclaims, “I transmit but do not innovate; I am truthful in what I say and devoted to antiquity” (Analects, 7:1). However, transmission of the past into the present is an invariably interpretive, creative, and critical endeavour. Fung calls this a process of, “creation through transmission” (1937, p. 48), which begins to shed light on the sub- tle nature of criticality in the Confucian tradition. Here, it is important to note that many ancient Chinese texts derive from oral traditions and appear in multiple versions before reaching their current common forms (Fung, 1937). Furthermore, I am drawing on these texts in English translation, which introduces another layer of interpretation. The herme- neutic process of transmission, translation, and interpretation itself constitutes an impor- tant form of ‘creation through transmission’ in Chinese traditions (Ames and Hall, 2003). However, the creative and critical elements of Confucius’ transmission of antiquity reach beyond mere translation and interpretation. Fung claims, “Although Confucius was con- servative as regards political change, he was in other respects revolutionary” (1937, p. 314). The primary thrust of this ‘revolution’ is the establishment of a scholarly class inde- pendent of ruling elites (Chan, 1963). The aspiration of a Confucian scholar is to become a junzi (gentleman).2 The use of the term junzi embodies elements of radicality, as the word, literally meaning ‘son of a royal’, is stripped of its hereditary and elite connotations and opened for the aspirations of common people (Fung, 1937). The scholarly class originating with Confucius opens the door for social status and public service based on merit instead of birth, no small feat for a society of his (or any) time. Confucius contends that achieving individual and social harmony requires following the dao (way) of the ancient Zhou Dynasty through the proper practice of li (ritual) and diligence in xue (study) to develop de (virtue), culminating in ren (benevolence/humanity) which facilitates action in accordance with yi (rightness) to become a junzi (gentleman). This requires a bit of unpacking. 2 Confucius’ philosophical and educational concerns focus on adult men. Removing the misogyny and age- ism of the time, it is evident the philosophy is applicable to all people. Confucian Criticality … once a practice has been mastered, in the sense that the requisite virtues have been fully developed, this mastery brings with it a certain independence from the rules that constitute the practice: the master is able to reflect upon the rules and may even choose to transgress or revise them… Practice mastery thus brings with it a type of transcendence: freedom to evaluate, criticize and seek to reform practice tradition itself (2001, p. 102). The practices under discussion include skilled activities like music, archery, and chari- oteering, along with the moral practices necessary to navigate the social world. Thus, the ‘rules’ of the Confucian tradition are not provided by fixed knowledge or rituals, but by self-cultivation of an adaptive moral intelligence, guided by ren, capable of changing learn- ing and ritual. This is a debatable interpretation. Some agree with the intentions but argue such an approach is psychologically infeasible (Slote, 2016). Others interpret Confucius as aiming to identify universal principles to guide ethical duty with a more deontological tilt (Roetz, 1993). Despite these interpretive debates, I argue the Confucian focus on reflex- ive and adaptive moral intelligence is substantiated by the evolution of Confucianism after Confucius, particularly through the work of Mencius (372–289 BCE), who claims “a great man will not observe a rite that is contrary to the spirit of the rites, nor will he perform a duty that goes against the spirit of dutifulness” (Mencius, 4B:6). Understanding this ‘spirit’ requires critical reflection on the practices intended to cultivate virtues, and the virtues themselves, which must guide adjustment of those practices. As others have argued, this is a necessarily critical process of reflexive self-cultivation (Sigurosson, 2017) and moral judgment (Tan, 2017). This reflexive process is essential to understanding, and facilitating, criticality within the Confucian tradition, as it creates the possibility for changes not only in practice, but for reinterpretation of how reasoning proceeds (rules of the game) from fundamental assumptions. p For example, Mencius employs ‘creation through transmission’ to reframe reasoning regarding obedience to social hierarchy. Some degree of allowance for disagreement and critique within hierarchy is necessary, as the idealised Zhou Dynasty from which Confu- cius draws his inspiration usurped power from a reigning emperor. This is a topic Confu- cius skirts around, but Mencius addresses at far more length, with powerful implications. Confucian Criticality Confucius conceives of dao as the ‘way’ of ideal human action, which is necessarily relational and ethical (Zhang, 1989). Acting in accordance with dao manifests as de (virtue). There are many ideal virtues, but the most important is ren, which can be seen as the virtue both containing and coordinating all others (Ames and Rosemont, 2011). Ren helps a person to discern yi (rightness), which is not fixed by rule but must be understood contextually (Slingerland, 2001). A Junzi, then, is a benevolent person that does the right thing in any given situation. The two key tools in becoming a junzi are study and ritual propriety. Confucius’ attention to xue (study) and li (ritual) can seem rather uncritical if taken as literal prescriptions of fixed rules. However, they are better understood as tools of (a type) of liberation than fixed rules guiding thought and action (Slingerland, 2001). This interpretation is not immediately evident as the Analects prescribes precise rituals and spe- cific studies. However, Confucius is clearly concerned with active thinking, not passive knowledge acquisition. He refuses to teach anyone who, after being shown “one corner of a square”, cannot come back with the “other three corners” through their own reason- ing (Analects, 7:8). Furthermore, while Confucius is exacting in the conduct of rituals, there are also examples of critical alterations (e.g., Analects 3:15, 16:22, 9:3). Ultimately, there is no intention for study of tradition to comprise the totality of all knowledge, nor for rituals to prescribe ‘correct’ action for every person in every situation (Ivanhoe, 2000). Instead, study and ritual are tools for cultivating the virtues that sustain an adaptive moral intelligence. Slingerland notes, 1 3 3 I. H. Normile 130 … once a practice has been mastered, in the sense that the requisite virtues have been fully developed, this mastery brings with it a certain independence from the rules that constitute the practice: the master is able to reflect upon the rules and may even choose to transgress or revise them… Practice mastery thus brings with it a type of transcendence: freedom to evaluate, criticize and seek to reform practice tradition itself (2001, p. 102). 1 3 Confucian Criticality In speaking with a local king Mencius asks, “If the Marshal of the Guards was unable to keep his guards in order, then what should be done about it?” The King naturally replies that he should be removed from office. Mencius then asks, “If the whole realm within the four bor- ders was ill-governed, then what should be done about it?” At this, the king “turned to his attendants and changed the subject” (Mencius, 1A:7). In the next passage, perhaps getting a bit worried, the King asks, “Is regicide permissible?” Mencius replies, “He who muti- lates benevolence is a mutilator; he who cripples rightness is a crippler; and a man who is both a mutilator and a crippler is an ‘outcast’. I have indeed heard of the punishment of the ‘outcast [of the slain king Tchou]’, but I have not heard of any regicide” (Mencius, A:8). Mencius is making the point that a ruler failing to rule with ren is not a legitimate ruler, thus their removal from power is in accordance with yi and does not amount to a disrup- tion of the socio-political order. This bit of ‘creation through transmission’ demonstrates a critical change in the criteriological framework guiding reasoning (rules of the game) with meaningful implications for thought and action. Mencius’ extends this critical development further while discussing the legitimacy of rulers and succession by arguing that while emperors must receive the ‘mandate of heaven’ (a traditional source of approval beyond human control), the disapproval of the people con- stitutes a sign that no such mandate has been given (Mencius, 5A:5). In a subtle yet highly 1 3 Exploring Criticality in Chinese Philosophy: Refuting… 131 critical move, Mencius shifts the very foundations for political legitimacy. The plight of a ruler is not merely a matter of birth or heavenly fate, but of human action (Chan, 1963). The same sentiment is expressed more explicitly when Mencius says, “the people are of supreme importance; the altars to the gods of earth and grain come next; last comes the ruler” (Mencius, 7B:14). These ideas diverge from the Analects, providing an example of ‘creation through transmission’ leading to criticality within the Confucian tradition. Funda- mental assumptions regarding the necessity of an emperor and a hierarchical social order remain intact, but the reasoning drawn from these assumptions is transformed with mean- ingful implications for thought and action. Confucian Criticality The ‘game’ remains the same, but some of the ‘rules’ have been changed. The subtle nature of this approach aims, and typically succeeds, at maintaining stability while still facilitating criticality. An example of more confrontational and explicit argumentation within the Confucian tradition is available through consideration of the most famous and perhaps more lasting of Mencius’ critical innovations, his conception of renxing (human nature) derived from the emotional experiences of familial relations and extension of empathy to provide a moral psychology capable of orienting and animating the pursuit of ren. Put more simply, Men- cius solidifies the Confucian assumption, still dominant today, that human nature is good, or at least contains the ‘sprouts’ of potentiality for goodness (Fung, 1937). Instead of elab- orating Mencius’ argument, made through a combination of thought experiment, anecdote, and analogy, it is more relevant to the purpose of this paper, aimed at shedding light on manifestations of criticality, to look at one of Mencius’ primary rivals, Xunzi (c. 310–235 BCE) who sharply critiques Mencius’ view on human nature. Chan claims Xunzi is “the most critical of ancient Chinese philosophers” (1963, p. 124). Graham sees Xunzi’s work as marking an important step forward in systematic and critical philosophy, remarking that: [Xunzi’s] attack on the Mencian theory of human nature illustrates the progress of argumentation in the Confucian school. Mencius’ case has to be re-assembled from scattered dialogues and discourses; [Xunzi] develops his in a consecutive essay… with Mencius as the named target, and a terminology clarified… by scrupulous defi- nitions (2003, p. 244). Xunzi’s critique of Mencius’ view of human nature is pointed. Book 23 of the Xunzi is titled “Human Nature is Bad” and sets out to make a reasoned argument for why this must be the case (Xunzi). One such argument is that if human nature were indeed good, and each individual capable of looking inward to cultivate ren, “then what use would there be for sage kings? What use for ritual and yi?” (Xunzi, 23:160). Ritual is necessary only because human nature must be shaped through conscious rational effort. In contrast to Mencius, Xunzi uses metaphors of craftmanship regarding the need to mould (or ‘mutilate’ as Men- cius would say) human nature as a craftsman bends wood or tempers metal. Xunzi values rationality over sentimentality and sees the need for mind and will to check the dangers of emotion and desire (Fung, 1937). Confucian Criticality This demonstrates an example of confrontational argu- mentative criticality within the Confucian tradition. Thus far, the focus has been on criticality within the Confucian tradition of the classi- cal era. Moving beyond this period sheds light on critical integration of traditions. After a brief but severe stint of repression under the Qin Dynasty (221–206 BCE), Confucianism is codified and institutionalised during the Han Dynasty (202 BCE-220 CE) with the schol- arly class of Confucians becoming an integral aspect of the aristocracy and government (Fung, 1953). The thrust of Confucianism shifts from a critical philosophy exploring how best to live, toward a scholarly bureaucracy of status, politics, and power (Bol, 2008). This 1 3 3 3 I. H. Normile 132 coincides with the arrival of Buddhism from India, beginning in the fourth century CE. There is immense diversity within early Chinese Buddhist thought and the tradition trans- forms significantly over time, with Chan Buddhism (which becomes Zen Buddhism upon migrating to Japan) eventually becoming the most dominant (Chan, 1963; Fung, 1953). The fundamental Buddhist aims of transcending the illusory nature of the phenomenologi- cal world based on the assumed existence of a ‘truer’ underlying reality challenge prevail- ing Confucian assumptions about the nature of the world, its relation to the mind, and the appropriate grounding of knowledge and morality (Chan, 1963). This requires Confucian- ism to face, and attempt to answer, new questions in new ways. Consequently, beginning in the Tang (619–907) and culminating in the Song (960–1279) and Ming (1368–1644) a diverse range of thinkers with varying and often incompatible perspectives address the more ‘metaphysical’ questions raised through contact with Buddhism, marking the origins of what later becomes broadly categorised (despite great internal diversity of ideas) as Neo- Confucianism (Angle and Tiwald, 2017). This is an excellent example of criticality within the Confucian tradition through critical integration of concepts from another tradition. In this process new concepts are integrated by reinterpreting existing ideas. One such old concept made new is that of li, typically translated as ‘Pattern’. This term appears very sparsely in the Confucian literature of the ancient period (Neo-Confucian li 理 is not to be confused with li 禮 as rite/ritual in ancient texts), and rarely war- rants detailed commentary (Zhang, 1989). Confucian Criticality However, by the Song and Ming period, li (along with a constellation of other revitalised concepts and terms) is central to Confu- cian debate (Angle and Tiwald, 2017). Chan says of li, “all things exist because of it and can be understood through it. It is universal truth, universal order, universal law” (1963, p. 519). This conveys the ambitious aims of articulating a theory of li, which are nothing short of understanding the Pattern giving rise to and underlying every aspect of reality. It also indicates a substantial shift from intensive focus on ancient rituals, normative ethics, and political bureaucracy toward a new set of concerns and concepts conducive to empirical investigation of the natural world and new theories of how the mind relates to reality (Chan, 1963). This shift is the result of critical integra- tion of questions and ideas encountered in a foreign tradition. Thus far, it is evident Confucianism contains the capacity to question and ‘rein- terpret’ fundamental assumptions in ways that change the ‘rules’ guiding reasoning and judgment. However, it remains difficult to gain a perspective from within the Confucian tradition capable of facilitating criticality of that tradition. Consequently, to understand criticality of tradition within Chinese philosophy it is helpful to look beyond Confucianism. Warring States Period (475 – 221 BCE) The Han historian Sima Qian recognises six philosophical ‘schools’ as having strong influence through the Warring States Period (Fung, 1937). Along with Confucianism, these include the Yangists, perhaps best understood as anarchist individualistic natural- ists advocating the renunciation of society and a return to simple, self-sufficient liv- ing (Graham, 2003). Simultaneously, the Logicians explore logical and metaphysical questions similar to those central in the Greek tradition (Chan, 1963). The Legalists integrate aspects of Xunzi’s Confucianism, with the argumentation of the Logicians to forge a highly pragmatic (and temporarily influential) political philosophy. The Mohists, espouse a kind of utilitarianism sharply attacking Confucian tradition at the most fun- damental level, arguing against filial piety, and for elimination of unnecessary rites and rituals (Johnston, 2013). Mohism is of particular interest because it is equal to, if not dominant over, Confucianism until the Han Dynasty (Chan, 1963). It also offers one of the strongest examples of criticality of tradition. However, the starting point for under- standing Chinese philosophy beyond Confucianism is with Daoism. ‘Daoism’ is not a school in any formal sense during the Warring States Period. None- theless, it exerts a powerful influence on philosophies of the time, and into the present (Zhang, 1998). The two most influential thinkers in early Daoist philosophy are Laozi, believed to have lived roughly contemporary to the time of Confucius in the sixth cen- tury BCE, and Zhuangzi, likely to have lived in the fourth century BCE (Chan, 1963). Laozi is traditionally credited with writing the Daodejing. However, like Confucius’ relationship to the Analects, it is unlikely he ever wrote anything (Fung, 1937). Instead, the text associated with his thought is compiled by subsequent followers in a variety of versions. The text bearing Zhuangzi’s name is also compiled, most likely over centuries, before reaching its current form. However, the first seven ‘Inner Chapters’ are generally agreed to have been written by one person (Graham, 2003).i g y p The first lines of the Daodejing state, “dao that can be put into words is not really dao, and naming that can assign fixed reference to things is not really naming” (p. 77). This oft-cited passage can be translated and interpreted in several ways. Beyond Confucianism This section explores philosophies directing criticality at the fundamental aims and assumptions of the Confucian tradition. This is done by looking at two chronologically disparate, but circumstantially similar periods of history. The first is that of the War- ring States Period (475–221 BCE), also known as the time of ‘100 schools of thought’, in which rival philosophies take direct aim at each other in confrontational critical debate. A similar such period occurs when the Qing Dynasty falls in 1911, begin- ning the Republican Era, during which China finds itself in a life-or-death struggle for 1 3 Exploring Criticality in Chinese Philosophy: Refuting… 133 modernisation, leading to eclectic and integrative philosophical thinking drawing on all types of criticality. Consequently, these are two exemplary periods for understand- ing criticality of tradition within Chinese philosophy. modernisation, leading to eclectic and integrative philosophical thinking drawing on all types of criticality. Consequently, these are two exemplary periods for understand- ing criticality of tradition within Chinese philosophy. Warring States Period (475 – 221 BCE) This is freedom from the prescriptions and fixed perspectives that divide and dim the power of unmediated experience. Zhuangzi is explicitly critical of Confucians and Mohists, the two most prominent philosophies of his time, stating, “what one calls right the other calls wrong; what one calls wrong the other calls right. But if we want to right their wrongs and wrong their rights, then the best thing to use is clarity (Zhuangzi, p. 39).” The ‘clarity’ Zhuangzi advocates comes from unburdening the mind from the preconceptions and habituations imposed by traditions. Only once freed from the preconceptions and constructs (includ- ing language) dividing the genuine flow of experience can one develop de and naturally (spontaneously) react, act, and interact with dao. Zhuangzi’s intention is to cut beyond simple recognition of differing opinions and point out the ‘groundlessness’ of any fixed opinion or perspective. He is not suggesting changes to the ‘rules’ of an existing game but suggesting the need for a ‘new game’ altogether. In a frequently quoted and vari- ously interpreted passage, Zhuangzi proclaims, Everything has its ‘that’, everything has its ‘this’. From the point of view of ‘that’ you cannot see it, ‘that’ comes out of ‘this’ and ‘this’ depends on ‘that’ – which is to say that ‘this’ and ‘that’ give birth to each other. But where there is birth there must be death; where there is death there must be birth (Zhuangzi, p. 39). Ziporyn argues this is Zhuangzi’s way of recognising the necessarily perspectival nature of indexical knowledge. That is, whether something is a ‘this’ or ‘that’ depends on the location of the perspective of reference (Ziporyn, 2009). While I am holding something, it is ‘this’, when I set that thing down and walk away, it becomes ‘that’. While there is obvious application of indexical perspectivism regarding perception and interaction with the physical world, Zhuangzi’s deeper point is moral (Slinger- land, 2003; 2014). Whatever ‘this’ a Confucian holds as ‘good’ can only be so in ref- erence to a ‘that’ which is ‘bad’. This is Zhuangzi’s way of recognising that not only are divergent perspectives relative, but any apparent contradiction or opposition is only superficial, because opposites form a necessary and indivisible unity. There can be no ‘this’ perspective without a ‘that’ perspective. Warring States Period (475 – 221 BCE) I follow Ames and Hall in taking this to indicate that as a process of constant transformation, dao is neither fixed nor constant, thus no fixed or constant practices (or language) can consist- ently align with or describe the true Way (2003). Consequently, knowledge and rituals drawn from the past are not the best means for gaining knowledge in the present. For example, chapter 38 of the Daodejing calls ritual propriety “the thinnest veneer of doing one’s best and making good on one’s word,” while referring to the Confucian claims of knowledge as “tinsel decorating the Way” (Daodejing, p. 136). While Confucianism and Daoism share concepts like dao and de, they are conceived of in meaningfully differ- ent ways. For example, the dao of Laozi is more naturalistic and cosmological, running contrary to what Tan calls, the ‘humanist’ Confucian conception of dao as ideal norma- tive behaviour (2017). This can be seen as a form of ‘creation through transmission’ of shared concepts predating both Confucian and Daoist thought. However, Laozi also cri- tiques Confucian methods for attaining dao, shifting focus from ritual and study toward intuition and experience. I argue these divergences constitute criticality of tradition. 1 1 3 3 3 134 I. H. Normile Daoist criticality is perhaps best exemplified in the Zhuangzi, which is a unique text for its time, and remains unique to this day. It is poetic and lyrical, filled with fantasti- cal stories of talking animals, magic, and mystical transformations. Confucius and his disciples appear as frequent characters (indicating Zhuangzi is well read in the clas- sics), often to espouse views contradictory to their own philosophies. The Zhuangzi is also filled with uncertainty and contradiction, leading many to speculate an intentional avoidance of precise articulation meant to facilitate interpretation as opposed to provid- ing explication (Kupperman, 1996). Watson suggests, if there is a central theme to the text, it is “freedom” (1968, p. 3). But exactly what kind of freedom is Zhuangzi striving for? It appears to be different from Laozi’s more explicit call toward withdrawal from the world (Slingerland, 2003; 2014). Furthermore, unlike later Buddhism, Zhuangzi does not see the material world as illusory or something that must (or even ought) be transcended (Chan, 1963). Instead, it is the human world of constructed meanings and arbitrary divisions that Zhuangzi aims to free people from (if he aims at anything at all). Warring States Period (475 – 221 BCE) Furthermore, drawing on observations from the natural world, in which opposites (e.g., night/day, summer/winter, birth/death) do not simply define each other in conceptual stasis, but through processual transforma- tion, any effort to maintain a fixed perspective in an always changing world is to strug- gle against dao. It is common to interpret the Zhuangzi as a collection of various types and degrees of skepticism and/or relativism (Kjellberg and Ivanhoe, 1996). Given that Exploring Criticality in Chinese Philosophy: Refuting… 135 criticality is necessarily a form of skepticism (to at least some degree) and the impor- tance of contextualised perspective within many contemporary philosophies and prac- tices, Zhuangzi should be recognised as one of the most critical and curious contribu- tors to Chinese philosophy.l criticality is necessarily a form of skepticism (to at least some degree) and the impor- tance of contextualised perspective within many contemporary philosophies and prac- tices, Zhuangzi should be recognised as one of the most critical and curious contribu- tors to Chinese philosophy.l p p y I shift now to the one of the most prominent and influential philosophies of the ancient period, that of Mohism. While Mozi (479–381 BCE) is the founding figure of Mohism, the school of thought develops continuously into the Han period, with substantial shifts from the earlier to later incarnations (Chan, 1963; Graham, 2003). Mohism applies a more logi- cal (and methodological) approach to articulating a systematic philosophy in distinct con- tradiction to Confucian and Daoist norms (Fung, 1937). This includes explicit critique of fundamental assumptions and aims underlying Confucian philosophy. For example, Chap- ter 39 of the Mozi, entitled ‘Against the Confucians’ explicitly attacks the idea of “follow- ing but not creating” by recognising that someone had to create the rituals Confucians so revere, thus showing the very sources of their reverence is for people that ‘created but did not follow’ (Mozi). Here, the Mohist seems to identify a kind of logical infinite regress in the normative foundations of Confucian theory. If current practices are based on the past, what were past practices based upon? This leads Mohists toward recognition of the need for a universal grounding for yi, con- ceived of as justice (Johnston, 2013). The central concept in this effort is the utilitarian idea of jian ai, often translated as ‘universal love’, though perhaps better understood as ‘equal concern for each person’ (Graham, 2003). Warring States Period (475 – 221 BCE) The implications of this cosmopolitan consequentialism are profound. Mohists rail against the most fundamental assumptions of Confucianism, namely the priority of filial piety as a source for cultivating positive moral psychology and the importance of rites and rituals in cultivating virtues capable of guiding action in accordance with yi (Mozi). They see the privileged and special bonds of fam- ily as problematically impeding establishment of jian ai, the excess of rituals as unnec- essarily wasteful, and the perspectivism of Daoism as unacceptably anarchic and morally relativistic (Chan, 1963). For the Mohist, society requires unity of purpose and values as the foundation for calculating and justly distributing benefit (Johnston, 2013). This is clear evidence of strong criticality of tradition. Importantly, Mohist critiques are addressed in the Mencius (e.g. 3B:9) and, at greater length throughout the Xunzi (e.g. Books 6 & 10), show- ing active and explicit critical debate within and between these traditions. Republican Era to the Present We jump forward now to the waning years of the Qing Dynasty (1636–1911). As part of an effort to overcome the challenges of internal division and external threats, the education system is overhauled, including elimination of the Imperial civil service exams in 1905, marking the end of a practice over 1000 years old (Bol, 2008). Six years later, the Qing dynasty falls, and the Republic of China is formed, marking the end of dynastic rule. Chan claims, “not since the third century B.C. have there been ‘one hundred schools’ of thought contending in China as in the twentieth century. The combination of Western thought and revolt against traditional heritage caused many intellectual currents to run in all directions” (1963, p. 743). The Republican Era brings about radically divergent perspectives, but all are grounded in, and aim to resolve, the same problem: how to modernise China (Cua, 2003). This is a time rife with criticality of all forms. In keeping with the current project, I only highlight a few prominent thinkers or ideas, which exemplify the various ways in which criticality manifests during this period in Chinese philosophical history. 3 I. H. Normile 136 At this juncture, thinking critically about fundamental philosophical aims and assump- tions becomes explicit. Some advocate total westernisation, others aim to integrate Chinese and Western ideas, while some argue for rectification and globalisation of Chinese traditions (Fung, 2010). Amidst these debates, Liang Qichao uses the term lixiang to represent “… the things that everybody imagines and are commonly taken as the most reasonable principles… inherited social customs of thousands of years (in Xiao, 2002, p. 19). He goes on to argue for transforming, and perhaps discarding, aspects of lixiang as essential to the survival of Chi- nese civilisation. This demonstrates the degree to which criticality of the Republican Era is marked by intentional effort to restore, reshape, or sometimes discard the fundamental aims and assumptions of Chinese philosophy. In this effort, many philosophers see the way forward as requiring critical integration of Chinese and Western philosophical traditions. For example, Zhang Dongsun draws heavily on Kant to articulate a theory of culturally contextual episte- mological pluralism intended to reconcile the seemingly incommensurate differences between Chinese and Western traditions (Jiang, 2002). Hu Shih, after studying at Columbia University with Dewey, is central to The New Culture Movement (Xinhe, 2002). Republican Era to the Present It is difficult to find more explicit criticality of tradition than a movement founded on the aim of forging a ‘new culture’. Hu draws on Nietzsche’s notion of ‘transvaluation of all values’ to espouse the need for a ‘critical attitude’ toward institutions and traditions (Hu, 2013). He aims to put Confu- cian and non-Confucian philosophies on equal footing to be analysed through the lens of con- temporary thought, claiming, “the future of Chinese philosophy would seem to depend much on the revival of those great philosophical schools that once flourished side by side with the school of Confucius in Ancient China” (Hu in Xinhe, 2002, p. 92). In this process he warns against imported ‘isms’ as necessarily ideological and dogmatic, thus not contributing to criti- cal philosophy or meeting the needs of the contemporary Chinese context (Xinhe, 2002). Ultimately, however, it is an -ism, that of Marxism-Leninism, that prevails in reuniting the Middle Kingdom into the People’s Republic of China in 1949. The critically integrated philos- ophies guiding the Communist revolution eventually give way to uncritical dogmatism in the form of Maoism, which reaches its heights during the Cultural Revolution and essentially par- alyzes philosophical development within Mainland China for several decades (Chan, 1963). Despite temporary devolution into uncritical dogmatism, there may be no better example of criticality than the creative integration of a foreign philosophical theory as the foundation for a new nation to preserve a culture facing the very real prospect of destruction. The deft manoeu- vrings of Deng Xiaoping navigating the transition from Maoism to the ‘opening’ of China, is perhaps one of the best modern examples of highly practical criticality within and between multiple traditions simultaneously (Vogel, 2011). Finally, it is important to recognise that Chi- nese philosophy is a dynamic and continually developing field, with an invariably comparative and/or integrative aspect (Cheng, 2002). For example, Mou Zongsan integrates Kant and Hei- degger with Buddhist and Daoist philosophy to ‘reconstruct’ Confucianism, while also mak- ing meaningful contributions to ‘Western’ theory (Lee, 2021). This might better be consid- ered ‘world’ philosophy (Mou, 2009). The Western imperialist legacy creates an imbalance of power that requires China to engage with Western philosophy in a way the West has not been forced to reciprocate. Implications for Practice This broad overview cannot do justice to the depth of specific concepts, theories, and debates within Chinese philosophy. However, it shows criticality within traditions, of traditions, and critical integration of different traditions. This should caution research- ers, practitioners, and students against making inferences about culture or students based on reductive essentialisations of ‘Confucianism’, which may fail to consider the complexity of actual Confucian philosophy. Importantly, this approach does not pre- clude the fact that some aspects of Confucian philosophy, particularly those ossified into socio-cultural traditions, may discourage development of critical capacities and the practice of criticality. My point is that this does not indict the entirety of Confu- cian philosophy as necessarily incompatible with critical thinking, nor does it obviate the potential of that philosophy for deeply meaningful criticality. Furthermore, Con- fucianism is not the only resource within Chinese philosophy with relevance to criti- cal thinking. Consequently, the research and practice of critical thinking may benefit from more nuanced consideration of Chinese philosophy. In the realm of practice, this could include attention to ongoing professional development for staff teaching critical thinking, whether embedded within a subject or more explicitly, particularly in contexts with large numbers of Chinese international students. Similarly, this paper may inspire reconsideration of reading and course content aimed at exploring or demonstrating criti- cality. Exploration of philosophies less prominent or dominant in contemporary theory and practice may help realise the critical potential of Chinese philosophy. For example, Xunzi, Mozi, and Zhuangzi may not exert as ‘measurable’ of an influence on contempo- rary culture and education, but their philosophies could contribute to understanding and potentially reimagining criticality. It could be instructive to investigate Xunzi’s forms of argumentation, the implications of jian ai for critical thinking, or explore the inter- play between embodied cognition, skeptical perspectivism, and criticality in the dao of Zhuangzi. I hope this paper inspires further inquiry into the broader critical resources within Chinese philosophy and their applications for theory and practice. In this paper I have also drawn attention to ‘creation through transmission’ as a method of criticality. This process does not contain the explicit argumentative form typical of much Western philosophy, and common in the teaching and practice of critical thinking. Recog- nition of this method of criticality corroborates conclusions drawn from philosophically informed empirical work exploring Chinese student engagement with critical thinking. Republican Era to the Present Many Western scholars have taken great interest in Chinese philosophy, but not with a sense of cultural survival on the line, as was the case in 19th and early twentieth century China. This is an imbalance contemporary philosophers should be working to elimi- nate (Rošker, 2020). Thankfully, the increasing internationalisation of education provides just such an opportunity. 1 3 1 3 Exploring Criticality in Chinese Philosophy: Refuting… 137 Implications for Practice For example, Tan argues, “… the image of a critical thinker as an independent and truth-driven champion of propositional knowledge, syllogism and adversarial debates is foreign to the Confucian traditions” (2017, p. 337). This leads her to suggest dialogical instead of dialec- tic critical engagement. My analysis substantiates this suggestion. However, I argue that this important observation fails to provide a complete picture. There are ample instances of explicit argumentative criticality within the history of Chinese philosophy. For example, Xunzi’s criticality within the Confucian tradition and Mohist criticality of the Confucian tradition, along with Zhuangzi’s criticality of Confucianism, Mohism, and skeptical ques- tioning of the foundations of morality. Furthermore, the past two centuries of Chinese phi- losophy have engaged in adversarial debate at a (literally) revolutionary scale, transforming both society and philosophy multiple times. What can be drawn from this is both a need to recognise the nuances of ‘creation through transmission’ as a dialogical form of critical- ity and the existence of resources within Chinese philosophy exemplifying explicit critical debate and action. 1 3 I. H. Normile 138 Finally, attention to the fundamental types of criticality identified in this paper may help educators and students better understand what is being asked within a particular con- text. If a tutor thinks they are engaging in criticality within tradition without recognising the requirement on students to grapple with criticality of their traditions, there is a risk of ‘speaking past’ each other instead of ‘speaking with’ each other. The same is true of students working diligently to filter learning into familiar traditions without confronting the possibility – perhaps the necessity – of questioning fundamental assumptions through criticality of those traditions. While this paper focuses on traditions of Chinese philosophy, understanding how aims and assumptions guide reasoning to form the boundaries of tra- ditions more generally (e.g., social, political, academic, professional) reveals the broader applicability of the types of criticality discussed here. Of course, not everyone can be an expert on every tradition. Thus, the onus is on all participants to explore the limits of their own assumptions in service of helping others do the same. And because it can be difficult to gain a perspective from within a tradition to facilitate criticality of that tradition, diverse educational contexts offer opportunities for engagement with perspectives conducive to expanding critical possibilities (MacAllister, 2016). Implications for Practice Such a process, however, requires explicit attention to differences between traditions to create meaningful critical dialogue. f This means universities must clarify the types of criticality they aim to teach and prac- tice. Do universities aim for criticality within tradition, of tradition, and/or to facilitate criti- cal integration between traditions? Given the increasingly diverse demographic of Western universities, it seems impossible to avoid criticality of traditions without severely limiting the scope of critical thinking. So, should Western universities engage with types of critical- ity not taught (or allowed) in Chinese higher education? After all, this may be one reason many Chinese international students choose to study abroad, and if not, it may be an oppor- tunity worth contemplation. Conversely, perhaps universities should bend toward critical aims better aligned with the backgrounds and likely future contexts of application for Chi- nese (and other) international students? The type of critical thinking drawn on in this paper would require all students from every background to seize the opportunity provided by diversity of traditions and perspectives to identify and critically reflect on their own aims and assumptions in any context that calls itself ‘critical’. 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https://openalex.org/W2586197949	https://www.frontiersin.org/articles/10.3389/fnbeh.2017.00020/pdf	English	null	Frontal Functional Connectivity of Electrocorticographic Delta and Theta Rhythms during Action Execution Versus Action Observation in Humans	Frontiers in behavioral neuroscience	2,017	cc-by	10,881	Citation: Keywords: subdural electrocorticography (ECoG), movement observation, movement execution, lagged linear connectivity, exact low-resolution brain electromagnetic tomography (eLORETA), frontal cortex, mirror neuron system Babiloni C, Del Percio C, Lopez S, Di Gennaro G, Quarato PP, Pavone L, Morace R, Soricelli A, Noce G, Esposito V, Gallese V and Mirabella G (2017) Frontal Functional Connectivity of Electrocorticographic Delta and Theta Rhythms during Action Execution Versus Action Observation in Humans. Front. Behav. Neurosci. 11:20. doi: 10.3389/fnbeh.2017.00020 Frontal Functional Connectivity of Electrocorticographic Delta and Theta Rhythms during Action Execution Versus Action Observation in Humans Claudio Babiloni 1,2†, Claudio Del Percio 3†, Susanna Lopez 1, Giancarlo Di Gennaro 4, Pier P. Quarato 4, Luigi Pavone 4, Roberta Morace 4, Andrea Soricelli 3,5, Giuseppe Noce 3, Vincenzo Esposito 1,4, Vittorio Gallese 6 and Giovanni Mirabella 1,4 Claudio Babiloni 1,2†, Claudio Del Percio 3†, Susanna Lopez 1, Giancarlo Di Gennaro 4, Pier P. Quarato 4, Luigi Pavone 4, Roberta Morace 4, Andrea Soricelli 3,5, Giuseppe Noce 3, Vincenzo Esposito 1,4, Vittorio Gallese 6 and Giovanni Mirabella 1,4 1Department of Physiology and Pharmacology, University of Rome “La Sapienza”, Rome, Italy, 2IRCCS S. Raffaele Pisana, Rome, Italy, 3IRCCS SDN, Naples, Italy, 4IRCCS Neuromed, Pozzilli, Italy, 5Department of Motor Sciences and Healthiness, University of Naples Parthenope, Naples, Italy, 6Department of Neuroscience, University of Parma, Parma, Italy We have previously shown that in seven drug-resistant epilepsy patients, both reaching- grasping of objects and the mere observation of those actions did desynchronize subdural electrocorticographic (ECoG) alpha (8–13 Hz) and beta (14–30) rhythms as a sign of cortical activation in primary somatosensory-motor, lateral premotor and ventral prefrontal areas (Babiloni et al., 2016a). Furthermore, that desynchronization was greater during action execution than during its observation. In the present exploratory study, we reanalyzed those ECoG data to evaluate the proof-of-concept that lagged linear connectivity (LLC) between primary somatosensory-motor, lateral premotor and ventral prefrontal areas would be enhanced during the action execution compared to the mere observation due to a greater ﬂow of visual and somatomotor information. Results showed that the delta-theta (<8 Hz) LLC between lateral premotor and ventral prefrontal areas was higher during action execution than during action observation. Furthermore, the phase of these delta-theta rhythms entrained the local event-related connectivity of alpha and beta rhythms. It was speculated the existence of a multi- oscillatory functional network between high-order frontal motor areas which should be more involved during the actual reaching-grasping of objects compared to its mere observation. Future studies in a larger population should cross-validate these preliminary results. ORIGINAL RESEARCH published: 07 February 2017 doi: 10.3389/fnbeh.2017.00020 Keywords: subdural electrocorticography (ECoG), movement observation, movement execution, lagged linear connectivity, exact low-resolution brain electromagnetic tomography (eLORETA), frontal cortex, mirror neuron system Edited by: Christoph M. Michel, University of Geneva, Switzerland Christoph M. Michel, University of Geneva, Switzerland Reviewed by: Marlene Meyer, University of Chicago, USA Shenbing Kuang, Institute of Psychology (CAS), China Correspondence: Claudio Babiloni claudio.babiloni@uniroma1.it †These authors have contributed equally to this work. Reviewed by: Marlene Meyer, University of Chicago, USA Shenbing Kuang, Institute of Psychology (CAS), China Correspondence: Claudio Babiloni claudio.babiloni@uniroma1.it Received: 22 July 2016 Accepted: 24 January 2017 Published: 07 February 2017 INTRODUCTION A bulk of magnetoencephalographic (MEG), electroencephalographic (EEG; Babiloni et al., 1999; Pfurtscheller and Lopes da Silva, 1999) and electrocorticographic (ECoG; Crone et al., 1998b; Miller et al., 2007) findings have shown that a desynchronization of alpha (i.e., 8–13 Hz) and beta (i.e., 15–25 Hz) oscillations, which constitute the so-called mu rhythm, occurs not only when A bulk of magnetoencephalographic (MEG), electroencephalographic (EEG; Babiloni et al., 1999; Pfurtscheller and Lopes da Silva, 1999) and electrocorticographic (ECoG; Crone et al., 1998b; Miller et al., 2007) findings have shown that a desynchronization of alpha (i.e., 8–13 Hz) and beta (i.e., 15–25 Hz) oscillations, which constitute the so-called mu rhythm, occurs not only when February 2017 \| Volume 11 \| Article 20 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 1 Functional Connectivity of Action Execution/Observation Babiloni et al. passive or active movements are performed, as a reflection of underlying somatomotor information processing, but also during the observation of actions performed by other people, as a reflection of underlying visual information processing (Pineda, 2005). Specifically, Gastaut and Bert (1954) reported that central EEG mu rhythm desynchronizes during the observation of films depicting biological motion (e.g., a bike race, boxing) as a function of the identification with the actor on screen. This finding was corroborated by further MEG and EEG evidence (for a review see Pineda, 2005). For instance, it has been reported that MEG and EEG beta synchronization following median nerve stimulation decreased in magnitude in a dipolar model of the activity in primary motor cortex during both movement execution and observation (Hari et al., 1998; Rossi et al., 2002; Muthukumaraswamy and Johnson, 2004a). Furthermore, EEG alpha and beta rhythms desynchronized over widespread frontal, central and parietal areas during the observation of movements performed by other people (Cochin et al., 1998, 1999; Babiloni et al., 2002; Martineau and Cochin, 2003; Neuper et al., 2009; Perry and Bentin, 2009). et al., 2004). To overcome these limitations, we recorded subdural ECoG activity from seven drug-resistant epilepsy patients, to assess whether both movement observation and execution induce parallel modulations of alpha, beta and gamma rhythms in task-relevant sensorimotor cortical regions (Babiloni et al., 2016a). In the two conditions (i.e., movement execution and observation), common objects implying power grips (e.g., cup, phone, glasses) were reached and grasped. INTRODUCTION Both arm-hand action execution and observation induced a desynchronization of alpha (8–12 Hz) and beta (16–24 Hz) rhythms in primary somatosensory (Brodmann area, BA, 1–2), primary motor (BA4), lateral premotor (BA6) and the inferior frontal gyrus (BA44 and BA45). This desynchronization was generally higher in amplitude during the execution than the observation of the actions, likely as a consequence of higher cortical activation during the former than during the latter condition. Action execution also induced a synchronization of gamma (36–44 Hz) rhythms in BA4 and BA6, as an additional sign of enhanced cortical activation. These results agree with previous evidence showing frontal alpha and beta desynchronization during the observation of movements performed by other people (Perry et al., 2010; Wriessnegger et al., 2013). As a novelty, Babiloni et al. (2016a) provided fine spatial details on the multiple oscillatory neurophysiological mechanisms underlying the binding of visual, premotor and prefrontal areas during the observation of an act as well as during the execution of the same act. Some other studies have shown a high degree of specialization of this mechanism, by demonstrating a strict functional relationship between the mu rhythm and movement parameters. For instance, it was found that the observation of a precision grip induced a stronger frontal and parietal alpha desynchronization when compared to observing a flat-hand extension or a precision grip gesture with no object to grasp (Muthukumaraswamy and Johnson, 2004b; Muthukumaraswamy et al., 2004). This finding can be considered as the ‘‘negative’’ counterpart (i.e., suppression of a background EEG oscillatory activity) of the binding processes that couple human non-primary frontoparietal ‘‘mirror’’ neurons and primary somatomotor neurons during movement observation (Pineda, 2005). In the same vein, frontal beta rhythms were associated with kinematic features of reaching and grasping objects executed by others, as an evidence of its strict relationship with visuomotor transformation processes (Avanzini et al., 2012). Beta rhythms were also related to kinematic features of observed movements in monkeys, as revealed by parietal local field potentials at beta frequencies (Kuang et al., 2016). During action observation, the desynchronization of the mu rhythm is not associated with a synchronization at 40 Hz (Pfurtscheller and Lopes da Silva, 1999; Perry et al., 2010; Wriessnegger et al., 2013), which can be considered as the ‘‘positive’’ counterpart of the above mentioned binding processes between mirror neurons. Frontiers in Behavioral Neuroscience \| www.frontiersin.org INTRODUCTION It may be argued that the above EEG and MEG results do not capture a main feature of human higher cognitive-motor functions, namely the linear functional or effective connectivity within long-range brain networks (Varela et al., 2001; Le Van Quyen et al., 2003; Börner et al., 2007). It can be speculated that most cortical processes related to action OBSERVATION and EXECUTION would be highly distributed and synchronized across several cortical and sub-cortical sensorimotor regions. Therefore, an ideal methodological approach would be the extraction of some indexes of functional brain connectivity from ECoG rhythms at electrode pairs. In this framework, linear indexes such as spectral coherence would provide information about the phase relationship between the EEG or ECoG rhythms recorded at electrode pairs (Rappelsberger and Petsche, 1988; Gerloff et al., 1998; Gevins et al., 1998). Other relevant linear indexes are lagged linear connectivity (LLC), Granger causality and related measures, and phase-amplitude cross-frequency coupling (CFC). LLC has the advantage to removing the zero-lag component of EEG/ECoG spectral coherence, which is likely due to the instantaneous physical propagation of the neural currents into the head as a volume conductor (Pascual-Marqui, 2007a,b,c). Granger causality and directed transfer function (DTF) would estimate the directionality of that EEG/ECoG functional coupling, suggesting the underlying effective ‘‘causal’’ connectivity (Babiloni et al., 2016a,b,c). Phase-amplitude CFC at a given electrode pair models the dependence between the phase of a low-frequency EEG/ECoG coupling and the amplitude of that coupling at higher EEG/ECoG frequencies (Canolty et al., 2006; Demiralp et al., 2007; Osipova et al., 2008; Cohen et al., 2009). However, non-invasive scalp EEG and MEG techniques have an insufficient spatial resolution to disentangling the effects of a movement observation on mu rhythms generated by contiguous primary somatosensory, primary motor and premotor areas. Therefore, this issue has been addressed by subdural ECoG recordings performed during the pre-surgical monitoring in drug-resistant epilepsy patients, which are characterized by high spatial and temporal resolution. One study, done just in one patient, has shown an alpha desynchronization in primary motor and Broca’s area during both execution and observation of aimless movements (Tremblay et al., 2004). No report concerning gamma synchronization was reported (Tremblay February 2017 \| Volume 11 \| Article 20 2 Functional Connectivity of Action Execution/Observation Babiloni et al. INTRODUCTION Therefore, the aim of the current exploratory study was to produce a proof-of-concept that event-related LLC (ErLLC) of ECoG rhythms would increase during the action execution with respect to the mere observation of the same act due to a higher flow of visual, executive motor commands and feedback somatosensory reafferent information. To this aim, the analysis of the ECoG rhythms was extended to delta/theta rhythms (<8 Hz). Previous ECoG studies in rodents have shown that these low-frequency rhythms were involved in the functional brain connectivity of distributed frontal, parietal and sub-cortical cognitive-motor networks (Jones and Wilson, 2005; Sirota et al., 2008), possibly to allow the integration of motor commands with incoming multimodal feedback sensory information (Caplan et al., 2003; Ekstrom et al., 2005). Similarly, studies on humans, unveiled that a desynchronization of theta (4–8 Hz) rhythms occurred both during the execution of reaching and grasping an object with the hand and during the observation of similar actions (i.e., the observation of video clips showing reaching and grasping an object with the hand; Frenkel-Toledo et al., 2013). Finally, it has also been shown that children exhibit higher theta desynchronization during the observation of a human movement compared to the observation of a non-biological movement (Cochin et al., 2001; Martineau et al., 2010). ECoG data acquisition was performed with a 400 Hz sampling and automatic anti-aliasing filter. All exploring electrodes were referenced to a reference electrode located at the extra- cephalic mastoid bones. Furthermore, electromyogram (EMG) of the flexor digitorum superficialis of the dominant hand, the electrocardiogram (EKG), and electrooculogram (EOG) were used for control purposes. During the recordings of ECoG, a patient was seated in his/her bed and two experimenters were near him or her. One did seat in front of the patient, while the other was placed on the right of the patient. In OBSERVATION or EXECUTION condition, each trial started when experimenter I positioned a common object that differed in size, shape and function (cup, glass, or phone) on the plate of a mechanical device, which was placed on a small table between the patient and the experimenter II. After placing the object, the experimenter I gave a verbal instruction, which was either ‘‘Observe’’ or ‘‘Execute’’. In the OBSERVATION condition, the experimenter II reached, grasped and lifted the object following its more common affordances, while the patient stayed still. INTRODUCTION In the EXECUTION condition, the same action was performed by the patient, while the experimenter II stayed still. In both conditions, the mechanical device detected the first lifting of the object on the plate sensor and released a TTL pulse (reduced in amplitude by a voltage divider and decoupled through an optoisolator chip) to the ECoG data acquisition system signaling the zero-time of each trial. Afterward, the object was put back to the table by the agent (i.e., patient or experimenter II) and took away by the experimenter I, who changed the objects trial-by-trial in a pseudorandom order. It can be estimated that the time gap between the verbal command given by the experimenter I and the so-called zerotime was of about 2 s. The inter-trial interval was never less than 15–20 s. The OBSERVATION and the EXECUTION condition included 60 trials each. Subjects Seven right-handed, adult volunteers (3 females, 4 males; age of 47.5 years ±5.9 standard error, SE) with intractable epilepsy were included in the experimental group. All ethical procedures were followed. Epileptogenic brain regions were localized following a validated procedure (Quarato et al., 2005). Apart epilepsy, no other neurological abnormality was observed. ECoG, EOG and EMG continuous records were divided in single epochs lasting from −5 to +5 s where zero time coincided practically with the contact between the subject’s hand and the object. The ECoG epochs with artifacts (ocular, muscular, others) were identified and rejected by the procedure validated in Moretti et al. (2003). Two experts validated that procedure. MATERIALS AND METHODS Details on the subjects, the cognitive-motor task, and ECoG recordings were reported in Babiloni et al. (2016a). In the following sections, we provide a short description of those methodological procedures. Estimation of Functional Connectivity of ECoG Rhythms: eLORETA LLC y All patients, but two had (subdural) ECoG electrode grids with 64 recording contacts while the remaining two patients had grids with 48 recording contacts. Kind and localization of grids were based exclusively on clinical considerations. Specifically, electrodes were placed in ventral prefrontal (BA44 and BA45, BA44/45), lateral premotor (BA6) and primary somatosensory- motor (BA1, BA2 and BA4, BA1/2/4) areas. The electrodes of interest for this study did not show epileptogenic foci and covered the neural networks coding for the execution of visual- guided movements and understanding of similar movements performed by others, as shown by previous fMRI studies (Buccino et al., 2004; Calvo-Merino et al., 2005, 2006; Cross et al., 2006). Subdural ECoG electrodes were localized as described elsewhere (Miller et al., 2007; Mattia et al., 2012). The exact low-resolution brain electromagnetic tomography (eLORETA) freeware (Pascual-Marqui, 2007a) is an evolution of LORETA and standardized LORETA package (Pascual- Marqui et al., 1994, 2002) and was used to estimate the functional linear connectivity of the ECoG rhythms in the present study. Specifically, we used the LLC function (Pascual- Marqui et al., 2011), which removes the zero-lag coherence of the functional connectivity typically due to the physical propagation of the neural currents in the head as a volume conductor (Pascual-Marqui, 2007b). Furthermore, the LLC minimizes the ‘‘common feeding’’ phenomenon (Pascual- Marqui, 2007c), namely the instantaneous coherence between February 2017 \| Volume 11 \| Article 20 Frontiers in Behavioral Neuroscience \| www.frontiersin.org Frontiers in Behavioral Neuroscience \| www.frontiersin.org 3 Functional Connectivity of Action Execution/Observation Babiloni et al. two cortical sources merely due to the effect of a third source. reducing the number of statistical comparisons given the small number of patients. In the principal analysis, the LLC of the ECoG rhythms was evaluated only between pairs of large cortical zones having direct anatomical connections. For this purpose, we preliminarily grouped cortical areas based on the classical distinction as ‘‘primary’’, ‘‘secondary’’ and ‘‘tertiary’’ (Luria, 1973). Specifically, the primary somatosensory (BA1/2) and motor (BA4) areas were collapsed into a primary somatosensory- motor ROI (BA1/2/4). Furthermore, the inferior prefrontal areas (BA44 and BA45) were collapsed into a ventral prefrontal ROI (BA44/45). Finally, the lateral premotor area was considered as a unique ROI. Afterward, the LLC was computed between the BA1/2/4 and BA6 pair (six patients) and between the BA6 and BA44/45 pair (six patients). Computation of Event-Related LLC (ErLLC) at a Given Electrode Pair and Frequency EXECUTION OBSERVATION BA- ROI pair ARF (Hz) BRF (Hz) ARF (Hz) BRF (Hz) BA1/2/4 and BA6 8.8 ± 0.4 19.8 ± 0.9 9.9 ± 1.0 19.9 ± 0.7 BA6 and BA44/45 10.8 ± 0.7 20.5 ± 0.5 9.2 ± 0.9 19.8 ± 0.7 See the “Materials and Methods” Section for an explanation of the meaning of the ARF and BRF and the description of the two experimental conditions. TABLE 1 \| Mean values ± standard error mean (SE) of the alpha “reactive” frequency (ARF) and the beta “reactive” frequency (BRF) peaks at two pairs of Brodmann area (BA) or cortical region of interest (ROI; i.e., BA1/2/4 and BA6 pair; BA6 and BA44/45 pair) for the EXECUTION and the OBSERVATION condition. Computation of Event-Related LLC (ErLLC) at a Given Electrode Pair and Frequency Table 1 reports the mean values (± standard error mean, SE) of the ARF and BRF for each BA-ROI pair and experimental condition (i.e., EXECUTION, OBSERVATION). For each BA-ROI pair, a t-test evaluated whether the ARF and BRF varied between the EXECUTION and the OBSERVATION condition (p < 0.05). No statistically significant difference was found (p > 0.05). For each BA-ROI pair, we computed an ErLLC value for each frequency band (i.e., delta-theta, alpha, beta and gamma), electrode pair, and experimental condition (i.e., EXECUTION or OBSERVATION). To take into account the inter-subject variability of the ECoG LLC, we identified a ‘‘reactive electrode pair’’ for a given BA-ROI pair, condition and frequency band. The reactive electrode pair was defined as the one showing the maximum ErLLC value in the BA-ROI pair for a given condition and frequency band. For example, for the BA1/2/4 and BA6 pair and a given condition, we identified (i) the delta-theta reactive electrode pair as the To take into account the inter-subject variability of the ECoG LLC, we used the index named ‘‘event-related lagged linear connectivity’’ (ErLLC). At a given electrode pair, ErLLC was defined as the arithmetical difference between the ECoG LLC value at the event period and the ECoG LLC value at the pre-event period. It should be remarked that the magnitude of ECoG ErLLC is smaller than the absolute ECoG LLC, as it results from the difference between coherence values. The ErLLC assumes positive values when the ECoG LLC is higher during the event than the pre-event period (i.e., enhanced ECoG LLC as a reflection of the enhanced functional connectivity of ECoG rhythms). On the contrary, the ErLLC is negative when the ECoG LLC is lower during the event than the pre-event period (i.e., reduced ECoG LLC as a reflection of the reduced functional connectivity of ECoG rhythms). TABLE 1 \| Mean values ± standard error mean (SE) of the alpha “reactive” frequency (ARF) and the beta “reactive” frequency (BRF) peaks at two pairs of Brodmann area (BA) or cortical region of interest (ROI; i.e., BA1/2/4 and BA6 pair; BA6 and BA44/45 pair) for the EXECUTION and the OBSERVATION condition. Estimation of Functional Connectivity of ECoG Rhythms: eLORETA LLC Both of them have direct anatomical connections according to previous investigations (Catani et al., 2012a,b; Thiebaut de Schotten et al., 2014). On the one hand, this choice was due to the need of reducing the number of statistical comparisons in the present small group of epilepsy patients. On the other hand, this approach, in principle, would unveil the modulation of a functional network from primary somatosensory-motor to lateral premotor areas, as well as from lateral premotor to prefrontal areas. A fixed delta-theta band ranged between 2 Hz and 6 Hz to avoid possible inflating effects of ECoG activity pass-banded to 0 Hz. For the alpha band, the alpha ‘‘reactive’’ frequency (ARF) was identified subject-by-subject for any BA-ROI pair (i.e., BA1/2/4 and BA6 pair; BA6 and BA44/45 pair). The ARF was defined as the frequency showing the maximum ErLLC (i.e., the maximum increase of LLC during the event period when compared to the pre-event period) within the band 7–13 Hz. All electrode pairs of a given BA-ROI pair were considered one-by-one. Based on the ARF, the alpha band ranged between ARF−2 Hz and ARF+2 Hz. For the beta band, the beta ‘‘reactive’’ frequency (BRF) was identified subject-by-subject for any BA-ROI pair. The BRF was the frequency showing the maximum ErLLC within the 16–24 Hz band. All electrode pairs of a given BA-ROI pair were considered one-by-one. Based on the BRF, the beta band ranged between BRF-2 Hz and BRF+2 Hz. A fixed gamma band varied between 36 Hz and 100 Hz (Crone et al., 1998a; Szurhaj et al., 2006; Miller et al., 2007, 2010). Noteworthy, the gamma frequency band of interest had the highest frequency limits well lower than the Nyquist frequency of 200 Hz, which is half of the sampling rate used for the digitalization of the EEG data. Computation of Event-Related LLC (ErLLC) at a Given Electrode Pair and Frequency The ECoG LLC for a given electrode pair and frequency was calculated during two different periods: (i) the pre-event period; and (ii) the event period. The event period was defined as the period of movement execution or observation (from 1 s before to 1 s after the zero-time, i.e., the lifting of the object), while the pre-event period was defined as the time interval between −4 s and −3 s before the zero-time. RESULTS Figure 1 shows that the BA-ROI pairs (i.e., BA1/2/4 and BA6; BA6 and BA44/45) and conditions (i.e., EXECUTION and the OBSERVATION) had positive ErLLC values at the delta- theta, alpha, beta and gamma bands, indicating that functional connectivity of the ECoG rhythms was enhanced during the event compared to the pre-event period. The results of the t-tests comparing the ErLLC between the two conditions showed a statistically significant difference only for the BA6-BA44/45 pair at the delta-theta band. Specifically, the ErLLC was greater during the EXECUTION compared to the OBSERVATION (t = 2.26; p = 0.04; one-tailed). Five additional analyses were conducted to control and corroborate the statistical effects of the principal analysis. First, we assessed whether the statistical effects could be due to some outliers in the delta-theta ErLLC in one or both conditions (Grubbs’ test; alpha value p = 0.05). Second, we verified whether the statistical effects could be merely due to the local event-related ECoG power density (using the pre-event period as a reference) reported in a mentioned reference study (Babiloni et al., 2016a). To address this last issue, a step-wise procedure was used. First, two t-tests (p < 0.05) evaluated possible statistically significant differences between the two conditions in the event-related ECoG power density computed at the BA-ROI most reactive electrodes the event-related ECoG power density was calculated with the formula of event-related desynchronization/synchronization (ERD/ERS- used in Babiloni et al., 2016a). Second, a correlation analysis (Pearson test, p < 0.05) was performed between the ErLLC at the BA-ROI pairs and the ERD/ERS computed at the same representative electrodes. The results of control analyses are reported in the following paragraphs. First, we evaluated whether the above statistical effect in the BA6- and BA44/45 pair would be due to some outliers in the delta-theta ErLLC Figure 2 plots the individual delta-theta ErLLC values for the BA6-BA44/45 pair and both conditions. No outliers were found even when using a more stringent statistical thresholds (i.e., alpha value p = 0.001). Second, we evaluated whether there was a statistically significant difference between the two conditions in the delta/theta event-related ECoG power density computed at the BA6 and BA44/45 at the most reactive electrodes. As shown in Figure 3, there was no difference between the two conditions (t-test, p > 0.15). Computation of ErLLC at Frequency Bands of Interest and Representative Electrode Pairs We performed the ECoG ErLLC analysis at the delta-theta, alpha, beta and gamma frequency bands. A finer analysis of alpha, beta and gamma sub-bands was not performed due to the need of February 2017 \| Volume 11 \| Article 20 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 4 Functional Connectivity of Action Execution/Observation Babiloni et al. one showing the highest delta-theta ErLLC value, (ii) the alpha reactive electrode pair as the one showing the highest alpha ErLLC value, (iii) the beta reactive electrode pair as the one showing the highest beta ErLLC value; and (iv) the gamma reactive electrode pair as the one showing the highest gamma ErLLC value. This procedure was followed for both experimental conditions (i.e., EXECUTION and OBSERVATION) and BA-ROI pairs (i.e., BA1/2/4 and BA6; BA6 and BA44/45). test this hypothesis, we used the eLORETA freeware to calculate the event-related phase-amplitude cross-frequency lagged connectivity (ErCFLC) in the BA-ROI pairs in both experimental conditions. The ErCFLC values were computed for two directions and three coupled frequency bands. The direction from one region to another indicated the modulation induced by the phase of the low-frequency ErCFLC in the first region on the amplitude of the ErCFLC in the second region and vice versa. The statistical significance of the effects was probed with t-tests (p < 0.05; two direction × three coupled frequency bands of interest). The hypothesis was that the ErCFLC would be greater in the EXECUTION compared to the OBSERVATION condition. Statistics In this study, the number of epilepsy patients (N = 7) was too low when compared to the factors and levels of an ideal ANOVA design using two conditions × connectivity in two pairs of regions × four ECoG frequency bands of interest (yielding to 16 contrasts). Therefore, the statistical design included only eight t-tests (exploratory alpha value set at p = 0.05) to evaluate whether the ErLLC of ECoG rhythms would differ between the EXECUTION and the OBSERVATION condition for two pairs of regions (i.e., BA1/2/4 and BA6 pair; BA6 and BA44/45 pair) and four frequency bands (i.e., delta-theta, alpha, beta and gamma). Due to problems occurred during ECoG recording, one subject were excluded from the sample, thus limiting the degree of freedom used for all the following statistical analysis to five. These statistical analyses were performed using the software named Statistica® (10.0 packages, StatSoft® Inc., Tulsa, OK, USA). Finally, we tested whether the ErLLC would be higher at the BA1/2/4 and BA44/45 pair (two ROIs not having known direct anatomical connections) during the EXECUTION than the OBSERVATION condition. This pair was represented by the most reactive electrode. To this aim, we performed four t-tests (alpha value p = 0.05, one-tailed), one for each frequency band of interest. Frontiers in Behavioral Neuroscience \| www.frontiersin.org RESULTS The correlation analysis performed between the delta/theta ErLLC at the BA6-BA44/45 pair and the delta/theta ERD/ERS computed at the same representative electrodes. Again, no statistically significant correlation was found (p > 0.2). These findings suggest that the regional ECoG power density did not affect the ErLLC values. Third, we used a t-test to evaluate whether the ECoG LLC in the BA-ROI pairs (the most reactive electrodes) would be greater during the EXECUTION than during the pre-event period (alpha value p = 0.05 one-tailed). Fourth, we evaluated whether the ErLLC effects in the BA-ROI pairs (the most reactive electrodes) would be related to a phase-amplitude coupling of the ECoG rhythms. Specifically, the hypothesis was that the phase of the delta-theta ErLLC in a cortical region would modulate the amplitude of alpha, beta and gamma rhythms in the other cortical region. To Third, we evaluated whether the delta-theta ECoG LLC in the BA6-BA44/45 pair was greater during the EXECUTION than during the pre-event period. As shown in Figure 4, the delta- theta ECoG LLC in the BA6-BA44/45 pair was significantly February 2017 \| Volume 11 \| Article 20 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 5 Functional Connectivity of Action Execution/Observation Babiloni et al. FIGURE 1 \| Mean (± standard error mean, SE) values of the event-related lagged linear connectivity (ErLLC) of subdural electrocorticographic (ECoG) rhythms for two pairs of cortical region of interest (ROI; i.e., BA1/2/4 and BA6 pair; BA6 and BA44/45 pair). The ErLLC values refer to four ECoG frequency bands of interest (i.e., delta-theta, alpha, beta, gamma) and two experimental conditions (i.e., EXECUTION, OBSERVATION). Noteworthy, the only signiﬁcant t-test effect was that the delta-theta ErLLC in the BA6 and BA44/45 pair was greater during the EXECUTION than the OBSERVATION condition (t = 2.26; p = 0.04; one-tailed). FIGURE 3 \| Mean (±SE) values of the delta-theta event-related desynchronization/synchronization (ERD/ERS) at the BA6 and BA44/45 most reactive electrodes for the EXECUTION and the OBSERVATION condition. Noteworthy, the t-test showed no statistically signiﬁcant difference in this ERD/ERS between the two conditions (p > 0.15). FIGURE 3 \| Mean (±SE) values of the delta-theta event-related desynchronization/synchronization (ERD/ERS) at the BA6 and BA44/45 most reactive electrodes for the EXECUTION and the OBSERVATION condition. Noteworthy, the t-test showed no statistically signiﬁcant difference in this ERD/ERS between the two conditions (p > 0.15). rhythms during the event than during the pre-event period. RESULTS In contrast, the ErCFLC values in the OBSERVATION condition were always near to zero. Therefore, for both directions and for all coupled frequency bands, the ErCFLC values were higher in the EXECUTION than in the OBSERVATION condition. The statistical significance of these phenomena was probed with six t-tests (two directions × three coupled frequency bands; p < 0.05). We found that the ErCFLC values were greater during the EXECUTION compared to the OBSERVATION only in the direction from BA6 to BA44/45 (delta/theta →alpha (t = 3.89; p = 0.005; one-tailed), delta/theta →beta (t = 3.00; p = 0.014; one-tailed) and delta/theta →gamma (t = 3.45; p = 0.009; one-tailed)). These findings supported the results obtained with the ErLLC, indicating a directional coupling and unveiling a modulation induced by the phase of delta-theta ErCFLC in the BA6 on the amplitude of the ErCFLC in the BA44/45. FIGURE 1 \| Mean (± standard error mean, SE) values of the event-related lagged linear connectivity (ErLLC) of subdural electrocorticographic (ECoG) rhythms for two pairs of cortical region of interest (ROI; i.e., BA1/2/4 and BA6 pair; BA6 and BA44/45 pair). The ErLLC values refer to four ECoG frequency bands of interest (i.e., delta-theta, alpha, beta, gamma) and two experimental conditions (i.e., EXECUTION, OBSERVATION). Noteworthy, the only signiﬁcant t-test effect was that the delta-theta ErLLC in the BA6 and BA44/45 pair was greater during the EXECUTION than the OBSERVATION condition (t = 2.26; p = 0.04; one-tailed). greater during the EXECUTION than the pre-event period (t = 4.75; p = 0.002, one tailed). Fourth, we evaluated whether the ErLLC values could be related to a phase-amplitude coupling. Specifically, we tested whether the delta/theta phase in one cortical region may modulate the alpha (delta/theta →alpha), beta (delta/theta →beta) and gamma (delta/theta →gamma) amplitudes in the other region. Figure 5 illustrates the mean values of this phase-amplitude coupling in the EXECUTION and in the OBSERVATION condition. The execution condition was always characterized by positive ErCFLC values reflecting an enhanced phase-amplitude cross-frequency connectivity of the ECoG Finally, we tested whether the ErLLC was higher in the EXECUTION than the OBSERVATION condition for the BA1/2/4-BA44/45 pair, two ROIs which do not have direct anatomical connections. As reported in Figure 6, ErLLC values were positive in both conditions at all frequency bands. RESULTS ErCFLC values were always positive in the EXECUTION condition, and near to zero in the OBSERVATION condition (see text for more details). Noteworthy, the t-tests showed that ErCFLC values were greater during the EXECUTION compared to the OBSERVATION only in the direction from BA6 to BA44/45 (delta/theta →alpha (t = 3.89; p = 0.005; one-tailed), delta/theta →beta (t = 3.00; p = 0.014; one-tailed) and delta/theta →gamma (t = 3.45; p = 0.009; one-tailed)). FIGURE 6 \| Mean (±SE) values of the ErLLC in the EXECUTION and the OBSERVATION condition for the BA1/2/4-BA44/45 pair and the four frequency bands of interest (i.e., delta-theta, alpha, beta and gamma). The ErLLC was computed at the most reactive electrode pair among the BA-ROI pairs. Noteworthy, the only statistically signiﬁcant t-test effect was that the beta ErLLC was greater during the EXECUTION than the OBSERVATION condition (t = 2.00; p = 0.03; one-tailed). FIGURE 6 \| Mean (±SE) values of the ErLLC in the EXECUTION and the OBSERVATION condition for the BA1/2/4-BA44/45 pair and the four frequency bands of interest (i.e., delta-theta, alpha, beta and gamma). The ErLLC was computed at the most reactive electrode pair among the BA-ROI pairs. Noteworthy, the only statistically signiﬁcant t-test effect was that the beta ErLLC was greater during the EXECUTION than the OBSERVATION condition (t = 2.00; p = 0.03; one-tailed). FIGURE 5 \| Mean values (±SE) of the event-related phase-amplitude cross-frequency lagged connectivity (ErCFLC) values in the BA6-BA44/45 pair in the EXECUTION and the OBSERVATION condition. These values were computed for two directions and three coupled frequency bands. ErCFLC values were always positive in the EXECUTION condition, and near to zero in the OBSERVATION condition (see text for more details). Noteworthy, the t-tests showed that ErCFLC values were greater during the EXECUTION compared to the OBSERVATION only in the direction from BA6 to BA44/45 (delta/theta →alpha (t = 3.89; p = 0.005; one-tailed), delta/theta →beta (t = 3.00; p = 0.014; one-tailed) and delta/theta →gamma (t = 3.45; p = 0.009; one-tailed)). cognitive tasks (Mukamel and Fried, 2012; Ritaccio et al., 2014; Daitch et al., 2016; Nourski et al., 2016). Compared to non-invasive EEG recordings from the human scalp, ECoG recordings have a higher spatial resolution, better gamma frequency resolution and enhanced signal-to-noise ratio (Mukamel and Fried, 2012). Furthermore, ECoG recordings are much less affected from eye movements and head muscle artifacts (Kovach et al., 2011). RESULTS However a significant difference was found only in the beta band FIGURE 2 \| Individual values of the delta-theta ErLLC at the BA6-BA44/45 pair for the EXECUTION and the OBSERVATION condition across all patients. Noteworthy, the Grubbs’ test showed no outlier (p < 0.0001; see text for more details). Lines represent the mean (±SE) values. FIGURE 4 \| Individual and mean (±SE) values of the delta-theta lagged linear connectivity (LLC) at the BA6 and BA44/45 pair for the pre-event and the event period in the EXECUTION condition. Noteworthy, the t-test showed that the LLC was signiﬁcantly higher during the EXECUTION than the pre-event period (t = 4.75; p = 0.002; one-tailed). Lines represent the mean (±SE) values. FIGURE 2 \| Individual values of the delta-theta ErLLC at the BA6-BA44/45 pair for the EXECUTION and the OBSERVATION condition across all patients. Noteworthy, the Grubbs’ test showed no outlier (p < 0.0001; see text for more details). Lines represent the mean (±SE) values. FIGURE 4 \| Individual and mean (±SE) values of the delta-theta lagged linear connectivity (LLC) at the BA6 and BA44/45 pair for the pre-event and the event period in the EXECUTION condition. Noteworthy, the t-test showed that the LLC was signiﬁcantly higher during the EXECUTION than the pre-event period (t = 4.75; p = 0.002; one-tailed). Lines represent the mean (±SE) values. FIGURE 4 \| Individual and mean (±SE) values of the delta-theta lagged linear connectivity (LLC) at the BA6 and BA44/45 pair for the pre-event and the event period in the EXECUTION condition. Noteworthy, the t-test showed that the LLC was signiﬁcantly higher during the EXECUTION than the pre-event period (t = 4.75; p = 0.002; one-tailed). Lines represent the mean (±SE) values. FIGURE 2 \| Individual values of the delta-theta ErLLC at the BA6-BA44/45 pair for the EXECUTION and the OBSERVATION condition across all patients. Noteworthy, the Grubbs’ test showed no outlier (p < 0.0001; see text for more details). Lines represent the mean (±SE) values. February 2017 \| Volume 11 \| Article 20 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 6 Babiloni et al. Functional Connectivity of Action Execution/Observation FIGURE 5 \| Mean values (±SE) of the event-related phase-amplitude cross-frequency lagged connectivity (ErCFLC) values in the BA6-BA44/45 pair in the EXECUTION and the OBSERVATION condition. These values were computed for two directions and three coupled frequency bands. RESULTS However, an important limitation of ECoG techniques is that epilepsy might induce some adaptive changes in the neurophysiological oscillatory mechanisms underpinning cognitive-motor functions even in the brain zones which are not producing epileptic activity. Therefore, the full understanding of these mechanisms requires the integration and comparison with findings coming from different approaches such as high-resolution scalp EEG, coregistration of scalp EEG and functional magnetic resonance imaging, MEG. FIGURE 5 \| Mean values (±SE) of the event-related phase-amplitude cross-frequency lagged connectivity (ErCFLC) values in the BA6-BA44/45 pair in the EXECUTION and the OBSERVATION condition. These values were computed for two directions and three coupled frequency bands. ErCFLC values were always positive in the EXECUTION condition, and near to zero in the OBSERVATION condition (see text for more details). Noteworthy, the t-tests showed that ErCFLC values were greater during the EXECUTION compared to the OBSERVATION only in the direction from BA6 to BA44/45 (delta/theta →alpha (t = 3.89; p = 0.005; one-tailed), delta/theta →beta (t = 3.00; p = 0.014; one-tailed) and delta/theta →gamma (t = 3.45; p = 0.009; one-tailed)). condition. These values were computed for two directions and three coupled frequency bands. ErCFLC values were always positive in the EXECUTION condition, and near to zero in the OBSERVATION condition (see text for more details). Noteworthy, the t-tests showed that ErCFLC values were greater during the EXECUTION compared to the OBSERVATION only in the direction from BA6 to BA44/45 (delta/theta →alpha (t = 3.89; p = 0.005; one-tailed), delta/theta →beta (t = 3.00; p = 0.014; one-tailed) and delta/theta →gamma (t = 3.45; p = 0.009; one-tailed)). (p < 0.05). Specifically, the beta ErLLC was greater during the EXECUTION with respect to the OBSERVATION condition (t = 2.00; p = 0.03; one-tailed). Frontiers in Behavioral Neuroscience \| www.frontiersin.org Delta and Theta Rhythms Underpin Functional Connectivity Between Lateral Premotor and Ventral Prefrontal Areas during Action Execution Compared to Its Mere Observation In the present exploratory study, we re-analyzed the ECoG data recorded in a small group of drug-resistant epilepsy patients (Babiloni et al., 2016a). As a novelty, here we tested the proof-of-concept that the ErLLC of ECoG rhythms would be enhanced in primary somatosensory-motor, lateral premotor and ventral prefrontal areas during the actual reaching and grasping of objects in the first person compared to the mere observation of those actions performed by another person. Results are discussed in the following sections. Of note, the interpretation of those results should take into account the typical methodological pros and cons of ECoG recordings done in drug-resistant epilepsy patients. The main aim of ECoG recordings is the localization of epileptogenic brain regions for the evaluation of possible surgical removal of epileptogenic tissues. Secondary to the clinical aim, ECoG recordings are also valuable in the field of research in order to investigate neurophysiological mechanisms underpinning cognitive and sensorimotor functions in brain areas outside the epileptogenic region, typically located around the epileptogenic foci monitored. This approach provided enlightening evidence on neurophysiological mechanisms underlying the execution of visuospatial, episodic memory, language, emotional and In the present study, the delta-theta (<6 Hz) ErLLC indicated that the event-related functional connectivity between the lateral premotor (BA6) and ventral prefrontal (BA44/45) areas was greater during the execution of transitive actions than during the mere observation of those actions performed by another agent. At this early stage of the research, we cannot provide a conclusive explanation for this finding, but we can provide a tentative explanation based on the available literature. In previous works, low-frequency ECoG oscillations (3–8 Hz) recorded from the mammalian brain has been conceived as a neurophysiological mechanism for the synchronization and integration of neural activity within distributed functional networks spanning thalamocortical-cortical systems and ascending brainstem and diencephalon-septal systems, both converging on February 2017 \| Volume 11 \| Article 20 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 7 Functional Connectivity of Action Execution/Observation Babiloni et al. entorhinal cortex and hippocampus for the formation of memory traces (Kirk and Mackay, 2003; Vertes et al., 2004). entorhinal cortex and hippocampus for the formation of memory traces (Kirk and Mackay, 2003; Vertes et al., 2004). accompanying movement initiation and sensory processing without any motor learning or memory demand (Cruikshank et al., 2012). Moreover, such modulation of theta rhythms was distinct from purely motor-related desynchronization of mu rhythm (Cruikshank et al., 2012). Delta and Theta Rhythms Underpin Functional Connectivity Between Lateral Premotor and Ventral Prefrontal Areas during Action Execution Compared to Its Mere Observation As scalp EEG primarily reflects neocortical sources activated from cortical and sub-cortical signals (e.g., brainstem, thalamus, basal ganglia and hippocampus; Nunez and Silberstein, 2000) and frontoparietal lesions impair sensorimotor information processing (Sirigu et al., 2004), it has been hypothesized that sensorimotor integration is an emerging cortical function (Ekstrom and Watrous, 2014). Sensorimotor-related EEG oscillations may manifest in the hippocampus via phase synchronization with cortical and subcortical brain areas (Ekstrom and Watrous, 2014). ) Previous ECoG studies in drug-resistant epilepsy patients have confirmed the importance of this mechanism based on theta oscillatory activity for short- and long-term memorization. Those ECoG studies showed that theta rhythms increased in amplitude in occipital, parietal and temporal cortical areas during short-term memory trials (Raghavachari et al., 2001). This increase in amplitude was coherent with that recorded at near cortical sites, suggesting the existence of local generating mechanisms of cortical theta rhythms (Raghavachari et al., 2006). Concerning the long-term memory, previous ECoG studies unveiled that the encoding of events to be recalled after hours was associated with an increase of frontal and temporal theta and gamma power (Sederberg et al., 2003). Furthermore, there was an increase of theta coherence between the hippocampus and rhinal-paleocortex (Fell et al., 2003), and among hippocampus, amygdala and occipital-temporal neocortex (Babiloni et al., 2009). In addition, an increase of low-frequency ECoG rhythms was observed across several cortical areas including temporal cortex during a spatial learning task implying the navigation into computerized mazes (Kahana et al., 1999). Finally, a widespread increase of cortical theta rhythms during the encoding of items subsequently recalled was also reported from scalp EEG recordings in healthy adults (Klimesch et al., 1996; Weiss et al., 2000; Sauseng et al., 2010). Taking the above theoretical framework, the present findings represent the first demonstration of a neurophysiological mechanism exploiting delta-theta synchronization for enhancing the functional connectivity between lateral premotor and ventral prefrontal areas during the execution of reaching and grasping movements with respect to the mere observation of the same actions performed by someone else. Another preliminary result of the present study indicated that the phase of the delta/theta rhythms in the lateral premotor area would entrain the amplitude of the alpha, beta and gamma rhythms in the ventral prefrontal cortex during the action execution, but not during its observation. Delta and Theta Rhythms Underpin Functional Connectivity Between Lateral Premotor and Ventral Prefrontal Areas during Action Execution Compared to Its Mere Observation This finding complements and extends previous ECoG evidence in epilepsy patients showing that the alpha or beta phases entrained the amplitude of gamma rhythms in the very early stages of movement preparation (Miller et al., 2012; Yanagisawa et al., 2012). Although this bulk of literature focused on the relationship between the brain theta rhythms and memory, the modulation of those rhythms in distributed functional networks is unlikely to represent a peculiar neural underpinning of episodic memory. Rather, neural synchronization at low-frequencies spanning delta and theta (<8 Hz) might be viewed as a general neurophysiological mechanism occurring when task demands increase (e.g., when the execution of a transitive action is contrasted with its mere observation), and information processing within distributed functional neural networks have to be enhanced. In this vein, it has been previously proposed that the neural synchronization at delta and theta frequencies (<8 Hz) might subserve a long-range coordination of distant brain regions, while neural synchronization at higher frequencies would subserve local interactions of neural assemblies (Kopell et al., 2000; von Stein and Sarnthein, 2000; Siegel et al., 2012). Furthermore, it has been proposed that cortical theta rhythms reveal integration of motor commands and incoming sensory information for an on-line correction of the motor output (Bland and Oddie, 2001; Caplan et al., 2003; Ekstrom et al., 2005), the so-called sensorimotor integration hypothesis. Frontiers in Behavioral Neuroscience \| www.frontiersin.org REFERENCES action observation. Curr. Biol. 16, 1905–1910. doi: 10.1016/j.cub.2006. 07.065 Avanzini, P., Fabbri-Destro, M., Dalla Volta, R., Daprati, E., Rizzolatti, G., and Cantalupo, G. (2012). The dynamics of sensorimotor cortical oscillations during the observation of hand movements: an EEG study. PLoS One 7:e37534. doi: 10.1371/journal.pone.0037534 Canolty, R. T., Edwards, E., Dalal, S. S., Soltani, M., Nagarajan, S. S., Kirsch, H. E., y g j et al. (2006). High gamma power is phase-locked to theta oscillations in human neocortex. Science 313, 1626–1628. doi: 10.1126/science.1128115 Caplan, J. B., Madsen, J. 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C., Singhal, A., Hueppelsheuser, M., and Caplan, J. B. (2012). Theta oscillations reflect a putative neural mechanism for human sensorimotor integration. J. Neurophysiol. 107, 65–77. doi: 10.1152/jn.00 893.2010 Calvo-Merino, B., Glaser, D. E., Grèzes, J., Passingham, R. E., and Haggard, P. (2005). Action observation and acquired motor skills: an FMRI study with expert dancers. Cereb. Cortex 15, 1243–1249. doi: 10.1093/cercor/bhi007 Daitch, A. L., Foster, B. L., Schrouff, J., Rangarajan, V., Kasikci, I., Gattas, S., et al. (2016). CONCLUSION In the present study, ErLLC of ECoG rhythms was estimated in a small group of drug-resistant epilepsy patients in order to assess the hypothesis that the functional relationships between sensorimotor cortical regions would be enhanced during reaching and grasping of objects in the first person compared to the mere observation of those actions performed by someone else. We found that the ErLLC of delta-theta rhythms between the ventral prefrontal and lateral premotor areas was greater during action execution than its observation. Furthermore, the phase of those rhythms entrained the amplitude of the coupling of ECoG rhythms with higher frequencies such as alpha, beta and gamma. y g g These results support the validity of our experimental approach based on pre-surgical ECoG recordings from epilepsy patients, and the computation of ErLLC of ECoG rhythms for probing functional cortical connectivity underpinning the execution and the observation of reaching and grasping movements in humans. All in all, we suggest that during the execution of transitive actions, neurophysiological low-frequency oscillatory mechanisms operating in a frontal neural network formed by BA6 and BA44/45 would underpin the integration of motor commands and visual-somatosensory feedback accompanying the own performance. Future studies The sensorimotor integration hypothesis is supported by several lines of evidence. Previous ECoG recordings in rodents have shown that during motor acts, theta rhythms are often coherent between the hippocampus and prefrontal cortex (Jones and Wilson, 2005) and between the hippocampus and parietal cortex (Sirota et al., 2008). Furthermore, a recent scalp EEG study in humans has reported increased theta rhythms February 2017 \| Volume 11 \| Article 20 8 Functional Connectivity of Action Execution/Observation Babiloni et al. will have to confirm this proof-of-concept exploiting a large sample of epilepsy patients and more conservative statistical models. writing article; GN and AS: data analyses, estimation of functional connectivity of ECoG rhythms; LP: localized electrode positions; GG, PPQ, RM and VE: patients selection and handling VG: project idea; GM: project idea, EEG data recording and writing article. ACKNOWLEDGMENTS The research leading to these results has received funding from the Italian Ministry of Health (project GR-2007-25 to GM) and Italian Ministry of University and Research (PRIN n. 2008- RBFNLH-005 to GM). Thanks to Dr. Fabrizio Vecchio for his very useful contribution to the development of a pilot data analysis with a traditional procedure for the computation of EEG spectral coherence including zerolag coupling. We also want to thank all patients who underwent the experiment. AUTHOR CONTRIBUTIONS CB: project idea and writing article; CDP and SL: data analyses, estimation of functional connectivity of ECoG rhythms and ETHICS STATEMENT This study was carried out in accordance with the recommendations of IRCCS Neuromed committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The study was approved by the IRCCS Neuromed committee, Pozzilli, Italy. 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https://openalex.org/W4396593102	https://www.medrxiv.org/content/medrxiv/early/2024/05/02/2024.05.01.24306640.full.pdf	English	null	Antidepressant Exposure and DNA Methylation: Insights from a Methylome-Wide Association Study	medRxiv (Cold Spring Harbor Laboratory)	2,024	cc-by	13,356	. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation: Insights from a Methylome-Wide Association Study Authors: Davyson E1,2, Shen X1, Huider F3,4, Adams M1, Borges K1, McCartney D2, Barker L5, Van Dongen J3,4, Boomsma D3,4, Weihs A6,7, Grabe H6,7, Kühn L6, Teumer A6,8, Völzke H8,9, Zhu T10,11, Kaprio J10, Ollikainen M10,11, David FS12,13, Meinert S14,15, Stein F13,16, Forstner AJ12,17,18, Dannlowski U14, Kircher T13,16, Tapuc A19,20, Czamara D20, Binder EB20, Brückl T20, Kwong A1,21, Yousefi P21,22,23, Wong CCY24, Arseneault L24, Fisher HL24,25, Mill J26, Cox S27, Redmond P27, Russ TC1,28,29, Marioni RE2, Wray NR5,30, McIntosh AM1* Authors: Davyson E1,2, Shen X1, Huider F3,4, Adams M1, Borges K1, McCartney D2, Barker L5, Van Dongen J3,4, Boomsma D3,4, Weihs A6,7, Grabe H6,7, Kühn L6, Teumer A6,8, Völzke H8,9, Zhu T10,11, Kaprio J10, Ollikainen M10,11, David FS12,13, Meinert S14,15, Stein F13,16, Forstner AJ12,17,18, Dannlowski U14, Kircher T13,16, Tapuc A19,20, Czamara D20, Binder EB20, Brückl T20, Kwong A1,21, Yousefi P21,22,23, Wong CCY24, Arseneault L24, Fisher HL24,25, Mill J26, Cox S27, Redmond P27, Russ TC1,28,29, Marioni RE2, Wray NR5,30, McIntosh AM1* Affiliations 1 the Netherlands 6Department of Psychiatry and Psychotherapy, University Medicine Greifswald, 17475 Greifswald, Germany 7 6Department of Psychiatry and Psychotherapy, University Medicine Greifswald, 17475 Greifswald, Germany 7 7German Center for Neurodegenerative Diseases (DZNE), Site Rostock/Greifswald, 17489 Greifswald, Germany 8 7German Center for Neurodegenerative Diseases (DZNE), Site Rostock/Greifswald, 17489 Greifswald, Germany 8 8German Centre for Cardiovascular Research (DZHK), Partner Site Greifswald, 17489 Greifswald, Germany 9 8German Centre for Cardiovascular Research (DZHK), Partner Site Greifswald, 17489 Greifswald, Germany Department SHIP/Clinical-Epidemiological Research, Institute for Community Medicine 9Department SHIP/Clinical-Epidemiological Research, Institute for Community Medicine, University Medicine Greifswald, 17475 Greifswald, Germany 10 University Medicine Greifswald, 17475 Greifswald, Germany y , , y 10Institute for Molecular Medicine Finland FIMM, HiLIFE, University of Helsinki, Helsinki, Finland. 10Institute for Molecular Medicine Finland FIMM, HiLIFE, University of Helsinki, Helsinki, Finland. NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. Affiliations 1 Affiliations 1Division of Psychiatry, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK 2Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK 3Department of Biological Psychology, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands 4Amsterdam Public Health Research Institute, Amsterdam, the Netherlands & Amsterdam Reproduction & Development, Research Institute, Amsterdam. the Netherlands 5Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. 6Department of Psychiatry and Psychotherapy, University Medicine Greifswald, 17475 Greifswald, Germany 7German Center for Neurodegenerative Diseases (DZNE), Site Rostock/Greifswald, 17489 Greifswald, Germany 8German Centre for Cardiovascular Research (DZHK), Partner Site Greifswald, 17489 Greifswald, Germany 9Department SHIP/Clinical-Epidemiological Research, Institute for Community Medicine, University Medicine Greifswald, 17475 Greifswald, Germany 10Institute for Molecular Medicine Finland FIMM, HiLIFE, University of Helsinki, Helsinki Finland. 11Minerva Foundation Institute for Medical Research, Helsinki, Finland 12Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany 13Department of Psychiatry and Psychotherapy, University of Marburg, Marburg, Germany 14Institute for Translational Psychiatry, University of Münster, Münster, Germany 15Institute for Translational Neuroscience, University of Münster, Münster, Germany 16Center for Mind, Brain and Behavior, University of Marburg, Marburg, Germany 17Institute of Neuroscience and Medicine (INM-1), Research Center Jülich, Jülich, Germany 18Center for Human Genetics, University of Marburg, Marburg, Germany 19Max Planck School of Cognition, Leipzig, Germany 20Max-Planck-Institute of Psychiatry, Department Genes and Environment, Munich, Germany 21Medical Research Council Integrative Epidemiology Unit at the University of Bristol, University of Bristol, Bristol, UK 22Population Health Science, Bristol Medical School, University of Bristol, Bristol, UK 23NIHR Bristol Biomedical Research Centre, University Hospitals Bristol and Weston NHS Foundation Trust and University of Bristol. 24Social, Genetic & Developmental Psychiatry Centre, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK 1Division of Psychiatry, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK 2 2Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK 3 Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK 3Department of Biological Psychology, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands sterdam Public Health Research Institute, Amsterdam, the Netherlands & Amsterdam roduction & Development, Research Institute, Amsterdam. the Netherlands itute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, 4Amsterdam Public Health Research Institute, Amsterdam, the Netherlands & Amsterdam Reproduction & Development, Research Institute, Amsterdam. the Netherlands 5Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. Amsterdam Public Health Research Institute, Amsterdam, the Netherlands & Amsterdam eproduction & Development, Research Institute, Amsterdam. Affiliations 1 11Minerva Foundation Institute for Medical Research, Helsinki, Finland 12 11Minerva Foundation Institute for Medical Research, Helsinki, Finland 12Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany Minerva Foundation Institute for Medical Research, Helsinki, Finland 12Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn Bonn Germany 12Institute of Human Genetics, University of Bonn, School of Medicine & University H i l B B G Hospital Bonn, Bonn, Germany 13 13Department of Psychiatry and Psychotherapy, University of Marburg, Marburg, Germany 14Institute for Translational Psychiatry, University of Münster, Münster, Germany 15Institute for Translational Neuroscience, University of Münster, Münster, Germany 16Center for Mind, Brain and Behavior, University of Marburg, Marburg, Germany 17Institute of Neuroscience and Medicine (INM-1), Research Center Jülich, Jülich, Germany 18Center for Human Genetics, University of Marburg, Marburg, Germany 19M Pl k S h l f C i i L i i G 13Department of Psychiatry and Psychotherapy, University of Marburg, Marburg, Germany 14 14Institute for Translational Psychiatry, University of Münster, Münster, Germany 15Institute for Translational Neuroscience, University of Münster, Münster, Germany 16Center for Mind, Brain and Behavior, University of Marburg, Marburg, Germany 17Institute of Neuroscience and Medicine (INM-1), Research Center Jülich, Jülich, Germany 18Center for Human Genetics, University of Marburg, Marburg, Germany Center for Mind, Brain and Behavior, University of Marburg, Marburg, Germany 17Institute of Neuroscience and Medicine (INM-1), Research Center Jülich, Jülich, Germany 18Center for Human Genetics, University of Marburg, Marburg, Germany y g g y 17Institute of Neuroscience and Medicine (INM-1), Research Center Jülich, Jülich, Germany 18Center for Human Genetics University of Marburg Marburg Germany 19Max Planck School of Cognition, Leipzig, Germany 20 20Max-Planck-Institute of Psychiatry, Department Genes and Environment, Munich, Germany 21 21Medical Research Council Integrative Epidemiology Unit at the University of Bristol, University of Bristol, Bristol, UK 22Population Health Science, Bristol Medical School, University of Bristol, Bristol, UK 23NIHR Bristol Biomedical Research Centre, University Hospitals Bristol and Weston NHS Foundation Trust and University of Bristol. 24 24Social, Genetic & Developmental Psychiatry Centre, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Affiliations 1 (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation 25ESRC Centre for Society and Mental Health, King's College London, London, UK 26University of Exeter Medical School, University of Exeter, Exeter, UK 27Lothian Birth Cohorts, Department of Psychology, University of Edinburgh, Edinburgh, UK 28Alzheimer Scotland Dementia Research Centre, University of Edinburgh, Edinburgh, UK 29Neuroprogressive and Dementia Network, NHS Research Scotland, Scotland, UK 29Department of Psychiatry, University of Oxford, Oxford, UK Corresponding author* Andrew M McIntosh Division of Psychiatry University of Edinburgh Royal Edinburgh Hospital Edinburgh EH10 5HF +44 131 537 6000 andrew.mcintosh@ed.ac.uk 25ESRC Centre for Society and Mental Health, King's College London, London, UK 26 25ESRC Centre for Society and Mental Health, King's College London, London, UK 26University of Exeter Medical School, University of Exeter, Exeter, UK 27Lothian Birth Cohorts, Department of Psychology, University of Edinburgh, Edinburgh, UK 28Alzheimer Scotland Dementia Research Centre, University of Edinburgh, Edinburgh, UK 29Neuroprogressive and Dementia Network, NHS Research Scotland, Scotland, UK 29Department of Psychiatry, University of Oxford, Oxford, UK University of Exeter Medical School, University of Exeter, Exeter, UK thian Birth Cohorts, Department of Psychology, University of Edinburgh, Edinburgh, Corresponding author* Andrew M McIntosh Division of Psychiatry University of Edinburgh Royal Edinburgh Hospital Edinburgh EH10 5HF +44 131 537 6000 andrew.mcintosh@ed.ac.uk 2 2 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation 3 Key Points Question Is antidepressant exposure associated with differential whole blood DNA methylation? Question Is antidepressant exposure associated with differential whole blood DNA Findings In this methylome-wide association study of 16,536 adults across Scotland, antidepressant exposure was significantly associated with hypermethylation at CpGs mapping to KANK1 and DGUOK-AS1. A methylation profile score trained on this sample was significantly associated with antidepressant exposure (pooled [95%CI]=0.196 [0.105, 0.288], p < 1x10-4) in a meta-analysis of external datasets. Meaning Antidepressant exposure is associated with hypermethylation at KANK1 and DGUOK-AS1, which have roles in mitochondrial metabolism and neurite outgrowth. If replicated in future studies, targeting these genes could inform the design of more effective and better tolerated treatments for depression. 3 3 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Abstract Importance: Understanding antidepressant mechanisms could help design more effective and tolerated treatments. Objective: Identify DNA methylation (DNAm) changes associated with antidepressant exposure. Objective: Identify DNA methylation (DNAm) changes associated with antidepressant exposure. Design: Case-control methylome-wide association studies (MWAS) of antidepressant exposure were performed from blood samples collected between 2006-2011 in Generation Scotland (GS). The summary statistics were tested for enrichment in specific tissues, gene ontologies and an independent MWAS in the Netherlands Study of Depression and Anxiety (NESDA). A methylation profile score (MPS) was derived and tested for its association with antidepressant exposure in eight independent cohorts, alongside prospective data from GS. Setting: Cohorts; GS, NESDA, FTC, SHIP-Trend, FOR2107, LBC1936, MARS-UniDep, ALSPAC, E-Risk, and NTR. Participants: Participants with DNAm data and self-report/prescription derived antidepressant exposure. Main Outcome(s) and Measure(s): Whole-blood DNAm levels were assayed by the EPIC/450K Illumina array (9 studies, Nexposed = 661, Nunexposed= 9,575) alongside MBD-Seq in NESDA (Nexposed= 398, Nunexposed= 414). Antidepressant exposure was measured by self- report and/or antidepressant prescriptions. 4 Results: The self-report MWAS (N = 16,536, Nexposed = 1,508, mean age = 48, 59% female) and the prescription-derived MWAS (N = 7,951, Nexposed = 861, mean age = 47, 59% female), found hypermethylation at seven and four DNAm sites (p < 9.42x10-8), respectively. The top locus was cg26277237 (KANK1, pself-report= 9.3x10-13, pprescription = 6.1x10-3). The self-report MWAS found a differentially methylated region, mapping to DGUOK-AS1 (padj = 5.0x10-3) alongside significant enrichment for genes expressed in the amygdala, the “synaptic vesicle 4 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. d by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; s://doi.org/10.1101/2024.05.01.24306640 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation membrane” gene ontology and the top 1% of CpGs from the NESDA MWAS (OR = 1.39, p < 0.042). Objective: Identify DNA methylation (DNAm) changes associated with antidepressant exposure. The MPS was associated with antidepressant exposure in meta-analysed data from external cohorts (Nstudies= 9, N = 10,236, Nexposed = 661, 0.196, p < 1x10-4). membrane” gene ontology and the top 1% of CpGs from the NESDA MWAS (OR = 1.39, p < 0.042). The MPS was associated with antidepressant exposure in meta-analysed data from external cohorts (Nstudies= 9, N = 10,236, Nexposed = 661, 0.196, p < 1x10-4). Conclusions and Relevance: Antidepressant exposure is associated with changes in DNAm across different cohorts. Further investigation into these changes could inform on new targets for antidepressant treatments. 5 5 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprin this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. d by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; s://doi.org/10.1101/2024.05.01.24306640 Antidepressant Exposure and DNA Methylation Introduction Major Depressive Disorder (MDD) is predicted to become the leading cause of disability worldwide by 20301, partly due to the limitations of current treatments2. Although antidepressants are commonly prescribed effective treatments3, they prove to be ineffective in a high proportion of cases, with an estimated 40% of those presenting with MDD developing treatment-resistant depression4,5. Furthermore, many treatments are commonly accompanied by side effects, including weight changes, fatigue and sexual dysfunction2. There is a need for more effective and better-tolerated antidepressant treatments and to target existing treatments to those most likely to respond. Advances are hampered by poor mechanistic understanding of both MDD itself1,16,17 and how currently prescribed antidepressants lead to therapeutic effects9. The mechanism of currently prescribed antidepressants is incompletely understood. Initial theories surmised that their therapeutic effects were due to an increase in monoamine brain synaptic concentrations10. However, antidepressant treatment has a delayed onset for symptomatic improvement, which does not reflect the immediate effect on monoamine levels7. This casts doubt on the simple role of monoamines as a causal factor in MDD7–9, although other experimental paradigms continue to suggest their importance11. Another prominent theory of antidepressant action suggests that their therapeutic mechanism involves increasing synaptic remodelling12 and neuronal plasticity9,13. The evidence for the effect of antidepressants on DNA methylation (DNAm) is growing14,15. In vitro studies found that the antidepressant paroxetine interacted with DNA methyltransferase (DNMT), a key enzyme involved in DNAm16. Furthermore, studies of chronically stressed rodent models have found 6 6 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation that stress-induced DNAm and behavioural changes are reversed through both antidepressant treatment17 and DNMT inhibitors18. that stress-induced DNAm and behavioural changes are reversed through both antidepressant treatment17 and DNMT inhibitors18. DNAm, the addition of a methyl group at a cytosine-phosphate-guanine (CpG) site, regulates gene expression and impacts cellular function19,20. Anxiety (NESDA). Fifth, the relationship between time in treatment and DNAm at Figure 1: Schematic of study design. Generation Scotland Generation Scotland: The Scottish Family Health Study (GS) is a family-based cohort study (N ~ 24,000), investigating the genetic and environmental factors influencing health within Scotland (eMethods)27,28. Data collection including biological sampling occurred between February 2006 and March 2011. Written consent was provided before any assessment and/or sampling took place. Ethical approval was provided by the Tayside Research Ethics Committee (REC reference 05/S1401/89). Introduction (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation Anxiety (NESDA). Fifth, the relationship between time in treatment and DNAm at significant CpG sites was investigated. Finally, a methylation profile score (MPS) for self- report antidepressant exposure was trained in GS and tested for an association with antidepressant exposure in eight independent external datasets: Finn Twin Cohort (FTC), Study of Health in Pomerania (SHIP-Trend), Lothian Birth Cohort 1936 (LBC1936), FOR2107, NTR, Avon Longitudinal Study of Parents and Children (ALSPAC), Munich Antidepressant Response Study/Unipolar Depression Study (MARS-UniDep) and the Environmental Risk (E-Risk) Longitudinal Twin Study, alongside a prospective sample of GS: Stratifying Depression and Resilience Longitudinally (STRADL) (Figure 1). Figure 1: Schematic of study design. Anxiety (NESDA). Fifth, the relationship between time in treatment and DNAm at Introduction In 2022, Barbu et al.21 performed a methylome-wide association study (MWAS) of self-reported antidepressant exposure in a subset of participants in Generation Scotland (GS, N = 6,428) and the Netherlands Twin Register (NTR, N = 2,449)21, identifying altered DNAm near to genes involved in the innate immune response in those exposed to antidepressants21. As self-report measures may be unreliable due to volunteer recall bias, a poor understanding of the medication nosology, and non-disclosure22–24, Barbu et al.21 also performed an MWAS of antidepressant exposure based on recorded antidepressant prescriptions in the last 12 months. However, this assumes continuous treatment, potentially overestimating exposure due to general low adherence to antidepressant medication25. Calculation of active treatment periods from consecutive prescribing events provides a potentially more reliable identification of antidepressant exposure26. In our study, we build upon previous analyses by Barbu et al.21 by analysing a larger sample of GS (N = 16,536), and by estimating active treatment periods from prescribing records to identify those exposed to antidepressants at DNAm measurement. First, an MWAS was performed on both the self-report and prescription-derived measures of antidepressant exposure. Second, to assess the potential confounding by MDD, the MWAS analyses were restricted to MDD cases only. Third, functional follow-up analysis of differentially methylated CpG sites was performed. Fourth, we investigated the enrichment of top CpGs in GS and an independent MWAS conducted in the Netherlands Study of Depression and In our study, we build upon previous analyses by Barbu et al.21 by analysing a larger sample of GS (N = 16,536), and by estimating active treatment periods from prescribing records to identify those exposed to antidepressants at DNAm measurement. First, an MWAS was performed on both the self-report and prescription-derived measures of antidepressant exposure. Second, to assess the potential confounding by MDD, the MWAS analyses were restricted to MDD cases only. Third, functional follow-up analysis of differentially 7 7 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Methylation data DNAm was profiled from baseline blood samples using the Illumina MethylationEPIC array for 19,390 individuals. Methylation typing was performed in 4 distinct sets between 2017 and 2021. Sets 1, 2 and 4 included related individuals within and between each set while all individuals in set 3 were unrelated to each other and to individuals in set 1 (genetic 8 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation relationship matrix (GRM) threshold <0.05). Following quality control (QC) (eMethods), the sample sizes were Set 1: 5,087, Set 2: 459, Set 3: 4,450 and Set 4: 8,873 individuals. The sets were combined and dasen normalisation was performed across all individuals29. In total, 752,741 probes and 18,869 individuals passed QC. Beta-values were transformed to M- values using the ‘beta2M()’ function in the lumi R package30, and standardised using the ‘std- probe’ flag in the OSCA software31. relationship matrix (GRM) threshold <0.05). Following quality control (QC) (eMethods), the sample sizes were Set 1: 5,087, Set 2: 459, Set 3: 4,450 and Set 4: 8,873 individuals. The sets were combined and dasen normalisation was performed across all individuals29. In total, 752,741 probes and 18,869 individuals passed QC. Beta-values were transformed to M- values using the ‘beta2M()’ function in the lumi R package30, and standardised using the ‘std- probe’ flag in the OSCA software31. Methylome-wide association study The MWAS were performed using a Mixed-linear-model Omics-based Analysis (MOA) in the OSCA software31. To account for relatedness within GS, each phenotype was regressed on a GRM33 using the Best Linear Unbiased Prediction (BLUP) tool in GCTA software34. The residuals were entered into a MOA model, which included a methylation omics- relatedness matrix as a random effect and age, sex, AHRR probe (cg05575921) M-values to proxy for smoking status, and predicted monocyte and lymphocyte cell proportions as fixed effects. Statistical significance was assessed using the p-value threshold 9.42x10-8, as recommended for case-control MWAS analyses35. Effect sizes represent a per-1 standard- deviation increase in CpG methylation M-values. recommended for case-control MWAS analyses35. Effect sizes represent a per-1 standard- deviation increase in CpG methylation M-values. Differentially methylated regions (DMRs) were identified using the dmrff R package36, Differentially methylated regions (DMRs) were identified using the dmrff R package3 Differentially methylated regions (DMRs) were identified using the dmrff R package36, which performs an inverse-variance-weighted meta-analysis of MWAS beta and standard- error estimates per region, adjusting for estimate uncertainty and the correlational structure between probes. Candidate DMRs are identified as sets (>2) of CpGs <= 500bp apart with nominal significance (P < 0.05) and consistent effect direction. DMRs achieving Bonferroni- corrected p-value < 0.05 were considered statistically significant. Antidepressant exposure phenotypes Prescription-derived antidepressant exposure was measured using antidepressant prescriptions from Public Health Scotland (eMethods), according to the British National Formulary (BNF) paragraph code ‘040303’, which largely corresponds to Anatomical Therapeutic Chemical (ATC) subclass ‘N06A’ (eTable 1) (Nprescriptions = 174,454, Npeople= 7,544) (eTable 2, eFigures 1-8). After removing ambiguous prescriptions (Nprescriptions = 5,484, Npeople = 171) (eMethods, eTable 3), active treatment periods were defined by consecutive dispensing of antidepressant medications (eMethods, eFigures 9-11). Those who had their blood-draw appointment ≥ 7 days after treatment initiation or < 7 days after the end of a treatment period were defined as exposed (Nexposed = 861) (eFigure 12). Those with no antidepressant prescriptions on record were defined as unexposed (Nunexposed = 7,090). Self-reported antidepressant exposure was derived from questionnaires sent 1–2 weeks before venepuncture (eMethods, eTable 4, eFigure 13). Those who did/did not self-report antidepressant use were defined as exposed and unexposed respectively (Nexposed = 1,508, Nunexposed = 15,028). Out of 6,473 individuals with both self-report and prescription-derived 9 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation and Statistical Manual, version IV (SCID)32 (prescription-derived: Nexposed= 380, Nunexposed = 412, self-report: Nexposed= 766, Nunexposed = 1,502) (eMethods, eFigure 15). and Statistical Manual, version IV (SCID)32 (prescription-derived: Nexposed= 380, Nunexposed = 412, self-report: Nexposed= 766, Nunexposed = 1,502) (eMethods, eFigure 15). Functional annotation Gene-sets for both MWAS were collated by annotating the top 100 CpGs (by p-value) by the Infinium MethylationEPIC BeadChip database37. Hypergeometric tests, using ‘phyper()’, were used to assess the overlap of CpGs and gene-sets from both analyses. The background set consisted of CpGs and genes on or annotated to the EPIC array (eTable 5). The 10 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation ‘GENE2FUNC’ analysis in functional mapping and annotation web-tool (FUMA) was used to assess enrichment of both gene-sets across 54 specific tissues in the Genotype-Tissue Expression (GTEx)38,39 Project. Both gene-sets were tested for enrichment in synapse-related GO terms using the SynGo web tool40 and for enrichment in GO: Biological Processes gene- sets (20 < ngenes < 500) in the msigdbr database41, using the ‘gsameth()’ function from missMethyl R package42 (eTable 6). Results The demographic features of both antidepressant exposure phenotypes in the GS are shown in Table 1. Table 1: Demographics of antidepressant exposed and unexposed individuals using the prescription-derived and self-reported measures in Generation Scotland. M = Mean, SD = Standard Deviation. Table 1: Demographics of antidepressant exposed and unexposed individuals using the prescription-derived and self-reported measures in Generation Scotland. M = Mean, SD = Standard Deviation. Methylation profile score A least absolute shrinkage and selection operator (LASSO) penalised regression model was applied using ‘cv.biglasso()’ from the glmnet R package43 on the GS sample. First, the self- report phenotype was regressed against the GRM (using BLUP), age, sex, AHRR probe M- values as a proxy for smoking status, batch, and white blood cell (monocyte and lymphocyte) proportions. The residuals were used as the dependent variable and the standardised CpG sites on both EPIC and 450K Illumina arrays (N = 365,912) were included as independent variables. Ten-fold cross-validation was applied and the mixing parameter was set to 1. The non-zero weighted CpGs identified in the LASSO model were used to calculate a weighted- sum MPS in external datasets (FTC, SHIP-Trend, FOR2107, NTR, LBC1936, ALSPAC, MARS-UniDep and E-Risk, Ntotal = 9,578, Nexposed = 619) and a prospective sample of GS (STRADL, Ntotal = 658, Nexposed = 42) (eMethods). The association between antidepressant exposure and the MPS was assessed using; generalised linear mixed models, generalised linear models and generalised estimation equations, depending on the cohort’s population structure (i.e., twin studies vs unrelated participants) and DNAm pre-processing (eMethods, eTables 7-9). Age, sex, batch (where applicable), white blood cell proportions/counts and M- values at the AHRR probe were included as covariates. A random-effects meta-analysis using 11 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation DerSimonian-Laird estimator was performed to assess the overall association between t MPS and antidepressant exposure, using the meta R package44. Methylome-wide association studies self-report MWAS (Figure 2A, Table 2) and prescription-derived MWAS (Figure 2B, The self-report MWAS (Figure 2A, Table 2) and prescription-derived MWAS (Figure 2B, eTable 10) identified seven and four hypermethylated CpGs respectively, in those exposed to antidepressants (eFigure 16, eFigure 17). The effect estimates from all CpGs in both analyses were significantly correlated (R = 0.54, p < 2.2x10-16) (eFigure 18). Differentially methylated regions There were 719,506 candidate regions considered in the dmrff analysis. The self-report MWAS had one significant DMR ( = 0.096, padj = 4.98x10-3) (eTable 12), consisting of two CpGs (cg01964004 and cg15071067), which maps to deoxyguanosine kinase antisense RNA1 (DGUOK-AS1) (eFigure 21). The prescription-derived MWAS identified no significant DMRs (eTable 13). Table 2: Eight CpGs associated with self-reported and/or prescription-derived Table 2: Eight CpGs associated with self-reported and/or prescription-derived antidepressant use. The EWAS catalog was searched using the ewascatalog R package45 for other studies (n > 1000) which report a significant CpG-trait association, accessed on 17/03/2024. Figure 2: Methylome-wide association study of self-reported (A) and prescription-derived (B) antidepressant exposure. (C) The standardised effect sizes and 95% confidence-intervals for 12 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. d by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; s://doi.org/10.1101/2024.05.01.24306640 Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation associated CpGs (p < 9.42x10-8) for the full sample (dark-green) and MDD-only sample (light-green). In the MDD-only self-report MWAS, only cg08527546 exhibited a significant association with antidepressant exposure (β = 0.050, p = 3.57x10-8); no CpGs were significantly associated in the MDD-only prescription-derived MWAS (eTable 11, eFigure 19). For both phenotypes, there was a significant correlation between CpG effect estimates in the full and MDD-only analyses (Rself-report = 0.57, pself-report < 2.2x10-16, Rprescription= 0.43, pprescription< 2.2x10-16) (eFigure 20). Notably, restricting the analyses to MDD cases did not attenuate the effect sizes of the significant CpGs (Figure 2C). 2.2x10-16) (eFigure 20). Notably, restricting the analyses to MDD cases did not attenuate the effect sizes of the significant CpGs (Figure 2C). Methylation differences by time in treatment To investigate the relationship of DNAm at significantly associated CpGs (n = 8) with the length of antidepressant exposure, a Spearman correlation test was performed between the DNAm levels and time in current treatment for those within a treatment period (N = 863). Two probes, cg15071067 (DGUOK-AS1) and cg26277237 (KANK1), showed a significant correlation between methylation and time in treatment (cg15071067:= 0.085, p = 0.012, cg26277237: = 0.087, p = 0.011) (eFigures 31-33, eTable 26), with the same direction of effect found in the MWAS. Enrichment analysis: Netherlands Study of Depression and Anxiety An MWAS of self-reported antidepressant exposure was performed in the NESDA cohort46 (N = 812, Nexposed = 398), with all participants having a recent (~ 6 months) MDD DSM-IV diagnosis (eResults). The DNAm assays were obtained from whole blood samples using methyl-CG binding domain sequencing (MBD-Seq)47–49 (eResults). Enrichment tests were performed to assess the overlap of top findings from GS and NESDA, using the shiftR R package50. For both MWAS’, CpGs were filtered to those which showed a concordant direction of effect and three thresholds (0.1, 0.5 and 1%) were used to define top (by p-value) findings. Results suggested a small (OR: 1.39) but significant (P < 0.042) enrichment between the top 1% of results from both MWAS’. Functional Annotation The most significant 100 CpGs from the self-report and prescription-derived MWAS were annotated to 77 and 83 genes respectively (eTables 14-17). There was a significant overlap between both the CpG-sets (Noverlap = 17, p = 1.95x10-48, eFigure 22) and the gene-sets (Noverlap = 16, p =1.3x10-25, eFigure 23). The self-report gene-set was significantly enriched for the genes expressed in the amygdala (eTable 18, eFigure 24), whilst the prescription- derived gene-set had no significant enrichment (eTable 19, eFigure 25). There was significant 13 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation enrichment of the self-report gene-set in GO:0008021;” synaptic vesicle membrane” (pFDR = 0.030, SF26-28, ST20-23). There was no significant enrichment for any GO:Biological Processes pathways tested for either gene set (eFigure 29-30, eTable 24-25) Methylation Profile Score 14 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. d by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; s://doi.org/10.1101/2024.05.01.24306640 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation There were 212 CpGs identified by the LASSO regression model (eFigures 34-36, eTable 27), used to calculate the MPS in external cohorts (Figure 3A, eFigures 37-46). All cohorts bar one (NTR) showed a positive relationship between antidepressant MPS and antidepressant exposure (FTC = 0.156, SHIP-Trend = 0.134, STRADL = 0.149, LBC1936 = 0.228, FOR2107 = 0.349, MARS= 0.263, ALSPAC= 0.170, ERISK= 0.342, NTR= -0.031) (eFigure 47, eTable 28). Nagelkerke’s pseudo R2 estimates ranged from 1.11x10-3 (NTR) to 0.03 (LBC1936) (Figure 3B). The random-effects meta-analysis found a significant association between antidepressant exposure and the MPS (pooled [95%CI]: 0.196 [0.105, 0.288], p < 1x10-4), with low heterogeneity between studies (I2 [95%CI] = 0% [0, 64.8%]) (eTable 29). There were 212 CpGs identified by the LASSO regression model (eFigures 34-36, eTable 27), used to calculate the MPS in external cohorts (Figure 3A, eFigures 37-46). All cohorts bar one (NTR) showed a positive relationship between antidepressant MPS and antidepressant exposure (FTC = 0.156, SHIP-Trend = 0.134, STRADL = 0.149, LBC1936 = 0.228, FOR2107 = 0.349, MARS= 0.263, ALSPAC= 0.170, ERISK= 0.342, NTR= -0.031) (eFigure 47, eTable 28). Figure 3: Antidepressant exposure ~ MPS in external cohorts. A) The sample sizes of each dataset, B) Nagelkerke’s pseudo R2, C) The effect sizes and confidence intervals of MPS ~ antidepressant exposure analysis in each cohort. Square size = study weight, diamond = meta-analysed effect. Discussion This study presents the largest investigation of the impact of antidepressant exposure on the methylome51. The results from self-report or prescription-derived antidepressant exposure were broadly consistent, corroborating previous findings26. There was evidence of hypermethylation at eight CpGs and a region on Chromosome 2 (BP: 74196550-74196572) in those exposed to antidepressants. The CpG with the highest significance and the largest effect size, cg26277237, mapped to KN motif and ankyrin repeat domains 1 (KANK1), was previously reported by Barbu et al.21 on a smaller sample of GS. The DMR analysis indicated antidepressant exposure is also significantly associated with hypermethylation near DGUOK- AS1, a long non-coding RNA (lncRNA). KANK1 facilitates the formation of the actin 15 15 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. d by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; s://doi.org/10.1101/2024.05.01.24306640 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation cytoskeleton and has an active role in neurite outgrowth and neurodevelopment52. A meta- analysis of copy-number variant association studies found a significant duplication in KANK1 in those with five different neurodevelopmental disorders, including MDD53. DGUOK-AS1 has an inhibitory role on the expression of a nearby gene DGUOK54, which encodes a mitochondrial enzyme involved in the production of mitochondrial DNA54, and has previously been implicated as a risk gene in schizophrenia55 and Alzheimer disease56. A recent review reported evidence that antidepressants do influence mitochondrial function, although the effects are heterogeneous between different types of antidepressants, independent of their current classification57,58. Seven of the CpGs significantly hypermethylated with antidepressant use have been reported previously to also be significantly hypermethylated with incident and/or prevalent type 2 diabetes (T2D)59 in GS. Discussion Previous epidemiological studies have indicated that antidepressant use leads to an increased risk of T2D onset in a time- and dose-dependent manner60,61. Future prospective and longitudinal research into the link between antidepressant use, DNAm and T2D, alongside the use of other independent datasets is required. The performance of the GS-trained MPS in discriminating antidepressant exposure across eight external datasets, demonstrates that this may be a generalizable biomarker indicative of antidepressant exposure and adds to a growing set of MPS that could potentially provide clinically relevant phenotypic information62–65. Accurate estimation of this exposure history could be highly valuable for epidemiological studies where prescribing data may not be available. Taken in combination with MPS for other risk factors, an MPS for antidepressant exposure may help provide a robust characterisation of an individual’s medical history. 16 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. d by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; s://doi.org/10.1101/2024.05.01.24306640 Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation There are several strengths of this study. The comparison of self-report and prescription- derived measures is valuable, as the former is often cheaper and easier to obtain in large-scale cohort studies25. Furthermore, the MDD-only analysis indicates that the hypermethylation associated with antidepressant use is not driven by MDD indication. Additionally, the significant association of an MPS trained in GS with antidepressant exposure in external datasets, and the significant enrichment with an independent MWAS consolidates our findings. This study has various limitations. Both measures of antidepressant exposure do not discriminate between antidepressant drugs, classes, or dosages. Munich Antidepressant Response Study / UniPolar Depression Study (MARS-UniDe We would like to thank all contributors to the research project including physicians, psychologists, study nurses, researchers and research assistants, and of course patients of the hospital of the Max Planck Institute of Psychiatry in Munich and psychiatric hospitals in Augsburg and Ingolstadt. Avon Longitudinal Study of Parents and Children (ALSPAC) We thank all the families who took part in this study, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. Discussion However, we anticipate the opportunity to investigate more medication-specific effects on the methylome using prescription-linkage data as biobanks increase in size. Additionally, all the cohorts used primarily consist of European ancestry. It is paramount that this analysis is conducted in non- European ancestral groups to further verify our findings and disentangle any ancestry-specific effects66–68. Finally, by design, this epidemiological study cannot directly address causality between antidepressant exposure and DNAm. The integration of DNAm analysis into randomised controlled trials of antidepressants is important to establish the exact nature of the association and to inform potential new targets for antidepressant therapy. This study indicates that antidepressant exposure is associated with hypermethylation at DGUOK-AS1 and KANK1, which have roles in mitochondrial metabolism and neurite outgrowth respectively. Future research should include more cohorts of non-European ancestry, alongside the incorporation of DNAm in randomised trials of antidepressants to further consolidate findings and establish causality. If replicated, targeting of these genes could inform the design of more effective and better tolerated treatments for depression. 17 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint A id E d DNA M h l i . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation FOR2107 We are deeply indebted to all study participants and staff. A list of acknowledgments can be found here: www.for2107.de/acknowledgements. E-risk Longitudinal Twin Study (E-risk) We are grateful to the E-Risk study mothers and fathers, the twins, and the twins’ teachers for their participation. Our thanks to Professors Terrie Moffitt and Avshalom Caspi, the founders of the E-Risk study, and to the E-Risk team for their dedication, hard work, and insights. Generation Scotland (GS) & Stratifying Resilience and Depression Longitudinally (STRADL) We thank the participants in Generation Scotland for making this research possible. We also acknowledge the team at Generation Scotland for collecting and preparing the data for analyses. Netherlands Twin Register (NTR) We warmly thank all twin families of the Netherlands Twin Register who make this research possible. Acknowledgments This work has made use of the resources provided by the Edinburgh Compute and Data Facility (ECDF, 2024) (http://www.ecdf.ed.ac.uk/). Funding ED was supported by the UK Research and Innovation (Grant No. EP/S02431X/1), UK Research and Innovation Centre for Doctoral Training in Biomedical AI at the University of Edinburgh, School of Informatics. 18 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation This work was supported by the United Kingdom Research and Innovation (grant EP/S02431X/1), UKRI Centre for Doctoral Training in Biomedical AI at the University of Edinburgh, School of Informatics. For the purpose of open access, the author has applied a creative commons attribution (CC BY) licence to any author accepted manuscript version arising. AMM is also supported by a UK Research and Innovation award (Grant No. MR/W014386/1) and by European Union Horizon 2020 funding (Grant No. 847776). Finnish Twin Cohort (FTC) Phenotype and genotype data collection in FT12 and FT16 studies of the Finnish twin cohort has been supported by the Wellcome Trust Sanger Institute, the Broad Institute, ENGAGE – European Network for Genetic and Genomic Epidemiology, FP7-HEALTH-F4-2007, grant agreement number 201413, National Institute of Alcohol Abuse and Alcoholism (grants AA- 12502, AA-00145, and AA-09203 to R J Rose; AA15416 and K02AA018755 to D M Dick; R01AA015416 to J Salvatore) and the Academy of Finland (grants 100499, 205585, 118555, 141054, 264146, 308248, 265240, 263278 to J Kaprio, 328685, 307339, 297908 and 251316 to M Ollikainen, and Centre of Excellence in Complex Disease Genetics grants 312073, 336823, and 352792 to J Kaprio), NIH/NHLBI (grant HL104125 to X Wang), and Sigrid Juselius Foundation to M Ollikainen and J Kaprio. GS & STRADL This work was supported by 3 Wellcome Trust grants (220857/Z/20/Z, 226770/Z/22/Z and 104036/Z/14/Z [AMM]). Funding from the Biotechnology and Biological Sciences Research Council and Medical Research Council (Grant Nos. MR/X003434/1 and MR/W014386/1) and the European Union (Grant agreement 847776) is also gratefully acknowledged. The Psychiatric Genomics Consortium (including AMM) has received major funding from the U.S. National Institute of Mental Health (Grant No. 5 U01MH109528-03). Study of Health in Pomerania (SHIP-Trend) SHIP is part of the Community Medicine Research net of the University of Greifswald which is funded by the Federal Ministry of Education and Research (01ZZ9603, 01ZZ0103, and 01ZZ0403), the Ministry of Cultural Affairs and the Social Ministry of the Federal State of Mecklenburg-West Pomerania. DNA methylation data have been supported by the DZHK (grant 81X3400104). The University of Greifswald is a member of the Caché Campus program of the InterSystems GmbH. MARS-UniDep The MARS cohort was sponsored by the Max Planck Society. The UniDep cohort was funded by the Bavarian Ministry of Commerce and by the Federal Ministry of Education and Research in the framework of the National Genome Research Network, Foerderkennzeichen 01GS0481 and the Bavarian Ministry of Commerce. DNA methylation analysis of a subset of both cohorts was financed by ERA-NET NEURON. NTR This work was supported by the Royal Dutch Academy for Arts and Science (KNAW) Academy Professor Award (PAH/6635) to DIB; the Netherlands Organization for Scientific Research (NWO 480-15-001/674) and Biobanking and Biomolecular Resources Research Infrastructure (BBMRI-NL: 184.021.007; 184.033.111). Lothian Birth Cohort 1936 (LBC1936) The LBC1936 is supported by the Biotechnology and Biological Sciences Research Council, and the Economic and Social Research Council [BB/W008793/1] (which supports SEH), Age UK (Disconnected Mind project), the Medical Research Council [G0701120, G1001245, MR/M013111/1, MR/R024065/1], the Milton Damerel Trust, and the University of Edinburgh. SRC is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (221890/Z/20/Z). Methylation typing of was supported by Centre for Cognitive Ageing and Cognitive Epidemiology (Pilot Fund award), Age UK, The Wellcome Trust Institutional Strategic Support Fund, The University of Edinburgh, and The University of Queensland. ALSPAC The UK Medical Research Council and Wellcome (Grant Ref: 217065/Z/19/Z) and the University of Bristol provide core support for ALSPAC. ASFK is funded by a Wellcome Early Career Award (Grant ref: 227063/Z/23/Z). A comprehensive list of grants funding is available on the ALSPAC website (http://www.bristol.ac.uk/alspac/external/documents/grant-acknowledgements.pdf). This publication is the work of the authors and ED, PY & ASFK will serve as guarantors for the ALSPAC contents of this paper. PY's work is supported by the National Institute for Health and Care Research Bristol Biomedical Research Centre and the Medical Research Council Integrative Epidemiology Unit at the University of Bristol (MC_UU_00032/4, MC UU 00032/6) The views expressed are those of the authors and not necessarily those of MC_UU_00032/6). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. FOR2107 The German multicenter consortium “Neurobiology of Affective Disorders. A translational perspective on brain structure and function” is funded by the German Research Foundation (Research Unit FOR2107). Principal investigators are Tilo Kircher (speaker FOR2107, DFG grant numbers KI588/14-1, KI588/14-2, KI588/20-1, KI588/22-1, KI 588/15-1, KI 588/17-1), Udo Dannlowski (co-speaker FOR2107; DA1151/5-1, DA1151/5-2, DA1151/6-1), Axel Krug (KR3822/5-1, KR3822/7-2), Igor Nenadic (NE2254/1-2, NE2254/2-1, NE2254/3-1, NE2254/4-1), Carsten Konrad (KO4291/3-1), Marcella Rietschel (RI 908/11-1, RI 908/11-2), 19 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation Markus Nöthen (NO 246/10-1, NO 246/10-2), Stephanie Witt (WI 3439/3-1, WI 3439/3-2). Tilo Kircher received unrestricted educational grants from Servier, Janssen, Recordati, Aristo, Otsuka, neuraxpharm. Conflicts of Interest REM is an advisor to the Epigenetic Clock Development Foundation and Optima Partners. HJG. has received travel grants and speaker honoraria from Fresenius Medical Care, Neuraxpharm, Servier, and Janssen Cilag, as well as research funding from Fresenius Medical Care. H.J.G. had personal contracts approved by the university administration for speaker honoraria and one IIT with Fresenius Medical Care. K received unrestricted educational grants from Servier, Janssen, Recordati, Aristo, Ots All other authors report no biomedical financial interests or potential conflicts of interest. All other authors report no biomedical financial interests or potential conflicts of interest. E-Risk The E-Risk Study is funded by grants from the UK Medical Research Council [G1002190; MR/X010791/1]. Additional support was provided by the US National Institute of Child 20 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint d d h l . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Health and Human Development [HD077482] and the Jacobs Foundation. Chloe C. Y. Wong was supported by the National Institute for Health and Care Research (NIHR) Maudsley Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London [NIHR203318]. Helen L. Fisher was supported by the Economic and Social Research Council (ESRC) Centre for Society and Mental Health at King’s College London [ES/S012567/1]. The views expressed are those of the authors and not necessarily those of the NIHR, the Department of Health and Social Care, the ESRC, or King’s College London. Data & code availability Summary statistics from the MWAS analyses are being submitted for open access on the EWAS catalog. All code and analysis scripts are available on GitHub (https://github.com/Elladavyson). 21 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint A id E d DNA M h l i . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation References 1. GBD 2017 Dis Injury Incidence Pr, James SLG, Abate D, et al. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. 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Nat Commun. 2020;11(1):6114. doi:10.1038/s41467-020-19791-w 27 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Antidepressant Exposure and DNA Methylation (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation 28 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Table 1 \| Demographics of antidepressant exposed and unexposed individuals using the prescription-derived and self-reported antidepressant exposure phenotypes in \| g p p p p g prescription-derived and self-reported antidepressant exposure phenotypes in Generation Scotland. M = Mean, SD = Standard Deviation. Prescription-derived phenotype Self-report phenotype Unexposed Exposed Unexposed Exposed N 7090 861 15028 1508 Age: M (SD) 47.6 (15.2) 51.1 (12.7) 46.6 (14.9) 50.2 (12.4) BMI: M (SD) 26.4 (4.78) 28.5 (6.11) 26.4 (5.01) 28.2 (6.05) Sex Female: N (%) 3746 (53) 631 (73) 8552 (57) 1151 (76) Male: N (%) 3342 (47) 230 (27) 6464 (43) 354 (23) Smoking behaviours Current: N (%) 963 (14) 214 (25) 2448 (16) 402 (27) Former: N (%) 1942 (27) 273 (32) 4183 (28) 481 (32) Never: N (%) 3913 (55) 328 (38) 8114 (54) 576 (38) Pack years: M (SD) 6.3 (13.1) 12 (17.1) 6.55 (13.3) 11.6 (16.9) Major Depressive Disorder (Lifetime status) Cases: N (%) 414 (5.8) 382 (44) 1502 (10) 766 (51) Controls: N 6514 (92) 373 (43) 12704 (85) 556 (37) Generation Scotland. M = Mean, SD = Standard Deviation. 1 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation (%) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation (%) 2 Table 2 \| Eight CpGs associated with self-reported and/or prescription-derived antidepressant use. The EWAS catalog was searched using the ewascatalog R package45 for other studies (n > 1000) which report a significant CpG-trait association, accessed on Table 2 \| Eight CpGs associated with self-reported and/or prescription-derived 2 2 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. d by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; s://doi.org/10.1101/2024.05.01.24306640 Antidepressant Exposure and DNA Methylation . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 2, 2024. ; https://doi.org/10.1101/2024.05.01.24306640 doi: medRxiv preprint Antidepressant Exposure and DNA Methylation Antidepressant Exposure and DNA Methylation 17/03/2024. 17/03/2024. 3 3 verity er’s erular 62, iabete Antidepressant Exposure and DNA Methylation SESR PSR PD SEPD PPD Other traits associated wit catalog) 45 0.0033 9.3x10-13 0.027 0.0044 6.1x10-10 C-reactive protein levels62 Chronic kidney disease (b 2 diabetes (basic model)59 glomerular filtration rate6 03:23:00 0.0036 2.35x10-10 0.034 0.0048 7x10-13 Alcohol consumption70, C (basic model)59, Sex71, Ag disease Braak stage73, Est filtration rate69, Schizophr 0.0039 6.46x10-8 0.027 0.0052 3.0x10-7 C-reactive protein levels ( Chronic pain (basic mode )59 62, Antidepressant Exposure and DNA Methylation (basic model)59, Sex71, Age72 0.0028 3.59x10-11 0.019 0.0037 5.17x10-7 Birthweight72, C-reactive protein level model)62, Gestational age, Chronic pain model)59, Type 2 diabetes (basic mode Age72 0.0032 9.84x10-9 0.020 0.0042 3.3x10-6 C-reactive protein levels (basic model) Incident chronic pain (basic model)59, Incident type 2 diabetes (basic & fully adjusted model)59, Chronic pain (self- report)59, Type 2 diabetes (basic model 0.0036 1.3X10-6 0.026 0.0047 3.0x10-8 C-reactive protein levels (basic model) Incident type 2 diabetes (basic & fully adjusted model)59, Chronic pain (basic model)59, Ischemic heart disease (basic model)59, Type 2 diabetes (basic mode ted y model)5901/05/2024 03:23:00 4 0.0034 3.7x10-7 C-reactive protein levels (basic model) Chronic kidney disease (basic model)59 2 diabetes (basic & fully adjusted mode 9 0.0035 3.85x10-8 C-reactive protein levels (basic model) Incident type 2 diabetes (basic model)5 Chronic kidney disease (basic & fully adjusted model)59, Chronic pain (basic 59, Type 2 diabetes (basic & fully adjus model)59
https://openalex.org/W2901666931	https://www.matec-conferences.org/10.1051/matecconf/201823201029/pdf	English	null	Construction of Information Integration System for RFID-based Urban Intelligent Building	MATEC web of conferences	2,018	cc-by	3,147	1 Introduction With the deepening of the sustainable development concept, intelligent building has become the goal of the urban construction industry. Intelligent building requires the integration of the whole life cycle supply chain in the construction process, but because of the lack of core automation technology, the data of the members of the supply chain are difficult to be integrated and unanimous, and thus the difficulty of integration is increased. At present, RFID (Radio Frequency Identification) has been widely used in many fields of automation management, such as industry and commerce. However, RFID is rarely used in the construction industry. The main reason is that the environment of the construction site is bad. Especially the information integration system of the RFID-based urban intelligent building does not have a complete theoretical system or implementation standard, which is unable to improve some management problems in the transportation and storage of urban intelligent building components, project progress control, and the information transmission in various stages of the construction life cycle. Therefore, such management problems have become key factors restricting the sustainable development of urban intelligent buildings. In order to solve these problems, we need to make a detailed research on the present situation of urban intelligent building in China, the application target, SWOT analysis and construction of the information integration system of RFID-based urban intelligent building. Through the research, a set of reasonable model framework is formed as well as relevant key technology and construction mechanism of the information integration system of RFID-based urban *Corresponding author: 1176184893@qq.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Construction of Information Integration System for RFID-based Urban Intelligent Building Wen HUANG Wuhan Business University, 430050Wuhan, China Wen HUANG Abstract.Under the background of sustainable development, urban intelligent building is the inevitable direction of building development. This paper has analyzed the current situation of urban intelligent building in China, and expounded the application target of urban intelligent building based on Radio Frequency Identification (RFID) in the perspective of sustainable development.In the meantime,this paper has also made a profound analysis of the SWOT of RFID-based urban intelligent building, and puts forward a model framework, relevant key technology and construction mechanism for constructiment, thus improving the construction efficiency of the intelligent building for controlling the the component information more accurately, helping to optimize the industrial structure and improve the supervision ability of the intelligent building, and ultimately finding the effective way to realize the value of the sustainable development of the urban intelligent building. intelligent building, and the corresponding standards are also formed. MATEC Web of Conferences 232, 01029 (2018) EITCE 2018 MATEC Web of Conferences 232, 01029 (2018) EITCE 2018 https://doi.org/10.1051/matecconf/201823201029 @qq EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 icenses/by/4.0/). 3 Application goal of RFID-based urban development from the perspective of sustainable development Architecture provides a relatively isolated space from nature. After the use of building technology, especially after the combination of high technology and architecture, it is bound to have new requirements for this space, i.e. the application goal of intelligent building technology must conform to people's needs of life and work, and the internal and external environment of the building should be a sustainable development space, and can play a certain role in disaster prevention. The main functions of the intelligent building include the comfortability of the user, the flexibility of the re- organization, the adaptability of the engineering, the energy saving of the system, the sustainability of the environmental protection, and the ability to greatly improve the handling of emergencies. At present, a very important goal of intelligent building is to save energy. The building energy saving mentioned in the white paper on China's Energy Status and Policy is one of the ten key points. Intelligent buildings have some close relationship with the following energy-saving measures: improving the energy saving of power and the efficiency of energy system, accelerating the application of high efficiency electrical appliances, green lighting, and so on. Weakness(W): (1) Because of industrial characteristics, architectural engineering is lack of RFID cross-domain motors, information, materials and professional personnel in other fields, resulting in the limitation on cross-domain RFID-related software and hardware facility and equipment research and development. (2) It is not easy to promote the development and application of industrial innovative technology by the non-governmental sector due to the lack of research and development functions and incentives. (3) Lack of relevant regulations, norms and standards on RFID in China results in the problem of impelling enforcement. Opportunity (O): (1) Internationally, the related applications and research of RFID are actively promoting in various industries, resulting in a trend, and China's construction industry needs to respond actively. (2) There are many kinds and large quantities of materials needed in the urban intelligent building industry. RFID has been gradually developed in material and logistics management and control, and can be promoted to the vast building material market. (3) In recent years, other domestic industries have actively invested in the application of RFID and related software and hardware manufacturing and research, and much attention will be paid to the promotion and development of RFID in the future. (4) The application of RFID in Wal-Mart has been initially successful. 4 SWOT analysis of urban intelligent building based on RFID 4.1 SWOT analysis Strength(S): (1) Colleges and universities and industry boast some higher- education civil engineering technical personnel who are engaged in research and development and practice work, and relevant material laboratories where researchers can be engaged in the experimental research of the RFID urban intelligent building industry application. (2) In addition to special construction projects, Chinese people are mature and experienced in the process of planning, design and construction of the general construction projects, and the upstream and downstream industries are quite complete. (3) Government departments actively promote the application of RFID in the public fields, and establish RFID promotion offices. 2 Present situation of urban intelligent building in China China's intelligent building has been developing for more than 10 years since it started in the 1990s. People were unfamiliar with intelligent buildings. Now, the intelligent system, like fire fighting and hydro power, is an indispensable thought change in large buildings, commercial buildings, all kinds of public buildings and even residential areas. On this basis, the intelligent building gradually goes from the simple emphasis on intelligence and blind control technology towards the expected goal of strengthening intelligent technology and improving building function based on practical needs. At present, the development of intelligent building in China (including western cities) has entered into a relatively mature stage of concept, market and technology. There are more than 2000 integrators in the field of intelligent building in China, and the total amount of intelligent engineering accounts for almost half of the world. But higher-level intelligent buildings are mostly concentrated in big public buildings (including office buildings) and commercial buildings; the intelligent degree of residential buildings is low, and these buildings have not reached the full range of popularization in intelligence. Especially in the process of constructing intelligent buildings, the standard is too high, which results in unbalanced development of regional structure and scale in China. For example, MATEC Web of Conferences 232, 01029 (2018) MATEC Web of Conferences 232, 01029 (2018) EITCE 2018 https://doi.org/10.1051/matecconf/201823201029 Beijing, Shanghai, Guangzhou, Shenzhen, Tianjin and Chongqing have taken the lead in entering the intelligent building, and other cities, such as Nanjing, Hangzhou, Shenyang, and Wuhan, are also advancing to the intelligent building. The western areas are late in the development of intelligent building in comparison with the developed areas in China. However, although the west is lagging behind, its starting point is high. Therefore, Chengdu, Chongqing and Xi'an are becoming the centers of higher education in China. At present, most of the construction intelligent projects in Shaanxi and Sichuan are undertaken by local integrators, and extend their business scope to adjacent areas of Sichuan and other provinces and cities in Northwest China. Building intelligent projects in Yinchuan, Xining and Guangxi are mostly contracted by external integrated companies. energy saving control on air conditioning, power and lighting. 3 Application goal of RFID-based urban development from the perspective of sustainable development With the gradual expansion of the amount of use, the low price RFID label will be available. It is foreseeable that it will be widely used in all walks of life to create additional value of goods and enhance the competitiveness of various industries. The above energy-saving measures are manifested in two aspects: one is power and air conditioning, the other is lighting.Using the intelligent system to transform the air conditioning and power is not only improving the hardware itself, but also the operation mode. The operation mode in accordance with the energy saving requirement is actually managed through the form of software, which is embodied in the automation system of the building. And there are many ways to achieve energy saving in lighting, such as the efficient and reasonable mode of energy saving according to its own light source, which includes different environmental conditions, different control equipment, e.g. “intelligent lighting”. A building automation system with perfect performance can achieve the goal of saving 20%-30% energy after Threat (T): (1) At present, the application of RFID has not reached scale economy. The high cost of its labels, software and hardware has impeded its development. (2) There are many related construction industries in China, but they are relatively small and have low willingness to invest in RFID. (3) Foreign 2 https://doi.org/10.1051/matecconf/201823201029 MATEC Web of Conferences 232, 01029 (2018) MATEC Web of Conferences 232, 01029 (2018) EITCE 2018 RFID technology has long been widely used in various industries for many years. The introduction and development of RFID technology in China are relatively later than those in other countries, many technology patents are attained only in foreign countries, and the domestic development is restricted. professional talents in other fields such as motors, information, and materials, the RFID-based urban intelligent building research should call for special talents in other fields to promote a link among industries, academic units and research institutions, integrate experts and scholars in different fields and domestic manufacturer resources, cooperate and develop the application and soft and hard equipment research of RFID in building industry, reduce the cost of related objects, and promote the development of the domestic urban intelligent building industry. (2) The government has listed the application of RFID as a priority promotion plan, but there is lack of relevant regulations, norms and standards. 4.2 Cross-strategy analysis of SWOT SO strategy: (1) On the one hand, relevant universities and civil engineering industry can integrate domestic talents and equipment, lead and promote the development and application of RFID in the urban intelligent building industry, helping to improve the efficiency of intelligent building engineering and reduce the cost of the related intelligent building. On the other hand, the cooperation with other RFID-related electronic industries in China should be taken into consideration to improve people’s willingness to invest in the construction industry. (2) The government has listed the relevant application of RFID as a priority promotion plan and established the promotion office. Through the research results of urban intelligent building based on RFID, we can develop and work out the relevant standards of RFID in the urban intelligent building industry; through Wal-Mart’s successful application experience and achievement in RFID, we can improve the investment in the urban intelligent building industry, finally achieving policy promotion, standard formulation, technology development and promotion of RFID application in urban intelligent building industry. 5 Construction RFID-based urban intelligent building information integration system from the perspective of sustainable development According to the above analyses, in order to achieve the main application goal of RFID-based urban intelligent building, it is necessary to integrate all the equipment in the intelligent building, so it is of practical significance to construct the information integration system of the RFID-based urban intelligent building. In the life cycle of urban intelligent building, RFID identification technology is introduced, and an effective storage method is set up, i.e. setting up a RFID Tag on each component, so that the intelligent building components have the ability to automatically provide information, and effectively integrate the information needs of each stage, so as to complete the construction of the RFID- based intelligent building demonstration house; and in the process of practical application, the correctness of the information link and the updating instantaneity of the component information are evaluated as the foundation for reanalyzing the construction safety when the needs of use change in the future, in order to more accurately control the information of the components, promote the construction efficiency of the intelligent building, facilitate the optimization of the intelligent building industry structure, and improve supervision ability. ST strategy: (1) Taking advantage of the domestic strength and the RFID-based urban intelligent building research results, we can integrate experts and scholars in different fields and domestic manufacturer resources, make co-operations on the application and soft and hard equipment research of RFID in the urban intelligent building industry, reduces the related cost, and promotes the development of the domestic urban intelligent construction industry. (2) The related innovative applications are available in China though many patents appear in foreign countries. Therefore, we can first list the research results of RFID-based urban intelligent building as a priority promotion plan through the application of RE-ID, then promote, guide and promote all these research results, and finally improve people’s willingness of the domestic urban intelligent building. WO strategy: (1) Through the research results of the application of RFID in intelligent building industry, relevant regulations, manuals and standards are formulated, and the successful application of RFID in Wal-Mart will be a major incentive to attract domestic investment in urban intelligent building industry. (2) The cross-domain integration among various fields is bound to become a trend, and so interdisciplinary professional talents should be integrated by RFID-based urban intelligent building, cooperate and develop the application and soft and hard equipment research of RFID in urban intelligent building industry. 3 Application goal of RFID-based urban development from the perspective of sustainable development Therefore, it is necessary to formulate the relevant regulations, norms and standards through the research results of the RFID-based urban intelligent building, and use the research results as an example of the application of RFID in the urban intelligent building industry to advocate, guide and promote all these research results, finally increase the investment willingness of domestic urban intelligent building industry. 5 Construction RFID-based urban intelligent building information integration system from the perspective of sustainable development The key technologies involved in this system are as follows: Production: After the designs are confirmed, the component manufacturers can purchase materials and produce components according to the design requirements. After the components are produced, the RFID labels can be attached to them before consignment. The basic information on the required designs can be obtained by using the material management function of the RFID label, which can replace the traditional paper WT strategy: (1) Because the urban intelligent building industry lacks RFID-based cross-field 3 3 MATEC Web of Conferences 232, 01029 (2018) EITCE 2018 https://doi.org/10.1051/matecconf/201823201029 the project, and the value of sustainable development of urban intelligent buildings can be truly realized. record method used by the original manufacturers when materials are fed or discharged, improving the efficiency of the material management and control, and saving the manpower needed for management and time. References 1. Z.W. Li. Computer Aided Design and Intelligent Building, 9,78(2011). 2. P.P. Wen . Enterprise Economy, 9,35(2012) . 2. P.P. Wen . Enterprise Economy, 9,35(2012) . 3. N. Wang. Modern Architecture Electric, 10, 1 (2011). 3. N. Wang. Modern Architecture Electric, 10, 1 (2011). 4. L. C.Wang ，Y. C. Lin ，P. H.Lin. Advanced Engineering Informatics, 4, 377(2011). 4. L. C.Wang ，Y. C. Lin ，P. H.Lin. Advanced Engineering Informatics, 4, 377(2011). 5. J. Patrick，I. L. Sweeney. Wiley Publishing Inc， 205(2012)． 5. J. Patrick，I. L. Sweeney. Wiley Publishing Inc， 205(2012)． 6. B.Zhang, Y. H.Yao. Value Engineering, 113(2014). 6. B.Zhang, Y. H.Yao. Value Engineering, 113(2014). 7. Q. Zeng, Y. Ouyang. China Economic Publishing House, 165(2013). 8. F. Nakib ．Conference on Technology & Sustainability in the Built Environment, 479(2012). The construction mechanism of RFID-based urban intelligent building information integration system should put forward in accordance with the features of the comfortability of the urban intelligent building, the flexibility of the re organization, the adaptability of the project, the energy saving of the system, the sustainability of the environmental protection, and the characteristics of the RFID-based function and management. In the process of implementing RFID- based urban intelligent building information integration system engineering, it is vital to clear the construction target of the intelligent building, adhere to various standards and standards, develop many mature products and systems, further standardize the market, strictly manage system testing and acceptance, and realize the sustainable development of the urban intelligent building. Acknowledgment Construction: The construction stage of the intelligent building follows the production stage. After the components leave the factory, they are transferred, stored, assembled and maintained. Then the information of each stage is integrated by the application of the RFID label, and the built engineering management system is used to monitor the assembly of the components. Foundation Programs.Technological Innovation Team Programs for Distinguished Young and Middle-aged Scholars of Hubei Higher Education of China(T201829); Technological Innovation Programs of Wuhan Business University of China (2016KC04); 2018 Academic Team Programs of Wuhan Business University of China (2018TD003) Building energy saving: Firstly, in the building the active RFID is used to control the central air conditioning equipment to make the indoor temperature, humidity and ventilation achieve the purpose of comfort and energy saving, establish the urban intelligent building sensor network, and carry out the simulation and related application research and development of the building environment for the demonstration house. Secondly, the urban intelligent building sensor network application can be utilized to simulate the environment of RFID-based urban intelligent building in order to revise the design of the best energy-saving demonstration house, test the construction ways of environmental protection and energy saving in the demonstration house (e.g. testing solar induction boards, temperature regulators and automatic sun shading devices), and ultimately realize the application of RFID to the urban intelligent building energy saving materials to enhance the sustainability of integration. 6 Conclusions In the background of sustainable development, the urban intelligent building is the inevitable direction of building development. The information integration system of RFID-based urban intelligent building provides the periodical information needed in every stage. It can not only construct the mode of the application of RFID technology in urban intelligent building, but also apply this kind of mode to every stage of the actual urban intelligent building engineering life cycle, thus helping to enhance the efficiency of the overall planning, design, construction, operation, maintenance and management of 4 4
https://openalex.org/W2144366108	https://europepmc.org/articles/pmc3211298?pdf=render	English	null	Synthesis, structure, and opto-electronic properties of organic-based nanoscale heterojunctions	Nanoscale research letters	2,011	cc-by	8,081	NANO REVIEW Open Access Open Access Synthesis, structure, and opto-electronic properties of organic-based nanoscale heterojunctions Bohuslav Rezek1, Jan Čermák1, Alexander Kromka1, Martin Ledinský1, Pavel Hubík1, Jiří J Mareš1, Adam Purkrt1,2, Vĕra Cimrová2, Antonín Fejfar1, Jan Kočka1 © 2011 Rezek et al; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Enormous research effort has been put into optimizing organic-based opto-electronic systems for efficient generation of free charge carriers. This optimization is mainly due to typically high dissociation energy (0.1-1 eV) and short diffusion length (10 nm) of excitons in organic materials. Inherently, interplay of microscopic structural, chemical, and opto-electronic properties plays crucial role. We show that employing and combining advanced scanning probe techniques can provide us significant insight into the correlation of these properties. By adjusting parameters of contact- and tapping-mode atomic force microscopy (AFM), we perform morphologic and mechanical characterizations (nanoshaving) of organic layers, measure their electrical conductivity by current- sensing AFM, and deduce work functions and surface photovoltage (SPV) effects by Kelvin force microscopy using high spatial resolution. These data are further correlated with local material composition detected using micro- Raman spectroscopy and with other electronic transport data. We demonstrate benefits of this multi-dimensional characterizations on (i) bulk heterojunction of fully organic composite films, indicating differences in blend quality and component segregation leading to local shunts of photovoltaic cell, and (ii) thin-film heterojunction of polypyrrole (PPy) electropolymerized on hydrogen-terminated diamond, indicating covalent bonding and transfer of charge carriers from PPy to diamond. Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Correspondence: rezek@fzu.cz 1Institute of Physics ASCR, v.v.i., Cukrovarnická 10, 16200 Prague 6, Czech Republic Full list of author information is available at the end of the article Introduction In the bulk-heterojunction design both materials (donor and acceptor) are prepared as a microscopic interpenetrating network (Figure 1b). Therefore, wherever the excitons are generated, an interface with the other material is always closer than the exciton diffusion length (in an ideal case). The remaining task is to ensure efficient charge carrier trans- port to electrodes. So far the best reported PV efficiency is the attainment of reaching 7.4% [8]. surface for grafting of more complex organic molecules on diamond [10,18-20]. In this study, we have chosen PPy because of its wide universality as a model of a chemically and optically sensitive organic dye. Its polymerization can be achieved by electrooxidation from a solvent [21], chemical vapor deposition [22], UV irradiation [23], or chemical poly- merization [24,25]. PPy is a well known yet still remains under intensive study in many fields of applications, like sensors [26-28], biosensors [29,30], fuel cells [31,32], corrosion protection [33], or rechargeable batteries [34,35]. For a long time, electronics has been the domain of inorganic materials. Even though electronic behavior of organic materials has been known for several decades, their applications are still limited. So far, the research and development activity of organic and inorganic elec- tronics has been strictly divided, although their combi- nation might be fruitful. An example of promising organic-inorganic (hybrid) systems is the dye-sensitized PV cell. In this so-called Grätzel cell [9], a photoexcited organic dye gives electrons to the electrode via porous inorganic TiO2. The missing electron is returned from the surrounding electrolyte, which restores the original state at the counter electrode. Although the PV power conversion of this type of PV cells is relatively high (10- 12%), their wider application is limited by the need of an electrolyte, which is commonly in liquid form. Organic-based electronic as well as optoelectronic devices are commonly made from several compounds and their mutual electronic cooperation at microscale is of key importance for the device properties. Therefore, revealing and understanding of microscopic structural, chemical, electronic, and optoelectronic properties is crucial for their further improvements. Such challenging task may be fulfilled by techniques based on local probe scanning. One of the prominent methods employing scanning probe is atomic force microscopy (AFM) with its diverse regimes of operation [36]. In AFM, a sharp tip mounted on a flexible lever (the so-called can-tilever) can detect both morphologic and electronic information with high lateral resolution. Introduction 1994 [3], organic integrated circuit by the Philips com- pany (1998)) until recently, which have seen application of organic displays in cell phones, and the start of com- mercial production of organic photovoltaic (PV) cells. Electronic devices nowadays are commonly based on inorganic semiconductors (e.g., silicon or germanium) and, thus, face the problem of high-cost industrial pro- cesses (high-vacuum technology, clean rooms, or high- purity source materials). A possible way to reduce costs is the use of organic materials with semiconducting or metallic properties. Although the first report on the conductivity of doped polypyrrole (PPy) was published in 1963 [1], the breakthrough is attributed to the studies on doped polyacetylene since 1977 by Heeger, Shira- kawa, and MacDiarmid [2], for which they were awarded the Nobel prize in 2000. Since then organic semicon- ductors have followed similar scientific evolution as inorganic ones (all-polymer field effect transistor in PV effect in organic materials is different from inor- ganic ones. The binding energy between photo-excited electron-hole pair is strong due to the low dielectric constant, typically in the range of 0.1-1 eV. Therefore, the excitons are not dissociated by thermal energy, which is approximately 26 meV at room temperature. Additional driving force is needed, which can be sup- plied by introducing a layer of a second organic material (the so-called double-layer cell, Figure 1a). Typical PV power conversion efficiencies of such devices are not higher than 0.1% [4,5] because of the short exciton dif- fusion length (around 10 nm [6]) compared to the total film thickness needed for efficient light harvesting (100- 200 nm). * Correspondence: rezek@fzu.cz 1Institute of Physics ASCR, v.v.i., Cukrovarnická 10, 16200 Prague 6, Czech Republic Full list of author information is available at the end of the article p Full list of author information is available at the end of the article Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Page 2 of 12 Figure 1 Schematic cross-sectional drawings of two main organic PV cell designs: (a) double-layer junction and (b) bulk-heterojunction. ss-sectional drawings of two main organic PV cell designs: (a) double-layer junction and (b) bulk-heterojunction. re 1 Schematic cross-sectional drawings of two main organic PV cell designs: (a) double-layer junction and (b) bulk- Significant improvement emerged with the bulk-het- erojunction design [7]. Introduction Another example of an organic-inorganic system, which is extensively studied, is diamond in combination with organics, e.g., fullerene [10,11]. Such systems are highly promising for bio-sensoric or opto-electronic applications, as a charge transport between the two materials is observed. However, the basic electronic properties at their interface still need to be fully understood. When the tip is in contact with the studied surface, a bias voltage can be applied between the sample and the AFM tip, and the induced electric current is dependent on the local conductivity at the position of the tip. As a result, maps of both topography and local conductivity are obtained at once in this so-called current-sensing AFM regime (CS-AFM) [37]. Applying this mode on organic materials is rather difficult, however, as the soft organic materials can be easily modified by the sharp AFM tip (radius typically 10-30 nm) under the applied force of typically several nN or more. From the electronic point of view, diamond is a wide bandgap (5.5 eV) semiconductor, which in its intrinsic form is electrically insulating. Apart from the bulk con- ductivity induced by doping [12], intrinsic diamond exhibits a special phenomenon of surface conductivity when a thin (10-20 nm) electrically conductive (p-type) layer is formed at the surface [13-15]. It is observed under ambient conditions when the diamond surface is terminated by hydrogen atoms. Various electrically con- ductive areas or channels can be patterned on the sur- face by changing the surface termination [16,17]. Hydrogen termination can also be used as a starting Application of an AC voltage between the sample and the AFM tip when the tip is at a certain distance from the surface creates a modulated electrostatic force that makes the cantilever oscillate because of contact poten- tial difference. These oscillations can be minimized to zero by applying an additional DC voltage to compen- sate the potential difference. If the tip surface potential is calibrated, then the absolute values of the surface potential (and then the work function) can be obtained. Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Page 3 of 12 This is the principle of the so-called Kelvin force micro- scopy (KFM) [16]. shown in Figure 2b. KFM detected the highest surface potential (up to 50 mV) in the central part of the den- drite and the lowest surface potential (as low as -150 mV) in its immediate surrounding. Introduction Farther surround- ings exhibit the potential between these two values. Typical value is around - 30 mV. Also fluctuations related with small clusters are seen in topography. As the layer is made of two materials, the observed varia- tions in surface potential most likely correspond to variations in local chemical composition. The two extreme potential levels may correspond to individual materials while the intermediate potential is related to the blend. In spite of being a powerful tool for resolving micro- scopic structural, mechanical, and electronic properties, AFM had not been able to provide direct chemical con- trast until very recently [38]. Such chemical detection in AFM makes use of adhesion differences on atomic scale and was, thus so far, applicable only to atomically flat surfaces. Spatially resolved chemical information can be obtained in more straightforward way by micro-Raman spectroscopy mapping, where the focused laser beam plays the role of the scanning probe. Although it is an optical method, the resolution can be satisfactory at sub-micrometer scale [39]. The resolution can be further enhanced using special metal tip in the so-called tip- enhanced Raman spectroscopy [40]. This method works well on resonant systems such as in the case of carbon nanotubes [41], but its general application is still highly challenging. This assertion is proven by micro-Raman spectroscopy and mapping. The spectrum collected on the dendrite showed a sharp peak at 1468 cm-1, which was attributed to fullerene C60, as well as a broad band at 1400 cm-1 [42,48]. Similar spectra, although with lower peak inten- sity, were detected on the small clusters in the sur- roundings. The map and the spatial profile of C60 characteristic Raman scattering peak at 1468 cm-1 are shown in Figure 2c. The image reveals higher concen- tration of C60 at the dendrite and low C60 Raman signal in its close surroundings. In this study, we employ and combine advanced scan- ning probe techniques as well as macroscopic character- istics to provide us significant insights into the correlation of microscopic structural, chemical, and opto-electronic properties of organic-based heterojunc- tions. Introduction We demonstrate benefits of such multi-dimen- sional characterizations on (i) bulk heterojunctions of fully organic composite films using fullerenes as electron acceptors, indicating differences in blend quality and component segregation leading to local shunts of PV cell [42,43], and (ii) thin-film heterojunction of PPy elec- tro-polymerized on hydrogen-terminated diamond, indi- cating covalent bonding and enhanced exciton dissociation in such systems [44-47]. Spectrum detected in the dendrite vicinity did not show the sharp peak, only a broad band around 1400 cm-1 [42]. Similar spectra were obtained on the smooth surface in the farther surroundings. This band is not present in typical C60 Raman spectrum [48]. Hence, we attribute it to a photoluminescence (PL) background due to Raman laser excitation (note that it cannot be directly compared with usual PL spectra). Spatially resolved map and the profile of Raman intensity at the 1400 cm-1 band are shown in Figure 2d. It is noteworthy that both Raman maps are qualitatively quite similar. Yet, quantitative comparison of the spatial profiles shows differences: the C60 signal is higher on the den- drite and small clusters, while the PL intensity prevails over C60 on the smooth surface and the very vicinity of the dendrite. We suggest that the map of sharp peak at 1458 cm-1 is related with segregated crystallized C60 on the dendrite and on small clusters in the surroundings. The broad band at around 1400 cm-1 may be attributed to C60 that is highly dissolved in the blend. This asser- tion is supported also by the AFM and KFM data. Advanced organic bulk heterojunction g j Based on the result with pure fullerene, more advanced bulk heterojunctions were prepared, with fullerene deri- vatives being used as electron acceptors: [6,6]-thie- nylC61 butyric acid methyl ester ([60]ThCBM), and [6,6]-thienylC71 butyric acid methyl ester([70]ThCBM). Poly(3-hexylthiophene-2,5-diyl) (P3HT) was used as electron donor. The blends were prepared in an inert atmosphere on ITO-covered glass substrates with a hole-conducting polymer (PEDOT:PSS) by spin coating from a 2.5% chlorobenzene solution [43]. Both blends were annealed at 125°C (the annealing improved the PV efficiency more than twice compared with non-annealed blends). Resulting thickness of the films was 140 nm, as measured by KLA-Tencor P-10 profiler. LiF (2 nm) and Al (60 nm) were thermally evaporated to make the sec- ond contact. Active area of the prepared PV cells was 6.5 × 2.5 mm2. The absolute values of the overall surface potentials shifted over time independently on the illumination. This effect is attributed to degradation of the blend films, as the AFM/KFM characterization was performed under ambient conditions. Degradation of the blends was also observed as a decrease in photo-response. There was almost no photo-response observed after 250 min in the case of P3HT:[60]ThCBM blend, and the same state is reached after 1000 min in the case of P3HT:[70]ThCBM. Figure 3a, b shows macroscopic (with both top and bottom planar contacts) I(V) characteristics of the two blend films measured under white light illumination in the inert nitrogen atmosphere (0 h) and after exposure to ambient air. The PV device made of P3HT:[70] ThCBM blend exhibits initial power conversion effi- ciency(h) of 0.71% which is reduced, after exposure to ambient air for 6 h, to h = 0.37%. The reduction is due to the decrease in short-circuit current (Isc) from 4.02 to 2.08 mA/cm2 while open-circuit voltage (Voc) remains 0.55 V. Similar decreases in both power conversion effi- ciency and Isc were observed also on the device made of P3HT:[60]ThCBM blend (initial: h = 0.86%, Isc = 4.05 mA/cm2; after 6 h: h = 0.42%, Isc = 1.8 mA/cm2; Voc remains 0.48 V). In other words, both blends exhibit similar PV performance as well as its development in time. I(V) and KFM characteristics indicate that the P3HT: [60]ThCBM degrades much faster than the other one. Basic organic bulk heterojunction Basic type of organic bulk heterojunction was based on fullerene C60 as electron acceptor. Composite blends made of poly[(2,7-(9,9-dihexa)fluorence)-co-(1,4-(2,5- didecylaminoketo) phenylene)] (VYP-120, developed at the Institute of Macro-molecular Chemistry, ASCR, v.v. i.) and fullerene C60 (Sigma Aldrich) were spincoated on indium tin oxide (ITO)-covered glass substrates and dried at 50°C under vacuum for 4 h. For characterizing the PV performance of the thin film, top Al electrodes were evaporated through a shadow mask. In spite of observed quenching of photoluminescence compared to polymer layer without the C60, the compo- site layer exhibited low PV power conversion efficiency (Isc ~ 2 nA, Voc ~ 5 mV, h ~ 0.06%) [42]. AFM mor- phology (Figure 2a) revealed a relatively flat and smooth surface (RMS roughness: 4 nm) which was covered with two types of clusters (lateral size either 100 nm or sev- eral μm) and dendrites (more than 10 μm). Electrical potential map obtained by KFM at the same area is In both Raman maps, there is a minimum in the vici- nity of the dendrite. This is not effect of thickness as the AFM shows even surface compared to surroundings. On the other hand, there is clear difference in the work function detected by KFM. This indicates that this area consists predominantly of the conductive polymer. In such a case, the dendrite vicinity is not of heterostruc- tural nature, and the conductive polymer may electri- cally shunt the device. Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Page 4 of 12 Figure 2 Multidimensional microscopic characteristics (images and spatial profiles) of the organic hetero fullerene C60 and poly[(2,7-(9,9-dihexa)fluorene)-co-(1,4-(2,5-didecylaminoketo) phenylene)]: (a) tapping-mode A (b) KFM surface potential, (c) micro-Raman intensity at 1468 cm-1, (d) micro-Raman intensity at 1400 cm-1, and (e) loc measured by CS-AFM (negative bias voltage applied on the sample). Positions of the profiles in the images are indica and arrows. Figure 2 Multidimensional microscopic characteristics (images and spatial profiles) of the organic heterostructure blend made of fullerene C60 and poly[(2,7-(9,9-dihexa)fluorene)-co-(1,4-(2,5-didecylaminoketo) phenylene)]: (a) tapping-mode AFM surface morphology, (b) KFM surface potential, (c) micro-Raman intensity at 1468 cm-1, (d) micro-Raman intensity at 1400 cm-1, and (e) local conductivity as measured by CS-AFM (negative bias voltage applied on the sample). Positions of the profiles in the images are indicated by the dashed lines and arrows. Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Page 5 of 12 diameter). Advanced organic bulk heterojunction The difference between the macroscopic and micro- scopic performance is most likely caused by the pre- sence of the metal electrode (in the macroscopic characterization), which partially seals the organic films. From this point of view, contact-less KFM characteriza- tion revealed different opto-electronic properties of the blends, not detected by the macroscopic I(V) characteristics. Basic organic bulk heterojunction These pits do not affect the electronic homo- geneity of the films. The films appear electronically uni- form as observed by KFM [43]. Therefore, to understand the differences in PV performance, we char- acterized dynamic opto-electronic response by modify- ing standard KFM technique. We stopped the physical motion of the AFM tip in one direction so that the cor- responding axis in the image rep-resents time axis. Dur- ing scanning in this modified regime we were switching on and off white light illumination repeatedly. To resolve the above effect, we characterized local electronic transport properties of the organic film by CS-AFM, so that its integrity was preserved, and its sur- face morphology was the same as in tapping mode. The image of local conductivity (Figure 2e: note that the applied bias voltage is negative) shows that the dendrite and farther surroundings are electrically insulating. Con- ductivity in the close vicinity of the dendrite (area lack- ing C60) is an order of magnitude higher. This observation corroborates the conclusion that such fea- tures can cause electrical shunting of the organic film and reduce its power conversion efficiency. Resulting data (KFM images and their cross sections) are shown in Figure 3c, d. Under the white light illumi- nation, the surface potentials of both blend films shifted. In the case of P3HT:[60]ThCBM blend, the shift of sur- face potential is slower. This indicates that, after the illumination is switched off, the film keeps negative charge. This behavior has already been observed also on BH PV cells containing PCBM [49] as well as all-poly- mer cells [50]. It was attributed to electron trapping in shallow traps [51]. Persisting negative charge may then limit the surface potential shift under repeated illumina- tion being switched on. In the case of P3HT:[70] ThCBM, the surface potential comes repeatedly back almost to the starting level during the short term, which indicates that the electron trapping in this blend is mini- mized, and therefore it is beneficial for PV applications. PPy-diamond heterojunction It is noticeable that the initial power conversion effi- ciency of the P3HT:[70]ThCBM blend is lower but its decrease in time is also slightly slower. Based on AFM topography (shown as inset images in Figure 3a, b), both blend films are smooth (RMS roughness around 2 nm) with shallow pits (10-20 nm deep, 100-200 nm in For creating organic-inorganic junction, we synthesized and grafted the PPy on diamond electrochemically from pyrrole (0.24 M) and NaCl (0.1 M) aqueous solution by the application of a constant current (current density -0.3 mA/cm2) and the employment of a hydrogen-termi- nated intrinsic monocrystalline diamond (synthetic IIIa Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Page 6 of 12 Figure 3 Macroscopic and microscopic opto-electronic characteristics of the organic blends. (a, b) Macroscopically measured current- voltage characteristics of the blend films under illumination (white light, 80 mW) in the nitrogen atmosphere (0 h) and after exposure to ambient air for 2 and 6 h. The inset images in (a, b) show AFM topography images of the blend films (1 × 1 μm2, height scale 30 nm). (c, d) Cross sections of the KFM images (shown as insets, 1 × 1 μm2, potential scale 40 mV) under repeated on/off switching of illumination. Figure 3 Macroscopic and microscopic opto-electronic characteristics of the organic blends. (a, b) Macroscopically measured current- voltage characteristics of the blend films under illumination (white light, 80 mW) in the nitrogen atmosphere (0 h) and after exposure to ambient air for 2 and 6 h. The inset images in (a, b) show AFM topography images of the blend films (1 × 1 μm2, height scale 30 nm). (c, d) Cross sections of the KFM images (shown as insets, 1 × 1 μm2, potential scale 40 mV) under repeated on/off switching of illumination. DNA molecules [20,52]. This indicates that the PPy molecules also establish covalent bonds with H-termi- nated diamond. CVD diamond) with conductive surface as a working electrode [44]. For the electronic transport measure- ments, we defined H-terminated conductive narrow channel (5 μm wide) on a monocrystalline diamond sur- face by selective oxygen plasma discharge treatment through a photolithographic mask. PPy was electroche- mically synthesized on the channel. Synthesis and elec- tronic measurement setups are shown schematically in Figure 4. Typical PPy growth curve is shown there as well. PPy-diamond heterojunction Surface potential measured by KFM on the place where the PPy film had been deposited and removed (Figure 6) is significantly lower (by about 0.1 V) than the potential on pristine H-terminated diamond surface. This change is similar to reports on DNA-diamond interfaces [53] and supports the assertion of PPy mole- cules being linked to diamond surface covalently while removing the H termination. We also observed that the surface conductivity disappeared after the synthesis and the removal of the PPy molecules from diamond surface, which is most likely caused by the missing H termination. Thickness of the PPy film was 25 ± 5 nm based on the height histogram of tapping-mode AFM image after local nanoshaving of the PPy film in contact mode AFM [44]. Then, we applied contact-mode AFM with increas- ing contact force. At certain threshold contact force, the sharp tip starts to penetrate and remove the organic film. This transition is evidenced in Figure 5. The threshold force deduced in this manner was about 40 nN. This value is comparable to the values observed on a system consisting of diamond with covalently grafted Covalent bonding between PPy and diamond was con- firmed also theoretically. Calculated interaction energy of about 6 eV per bond pointed to a covalent character of the bonds formed at the one- and multi-bond con- tacts between PPy and diamond [54]. Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Page 7 of 12 Figure 6 Average surface potential obtained by KFM on PPy- diamond, H-diamond, and diamond after PPy removal. The potential values are referenced to the grounded Au contact pad. Figure 4 Design and electrochemical synthesis of PPy-diamond heterojunction: (a) Schematic cross-sectional drawing of experimental setup for electrochemical synthesis of PPy on diamond device structure. (b) Schematic top view of PPy-diamond device connected for electrical measurements. (c) Voltage as a function of time as detected during the electrochemical synthesis in galvanostatic regime. Based on the above experimental and theoretical results, we propose a model that, during the electroche- mical synthesis of PPy on diamond, the hydrogen atoms are removed and, instead, PPy molecules establish cova- lent bonds with carbon atoms of diamond. To study optoelectronic properties at the PPy-dia- mond interface we first removed PPy from a small area (10 × 10 μm) using the AFM nanoshaving. Micro- Raman spectroscopy confirmed the removal of PPy [45]. PPy-diamond heterojunction This area and its surroundings were then studied by KFM in the dark and under the white light illumination. Similar to the experiments on advanced bulk hetero- junctions, the KFM measurement was performed with one-scan direction disabled, and the illumination was repeatedly switched on and off (Figure 7a). Figure 7b shows temporal potential profiles that were obtained via KFM scanning across PPy-diamond and bare diamond surface (where PPy was removed) under repeated illumi- nation switching. The positions of profiles are indicated by lines and arrows in the image. The changes of poten- tial are relatively fast (<1 s, limited by KFM scan speed), reproducible, and well defined. Figure 4 Design and electrochemical synthesis of PPy-diamond heterojunction: (a) Schematic cross-sectional drawing of experimental setup for electrochemical synthesis of PPy on diamond device structure. (b) Schematic top view of PPy-diamond device connected for electrical measurements. (c) Voltage as a function of time as detected during the electrochemical synthesis in galvanostatic regime. Based on the surface potentials combined with data from the literature for both materials, we were able to assemble the energy band configuration of the PPy-dia- mond system (Figure 8). Detailed description of the pro- cedure for band diagram assembly is given in [45]. Theoretical calculations showed that, for the PPy-C:H interface, the charge neutrality level is located below the Fermi level, so the transfer of electrons occurs from an electrode to the PPy molecules [54]. This confirm down- ward band bending of PPy bands at the junction to diamond. Figure 5 Scratching of the PPy film by AFM tip with increasing contact force. Forces reaching 40 nN are strong enough to remove PPy molecules from diamond substrate. Under illumination, the observed positive shifts of sur- face potentials correspond to a decrease of work func- tion in both PPy and diamond as indicated in Figure 8. These shifts of surface potentials were attributed to the SPV effects. Based on the SPV theory, a model of charge transfer between diamond and PPy was established [45]. This model suggests that under illumination strongly Figure 5 Scratching of the PPy film by AFM tip with increasing contact force. Forces reaching 40 nN are strong enough to remove PPy molecules from diamond substrate. Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Page 8 of 12 Figure 7 Surface potential of PPy-diamond heterojunction as function of illumination and time. PPy-diamond heterojunction (a) Three-dimensional representation of surface potential map, and (b) temporal potential profiles that were obtained via KFM scanning across PPy-diamond and bare diamond surface (where PPy was removed) under repeated illumination switching. The positions of profiles are indicated by lines and arrows in the image. Figure 7 Surface potential of PPy-diamond heterojunction as function of illumination and time. (a) Three-dimensional representation of surface potential map, and (b) temporal potential profiles that were obtained via KFM scanning across PPy-diamond and bare diamond surface (where PPy was removed) under repeated illumination switching. The positions of profiles are indicated by lines and arrows in the image. conductive (approximately 100 pA when 1 V is applied, see Figure 9a). Such changes of electrical current occur within 1 s and are reproducible [46]. This is in agree- ment with the fast changes in the surface potential as shown in Figure 7b. bound excitons are generated in PPy. The excitons can be split at the interface with diamond, and the holes are trapped in diamond surface/interface states. The manner in which these holes contribute also to sub-band gap optical excitation of free holes in diamond depends on many factors [45]. Therefore, we character- ized electronic transport properties of the PPy-diamond system by I(V) measurements both in the dark and under the white light illumination. In the dark, the dia- mond channel with grafted PPy on top was highly elec- trically insulating as expected due to the missing H termination. The conductivity does not recover even when we remove PPy and expose the diamond surface to the ambient air (adsorbates) again. Hence, the adsor- bates play no role in this effect. Under the white light illumination, the channel with PPy turned electrically We can explain this illumination-induced conductivity by the charge transfer of holes from PPy to diamond as suggested above. The electronic transport measurements indicate that the holes accumulated in diamond-trap states are excited to diamond valence band, where they support the in-plane conductivity [46]. Such concept is schematically illustrated in Figure 9b. Relatively quick response to the change of illumination can be explained by a quick re-trapping of photogenerated holes in dia- mond and their recombination with photo-electrons remaining in the vicinity of the PPy-diamond junction Page 9 of 12 Rezek et al. PPy-diamond heterojunction Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Figure 8 Energetic scheme of the PPy-diamond system in equilibrium in the dark (full line) and under white light illumination (dashed line). Figure 8 Energetic scheme of the PPy-diamond system in equilibrium in the dark (full line) and under white light illumination (dashed line). Figure 8 Energetic scheme of the PPy-diamond system in equilibrium in the dark (full line) and under white light illumination (dashed line) due to downward band bending of PPy LUMO. The response rate is comparable with the organic blend [70] ThCBM:P3HT shown in Figure 3c. Also, we suggest that the photo-electrons are not persistently trapped. Hence they can support the quick changes in potential and current. They may also form another transport channel along the heterojunction but this still remains to be investigated. due to downward band bending of PPy LUMO. The response rate is comparable with the organic blend [70] ThCBM:P3HT shown in Figure 3c. Also, we suggest that the photo-electrons are not persistently trapped. Hence they can support the quick changes in potential and current. They may also form another transport channel along the heterojunction but this still remains to be investigated. The observed enhancement of in-plane conductivity under illumination might be explained also by the brid- ging effect via the semi-conductive and photosensitive PPy. To resolve this issue, we have measured mobility of charge carriers using Hall effect. Mobility of holes in PPy and diamond differs by at least five orders of mag- nitude (H-terminated diamond: 100-102 cm2/Vs, PPy: 10-5-10-10 cm2/Vs). We prepared microscopic Figure 9 Electronic transport characteristics and scheme of PPy-diamond hetero-junction. (a) Current-voltage characteristics of the PPy- diamond system in the dark and under white light illumination. (b) Conceptual drawing of charge transfer of photogenerated holes from PPy to diamond where they support the in-plane conductivity. Figure 9 Electronic transport characteristics and scheme of PPy-diamond hetero-junction. (a) Current-voltage characteristics of the PPy- diamond system in the dark and under white light illumination. (b) Conceptual drawing of charge transfer of photogenerated holes from PPy to diamond where they support the in-plane conductivity. Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 Page 10 of 12 Page 10 of 12 0.2 T. The mobility was evaluated from the Hall voltages to 7 cm2/Vs [47]. However, the Hall voltages were slowly drifting in time, and thus the mobility was obtained with high error bar of 20 cm2/Vs. was proposed. The model was supported by in-plane I(V) and Hall effect measurements. time, and the difference between values for positive and negative magnetic field, which should correspond to the true Hall voltage, is decreasing. We assign this effect to temperature gradients (up to several kelvins) arising in the measuring chamber because of heat flow from the magnet coils. These gradients create thermal and thermomagnetic electromotoric forces (EMFs) [55] of the order of 1 mV modifying the measured voltage. In more conductive sam- ples, these EMFs can be partially suppressed by alternating the direction of the measuring current. Unfortunately, in our case, the necessary long times of RC relaxation and integration (tens of minutes) prevent a successful applica- tion of this procedure. However, after leaving the magnet to cool down to room temperature, the repeated measure- ment shows a similar dependence, confirming in this way that we really observed the Hall voltage. Abbreviations AFM i f AFM: atomic force microscopy; CS-AFM: current-sensing AFM regime; EMFs: electromotoric forces; ITO: indium tin oxide; KFM: Kelvin force microscopy; PPy: polypyrrole; SPV: surface photovoltage. Acknowledgements The authors appreciate technical assistance of Z. Poláčková, J. Potmĕšil, V. Jurka, K. Jurek, and J. Zemek. The authors also acknowledge the financial support of the Academy of Sciences of the Czech Republic, (project No. KAN400100701, and the Fellowship J.E. Purkynĕ), the Ministry of Education, Youth and Sports of the Czech Republic (grants No. LC06040, LC510, 1M06031, AV0Z10100521), and the Grant Agency of the Czech Republic (grants No. 202/09/H041, P204/10/0212). Conclusions In this article, we showed that employing and combining advanced scanning probe techniques can provide signifi- cant insight into the correlation of structural, chemical, and opto-electronic properties of organic-based hetero- junctions on the microscopic scale. We presented specific results obtained on fully organic bulk heterostructures and on heterojunction of diamond with electrochemically synthesized organic dye. Although these systems seem to be different, from the optoelectronic point of view, they behave in a similar way. In both systems, strongly bound excitons are created which dissociate at the interface with the other material. Correlation of advanced scan- ning probe techniques (AFM, KFM, and CS-AFM) with micro-Raman mapping identified the reasons for low PV efficiency and could resolve differences in optoelectronic quality and stability by contact-less monitoring of dynamic photo-response of the organic blends. We demonstrated the applicability of the above methodology also to a hybrid organic-inorganic system. By correlating AFM nanoshaving, KFM surface potentials, as well as I (V) characteristics, we proved that during the electroche- mical synthesis a covalent bond between diamond and PPy is formed. Such conclusion is difficult to make based on commonly applied techniques such as X-ray photo- electron spectroscopy because here we have all-carbon systems (polymer-diamond). Furthermore, based on microscopic KFM and SPV measurements, the model of charge transfer from PPy to diamond under illumination participated in the experimental setup development. JK participated in discussions of electronic transport and overall results. participated in the experimental setup development. JK participated in discussions of electronic transport and overall results. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 1 October 2010 Accepted: 18 March 2011 P bli h d 18 M h 2011 Received: 1 October 2010 Accepted: 18 March 2011 Published: 18 March 2011 Received: 1 October 2010 Accepted: 18 March 2011 Published: 18 March 2011 Authors’ contributions BR conceived the idea of PPy-diamond, designed, and coordinated the study, participated in scanning probe experiments and PPy synthesis, participated in the experimental setup development, participated in discussions and analysis, and prepared the manuscript. JC carried out scanning probe experiments, fabricated device structures, participated in the experimental setup development and discussions, helped in drafting the manuscript. AK provided diamond specimens and their surface terminations. ML carried out micro-Raman spectroscopy and participated in discussions. PH performed Hall measurements and data analysis. JM assisted with Hall measurement and discussions. AP prepared organic blends and measured I (V) characteristics of the blends. VC participated in the design of the organic blends study and discussion; built up the experimental setup for preparation and macroscopic study of organic blends. AF conceived the scanning probe study of organic blends and the idea of comparing Hall mobility; Author details 1 1Institute of Physics ASCR, v.v.i., Cukrovarnická 10, 16200 Prague 6, Czech Republic 2Institute of Macromolecular Chemistry ASCR, v.v.i., Heyrovského nám. 2, 16206 Prague 6, Czech Republic The same Hall voltages plotted as a function of mag- netic field are shown in Figure 10c. If an arithmetic mean is performed for each applied magnetic field and these values are used for evaluation, then the resulting Hall mobility is 16 ± 10 cm2/Vs. This revised value of mobility with much lower relative error supports the idea of charge transport via diamond and thus transfer of charge from PPy to diamond. PPy-diamond heterojunction The model was supported by in-plane I(V) and Hall effect measurements. PPy-diamond heterojunction conductive square (10 × 10 μm2) with electric leads at the corners by selective hydrogen and oxygen termina- tions. A resin layer with the opening at the position of the square has been prepared by UV lithography to restrict the active area for electrochemical deposition. PPy was electrochemically synthesized under similar conditions as described in the previous sections. The Hall voltage was detected by two electrometers, and the current of 2 pA was supplied by Keithley 220 source. The magnetic field applied during the experiment was ± To reduce the effect of Hall voltage drift, we re-designed electrical connection of the PPy-diamond structure as shown in Figure 10a and also increased the magnetic field. The obtained diagonal Hall voltage under white light illu- mination (cold light source, 40 klx) is plotted as a function of time in Figure 10b. The Hall voltage is still varying in Figure 10 Hall effect measurements on PPy diamond heterojunctions (a) Schematic top view of the diamond in plane mesa structure for Figure 10 Hall effect measurements on PPy-diamond heterojunctions. (a) Schematic top view of the diamond in-plane mesa structure for Hall effect measurements showing a bare H-terminated mesa structure and its transformation into PPy-diamond mesa structure. Mesa surroundings are electrically insulated by oxidation of diamond surface. The resin encapsulation is used to confine PPy growth to the mesa area. (b) Hall voltage on the PPy-diamond mesa structure measured under +0.3 T (triangles) and - 0.3 T (squares) as a function of time. (c) The same Hall voltage plotted as a function of magnetic field. All measurements were done under the cold light illumination. Figure 10 Hall effect measurements on PPy-diamond heterojunctions. (a) Schematic top view of the diamond in-plane mesa structure for Hall effect measurements showing a bare H-terminated mesa structure and its transformation into PPy-diamond mesa structure. Mesa surroundings are electrically insulated by oxidation of diamond surface. The resin encapsulation is used to confine PPy growth to the mesa area. (b) Hall voltage on the PPy-diamond mesa structure measured under +0.3 T (triangles) and - 0.3 T (squares) as a function of time. (c) The same Hall voltage plotted as a function of magnetic field. All measurements were done under the cold light illumination. Page 11 of 12 Rezek et al. Nanoscale Research Letters 2011, 6:238 http://www.nanoscalereslett.com/content/6/1/238 was proposed. The model was supported by in-plane I(V) and Hall effect measurements. was proposed. References Binning G, Quate C: Atomic force microscope. Phys Rev Lett 1986, 56:930-933. 12. 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Härtl A, Schmich E, Garrido J, Hernando J, Catharino SC, Walter S, Feulner P, Kromka A, Steinmüller D, Stutzmann M: Protein-modified nanocrystalline diamond thin films for biosensor applications. Nat Mater 2004, 3:736-742. 43. Čermák J, Rezek B, Cimrová V, Fejfar A, Purkrt A, Vanĕček M, Kočka J: Time- resolved opto-electronic properties of poly(3-hexylthiophene-2,5-diyl): fullerene heterostructures detected by Kelvin force microscopy. Thin Solid Films 2010, 519:836-840. pp 20. Rezek B, Shin D, Nebel C: Properties of hybridyzed dna arrays on single- crystalline undoped and boron-doped (100) diamonds studied by atomic force microscopy in electrolytes. Langmuir 2007, 23:7626-7633. 44. Čermák J, Rezek B, Kromka A, Ledinský M, Kočka J: Electrochemical synthesis and electronic properties of polypyrrole on intrinsic diamond. Diam Relat Mater 2009, 18:1098-1101. y y 21. 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https://openalex.org/W2774936818	http://journal.walisongo.ac.id/index.php/walisongo/article/download/1329/pdf	English	null	Religious Freedom and the Idea of Establishing Islamic State	Jurnal Penelitian Walisongo/Walisongo: Jurnal Penelitian Sosial Keagamaan	2,017	cc-by-sa	9,068	Walisongo: Jurnal Penelitian Sosial Keagamaan Vol. 25 No.1 (2017) pp. 63-90 Doi: 10.21580/ws.25.1.1329 RELIGIOUS FREEDOM AND THE IDEA OF ESTABLISHING ISLAMIC STATE A B U H A P S I N 1 Universitas Islam Negeri Walisongo Semarang Corresponding author; email: 1abu_hapsin@walisongo.ac.id ISSN 0852-7172 (p) 2461-064X (e) © 2017 Walisongo: Jurnal Penelitian Sosial Keagamaan http://journal.walisongo.ac.id/index.php/walisongo 2 This translation is quoted from (Asad 1980, 57) A B U H A P S I N formal tentang keadilan. Dengan menggunakan penelitian kuali- tatif dan kajian pustaka penelitian ini mencoba mengungkapkan: gagasan Gamwell tentang kebebasan beragama, ciri-ciri Negara Islam seperti yang dijelaskan oleh Abdul Rauf, dan konsep Gam- well tentang kebebasan beragama, serta gagasan untuk mendiri- kan Negara Islam yang dianjurkan oleh Abdul Rauf. Keywords: Gamwell; religious freedom; Islamic state; democratic resolution. Keywords: Gamwell; religious freedom; Islamic state; democratic resolution. Abstract The idea of Gamwell on “religion as rational” was based on the concept that religious freedom is nothing other than a political discourse that can be figured out only through a democratic reso- lution. Changing paradigm from “religion as non-rational” to “re- ligion as rational” is a necessary condition for entering a public debate. Yet, the sole public debate or public view is not enough to solve the modern political problematic. The public debate must be guided by a constitutional procedure affirmed by the body politic so that it fulfills the criteria of formal claim about justice. Apply- ing qualitative research and literature review this research tried to reveal: Gamwell’s idea of religious freedom, the features of the Islamic State as described by Abdul Rauf and Gamwell’s concept of religious freedom and the idea of establishing the Islamic State advocated by Abdul Rauf. Gagasan Gamwell tentang “agama itu rasional” didasarkan pada konsep bahwa kebebasan beragama tidak lain adalah wacana poli- tik yang hanya bisa diraih melalui resolusi demokratis. Mengubah paradigma dari “agama sebagai tidak rasional” menjadi “agama sebagai rasional” adalah syarat yang diperlukan sebelum mema- suki debat publik. Namun, debat publik atau pandangan publik saja tidak cukup untuk memecahkan masalah politik modern. Per- debatan publik harus dipandu oleh prosedur konstitusional yang ditegaskan oleh badan politik sehingga memenuhi kriteria klaim 63 63 Introduction “There is no compulsion in religion; truly the right way has be- come clearly distinct from error; therefore, whoever disbelieves in the Shaitan and believes in Allah he indeed has laid hold on the firmest handle, which shall not break off, and Allah is Hearing, Knowing” (al-Quran, al-Baqarah: 256).2 “There is no compulsion in religion; truly the right way has be- come clearly distinct from error; therefore, whoever disbelieves in the Shaitan and believes in Allah he indeed has laid hold on the firmest handle, which shall not break off, and Allah is Hearing, Knowing” (al-Quran, al-Baqarah: 256).2 The explanation of this verse stated that religion (din) here signifies the belief about God embodied in the above ‘Verse of the Throne’ and the entire system of life which rests upon it. The verse means that the system of Islam, embracing belief, mor- als and practical conduct cannot be imposed by compulsion. These are not things to which people can be yoked forcibly (Ab- dul-Rahman 2009). The Muslim thinkers seem to agree unanimously that the above verse clearly and decisively indicates that Islam offers re- ligious freedom, so that every time they discuss the concept of religious freedom, they always refer to that verse. Ibn Kathir, one of those scholars interpreted the above verse that meant for “not to force anyone to become Muslim, for Islam is plain and clear, and its proofs and evidence are plain and clear. Therefore, there is no need to force anyone to embrace Islam. Rather, whoever Allah directs to Islam, opens his heart for it and enlightens his 64 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . mind, will embrace Islam with certainty. Whoever Allah blinds his heart and seals his hearing and sight, then he will not benefit from being forced to embrace Islam (Abdul-Rahman, 2009) One of the several contemporary Muslim thinkers who has tried to describe the concept of religious freedom in Islam and refers to the above verse is Tan Sri Muhammad Abdul Rauf. In his book, The Concept of Islamic State: with Particular Ref- erence to Treatment of Non-Muslim Citizens (1988) he elabo- rates the features of the Islamic State and how it should treat the non-Muslim citizens. Walisongo: Jurnal Penelitian Sosial Keagamaan Introduction Apropos of the rights of the non-Mus- lims, especially that related to religious freedom, I find it very convincing and fair. This was clearly described and analyzed by Akram D. al-Umari in his article, Medinan Society at the Time of the Prophet (1991), in which stated how Muslims treated well the non-Muslim citizens. However, no matter how just the treatment of the Islamic State in dealing with the non-Muslims would be, it raises at least two fundamental questions: what is the meaning of religious freedom? and, can the concept of religious freedom be defined only by one side, namely by the Muslims?I believe that the con- cept of religious freedom can only make sense in so far as it is defined or formulated by both sides, by the Muslims and the non-Muslims (Audi and Wolterstorff 1997). If Islam legitimates the plurality of religions as the above verse claims, logically the Islamic State should let the legitimate religious convictions de- fine their own religious freedom. In view of this, I would like to discuss whether the claim of religious freedom and the idea of establishing an Islamic State proposed by Abdul Rauf is a coherent political principle or not. In discussing his ideas, I would like to use the concept of reli- gious freedom advocated by Franklin I. Gamwell in his work, The Meaning of Religious Freedom, Modern Politics and the 65 Walisongo: Jurnal Penelitian Sosial Keagamaan A B U H A P S I N Democratic Resolution (1995). This is based on the reason that, as far as I am concerned, this work offers a coherent resolution for figuring out the modern political problems related to reli- gious freedom. The discussion will be divided into three parts. The first part is that related to the Gamwell’s idea of religious freedom. This will be used as the theoretical basis for analyzing the idea of establishing the Islamic State as proposed by Abdul Rauf. The second part will reiterate the features of the Islamic State as de- scribed by Abdul Rauf. The last part will discuss the application of Gamwell’s concept of religious freedom and the idea of estab- lishing the Islamic State advocated by Abdul Rauf. The Meaning of Religious Freedom Religious freedom and religious liberty are often used inter- changeably. Both of them have a synonymous meaning, express- ing the absence of coercion of someone mind in expressing his or her belief or conscience. Yet in the context of political and legal rights, the term “religious freedom” is more often used than the term “religious liberty” (Koshy 1992, 23; Attabani 1995). Carilo de Albornoz, as quoted by Koshy, suggests that reli- gious liberty or religious freedom has four main aspects, namely; liberty of conscience, liberty of religious expression, liberty of religious association and, the last, corporate and institutional religious freedom. Among these four aspects, freedom of con- science is the most genuine and an absolute right in the sense that inalienability transcends the other three aspects. Since the freedom of conscience is an absolute right, religious freedom should encompass freedom to choose or not to choose a certain religion. Personal truth must be regarded as the supreme value. It entails self-commitment and self-responsibility. It transgresses the commitment to other agents such as government and even 66 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . god (Koshy 1992, 22). It is in this sense that Gamwell defines religion and only in this sense should the meaning of “authentic human being” be defined. Thus, in broader terms, religion is de- fined as a self commitment to personal truth since it is the foun- dation to determine the authenticity of a human being (Gamwell 1995, 30–31).3 The freedom of conscience constitutes the inner dimension, while the expression or the manifestation of the conscience, ei- ther manifested personally or institutionally, constitutes the ex- ternal dimension of religious freedom (Koshy 1992, 24). The division of religious freedom into the internal and the external dimensions finally leads to an idea that the later is not absolute in nature. This means that the right to manifest the religious conscience is dependent on the other agents such as government and other elements of body politics. Even though for example, one’s personal truth or personal conscience says that committing suicide is a noble act, he cannot manifest it freely by simply say- ing “this is my right”. The manifestation of religious conscience is subject to public debate, discussion and assessment from the other agents. 3 In the other parts of hisbook, Gamwell criticizes the idea of Sidney E. Mead because of his inclusive definition of religion. Mead defines religionon- ly that belongs to theistic religion. According to Gamwell, this is not sensible since this will eliminateother people’s right to express his or her self-authen- ticity, see Gamwell (1995, 147). Walisongo: Jurnal Penelitian Sosial Keagamaan The Meaning of Religious Freedom Thus, religious conscience or religious conviction is subject to public debate when it is manifested in a political arena. It is in this context that the idea of religious freedom is closely linked with politics. According to Franklin I. Gamwell, the assessment of reli- gious freedom in relation to politics can be described in two ways; religion as rational and religion as non-rational. The the- sis of religion as rational suggests that every religious conviction is allowed to enter into a political arena. In a democratic dis- 3 In the other parts of hisbook, Gamwell criticizes the idea of Sidney E. Mead because of his inclusive definition of religion. Mead defines religionon- ly that belongs to theistic religion. According to Gamwell, this is not sensible since this will eliminateother people’s right to express his or her self-authen- ticity, see Gamwell (1995, 147). 67 67 Walisongo: Jurnal Penelitian Sosial Keagamaan A B U H A P S I N course, no one is allowed to proscribe other people to express their self-authenticity which is found in their religious conviction. But, as a consequence, the religious conviction must be ready to be debated and assessed publicly. This is why, he criticizes the political approaches which are based on the thesis of religion as non-rational like that developed by John Rawls, the approach of the partisans promoted by John C. Murray and the approach of the pluralist from Kent Greenawalt (1988).4 Gamwell is quite sure that the assessment of religion as non-rational will only end up with non-coherent resolution and will only produce non-co- herent political principles. In brief, the assessment of religion as non-rational will not be able to solve what Gamwellcalls “the modern political problematic.” The modern political problematic refers to a condition in which religious authority loses its domination over the state and, at the same time, there is an increasing freedom to choose a given religious conviction within the political community. The erosion of the religious authority finally leads to the state to a legitimate plurality of religious convictions. The state can no more prescribe politically to limit the emergence of religious convictions. Thus, the modern political problems are character- ized by an indeterminate number of religious convictions. The limitation of the number of religions can possibly be done only by force (Gamwell 1995, 5–10). 4 To know how Gamwell criticizes the approaches which are based on the thesis of religion as non-rational, seeGamwell (1995, 47–115). Walisongo: Jurnal Penelitian Sosial Keagamaan The Meaning of Religious Freedom However, the limitation by force is not a good resolution since it does not run parallel with the idea of democracy. The democratic resolution to the modern political problems can work out only by letting the political community exercise their activities which is constituted or represented by their self-under- standing. The self-understanding is, however, the choice of their Vol. 25, No.1 (2017) 68 R E L I G I O U S F R E E D O M A N D T H E I D E A . . . purpose for the sake of searching and determining their self-au- thenticity as a human being (Gamwell 1995, 17). Even though our activities choose and decide our purpose with understanding, “it does not mean that our understanding is always explicit (Gamwell 1995, 17).” This means that our pur- pose is not always chosen by our activities through a conscious thought. Since our activity is not always conscious and under- stands explicitly of all things completely, our activity also in- cludes or is represented by“implicit understanding”, that is the understanding which is excluded from conscious thought but it is so decisive that our explicit understanding dependent on it and without it our explicit understanding “could not be what it is” (Gamwell 1995, 17, 217). Distinguishing the implicit understanding from the explicit understanding is important in order to know the formal dis- tinction between religious and political activity.Since both reli- gious and political activities are forms of human activities, both can only be differentiated from their explicit understanding, not from their implicit understanding (Gamwell 1995, 19). For ex- ample, a state stipulates the marital law which is based on the principle of monogamy. This activity is, of course, directed to an- swer explicitly the question about the state’s policy, and hence, it is a political activity. But implicitly, it is religious activity since it implies that the state supports and implements the Christian understanding of the principle of marriage. Because an understanding provides an answer to a given question, human activities are also distinguished by the ques- tion which is explicitly asked. Thus, religious activities are distinguished from political activities because of the question which is explicitly asked. On this account, Gamwell defines re- ligion as “the primary form of culture in terms of which the comprehensive question is explicitly asked and answered. The Meaning of Religious Freedom The 69 A B U H A P S I N term “form of culture” here means that a religion is “a set or system of concepts and symbols in terms of which human ex- plicitly understand themselves” (Gamwell 1995, 23) while, the term “primary” indicates that there is also the secondary form of cultures such as theology, philosophy, and philosophical the- ology. If the “primary form” asks and answers to the compre- hensive question explicitly and decisively, the “secondary form” asks and answers to the comprehensive question critically. Thus, theology and philosophy are forms of religious activity in order that the religious activity becomes self-critical (Gamwell 1995, 29). Meanwhile, the term “comprehensive” in the above defini- tion refers to the purpose of life (Gamwell 1995, 18). Thus, the question about the religious activity might be formulated: what should the activities of the religion be or what should a religion do to fulfill the purpose of life? If the religion asks and answers explicitly to the comprehen- sive question, the politics asks and answers explicitly to the ques- tion about the state. This is because the politics is only a form of association, not a form of culture. Hence, a political activity is not directed to ask and answer explicitly to the comprehensive question, but it is limited only to ask and answer explicitly to the question of the state. Therefore, the question about the political activity might be formulated: “what should the activities of the state be and what should the state do?” (Gamwell 1995, 32). Based on the above explanation it is clear that our activity is constituted by both explicit and implicit self-understanding. If our activity is intended to understand the purpose of life, it is constituted by our “comprehensive self-understanding (Gamwell 1995, 18).” That is why, the question about the purpose of life should also be constituted by“comprehensive question”, and it is the answer to the comprehensive question that the authentici- ty of a human being can be found(Gamwell 1995, 35–36). Since 70 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . it is a religion that provides the comprehensive understanding and provides the answer to the comprehensive question, it is not wise to discard religion from the political arena. Walisongo: Jurnal Penelitian Sosial Keagamaan The Meaning of Religious Freedom But, as a con- sequence, the religious conviction has to be ready to enter into a free and full public debate. The above statements indicate that, for Gamwell, the re- ligious conviction must be constituted in the political expres- sion. But, before it becomes the state’s policy, it must be tested through the public debate or public view. This public view is important for the sake of determining the validity of a given re- ligious conviction. In view of this human experience and reason is, of course, very significant to see whether that religious con- viction is valid or not (Gamwell 1995, 190). This idea is in line to what Mayer discussed related to the Islam and Human Rights Tradition and Politics (Mayer 1994, 173–76) in which she offers the concept of“Islamic human rights schemes” in the scheme of political way. These are the attempts done by conservative reli- gious scholars and intellectuals to produce an Islamic version of human rights law–typically in the form of an Islamic “bill of rights”. The schemes resulted sometimes replicate provisions found in documents like the 1948 Universal Declaration of Hu- man Rights. More often, however, they modify these provisions in accordance with Islamic criteria or introduce some alternative sets of rights which are not found in the contemporary interna- tional law. Mayer also offers to set up any discussion related to Islamic human rights. The full and free debate is only possible to be done if we change our perspectives from the thesis of religion as non-ra- tional to the thesis of religion as rational. This is the first and foremost step to be taken into consideration when we would like to follow Gamwell’s ideas in overcoming the political prob- lems related to religious freedom. It is a necessary condition for 71 71 A B U H A P S I N the following step proposed by him, namely “democratic res- olution.” Thus, before we come to a political arena, our mind should agree upon the idea that religious conviction is subject to public debate and discussion. The democratic resolution of religious freedom entails a democratic constitution. The democratic constitution should en- compass and answer both politically and religiously formulated problems. The formulation of the political problem will ask: is a given constitution consistent with the plurality of legitimate religions? The Meaning of Religious Freedom In contrast, the formulation of the religious problem will ask: can a given constitution be affirmed by adherents of a plurality of religion (Gamwell 1995, 161). These two prob- lematic formulations are of great significance for determining whether the public view or public debate can work out or not. A given constitution that does not encompass the answer to these two problematic formulations is not the democratic constitu- tion, and hence, one without religious freedom. The democratic constitution must also identify the partici- pants in the discussion or debate and their rights in order that the discussion could be fully free. The procedure for making decisions, the procedure for executing decisions, the procedure for enforcing the prescription of the constitution, the procedure for adjudicating differences in relation to both decisions and the constitutional prescriptions must b included in the constitution. In order to be a fully free-debate, the constitution itself must be subject to continual assessment. Thus, the procedure for chang- ing or amending the constitution must be explicitly stated in the constitution (Gamwell 1995, 163). In brief, the democratic con- stitution should reflect the affirmation of all religious adherents. That is why the constitution that explicitly describes a certain comprehensive understanding is not a democratic constitution. The democratic constitution must be neutral from any explicit 72 72 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . claim of any comprehensive understanding. The democratic decision produced by the democratic con- stitution might not be in line with other comprehensive under- standings or does not satisfy the other religious adherents. But insofar as the procedure reflects justice, in the sense that that procedure has been stipulated trough agreement before the de- cision emerges, there is no reason for the other adherents of re- ligion not to obey that decision. In other words, they might be in disagreement with that decision, but they have to obey and respect it as long as it is stipulated through an agreed procedure. The inconsistency to the democratic procedure means inconsis- tency to the overriding commitment and inconsistency to the idea of religious freedom itself (Gamwell 1995, 164–67). The feeling or the stance that a given constitution is just might change from time to time. Walisongo: Jurnal Penelitian Sosial Keagamaan The Meaning of Religious Freedom A given religious adherent might feel that the decision making procedure written explicitly in the constitution is just and, therefore, they are bound to a commitment to it. But, later, after undertaking a series of de- bates, they find the decision-making procedure unjust. As a re- sult, they want to change their previous commitment. It is very relevant to mention the research result of Charles M. North and Carl R. Gwin (2004, 103–17) that was done in 59 countries and revealed that the establishment of state religion and consti- tutional protection of religion had a very significant opposing effect. The existence of a state religion reduces attendance of religious practices, whereas each decade of constitutional pro- tection increase attendance of religious practice significantly. It was also found that other measures of religious regulation have a negative effect on attendance. Besides that, it was found also that ironically the motive behind the establishment of a partic- ular state religion is to strengthen that religion, but the effects are ultimately undermine the vitality of the established religion. 73 A B U H A P S I N A B U H A P S I N The democratic discourse, however, does not regard changing commitment as an aberration of the commitment itself. Chang- ing commitment to the overriding decisions is justifiable. Those who disagree must have a room in the constitution in order to persuade the body politic, say the other religious adherents, that a certain decision-making procedure written in the constitution must be changed.While they have the opportunity to persuade the other religious adherents to change their commitment to the overriding decisions, at the same time they have to respect and comply with the decisions produced through that decision-mak- ing procedure with which they now disagree (Gamwell 1995, 167). With the above description, Gamwell stresses a distinction between formal and material claims about justice. The formal justice is a justice which is reflected by and manifested in the constitutional procedure that overrides the other decisions. As long as that overriding constitutional procedure is affirmed by the body politics, there has been the claim about justice. Thus, the affirmation of the body politic to the overriding constitu- tional procedure is a decisive factor to determine that the formal claim about justice has been fulfilled. While the material justice is a justice which is manifested in the decisions which are pro- duced through the constitutional procedure. In the democratic discourse, what has been at stake is the formal claim about justice. The material claim about justice might be different from one to another adherent of various re- ligious convictions. But, as long as the formal claim about jus- tice manifested in the procedure of making, executing, enforcing and changing decisions is affirmed by the adherents of all reli- gious convictions the democratic discourse can work out (Gam- well 1995). Thus, even though the material claim about justice manifested in, for instance, a given ordinance cannot satisfy the 74 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . whole body politic, all must obey that ordinance since it is stip- ulated through the overriding constitutional procedure that has fulfilled the criteria of the formal claim of justice. This is what has made Gamwell quite sure that the democratic resolution is the only way to solve the modern political problems related to religious freedom. Walisongo: Jurnal Penelitian Sosial Keagamaan A B U H A P S I N The full and free debate is a coherent consti- tutional principle which is able to unite a political community through a formal claim about justice (Gamwell 1995, 183). Although the democratic constitution emphasizes the im- portance of the procedural justice or formal claim about justice, this does not mean that the material justice can be neglected. The material claim about justice manifested in a given ordinance must also be tested. In other words, the comprehensive under- standing or the religious conviction that now becomes an or- dinance must be validated by appealing to human experience and reason (Gamwell 1995, 190). For instance, the body politic stipulates an ordinance allowing the practice of slavery. The or- dinance is, finally, stipulated through voting after having been debated freely and in accord with the affirmed constitutional procedure or procedural justice. Although this ordinance is stip- ulated through the procedural justice, it is not in line with the idea of democratic discourse since human reason and experience says that practicing slavery is inhuman. Thus, a religious convic- tion which is represented in the ordinance contradicts the idea of religious freedom. This ordinance implicitly indicates that there is no religious freedom. From the above explanation, it is clear that the democratic resolution entails the involvement of various legitimate compre- hensive understandings or various religious convictions to the play in a political arena. As a result, the democratic resolution of religious freedom as proposed by Gamwell can work out only if we change our paradigm from the thesis of “religion as non-ra- 75 A B U H A P S I N tional” to the thesis of “religion as rational”. The term “religion as rational” here means that every religious conviction must be ready to be freely and fully debated. Changing paradigm from the thesis of religion as non-ra- tional to religion as rational is a very significant precondition without which there would be no democratic discourse. Howev- er, the only public view or public debate is not enough to settle the modern political problems related to religious freedom. The public debate must be guided by a democratic constitution in or- der that the public debate could produce a democratic decision. The democratic constitution must fulfill the criteria of the formal claim about justice, that is a constitutional procedure that has had the affirmation of the body politic. A B U H A P S I N So important is the affirmed constitutional procedure that there would be no democratic discourse without it. Yet, the sole just constitutional procedure is not enough unless the decisions produced through that constitutional procedure also fulfill the criteria of the mate- rial claim about justice. The Features of the Islamic State The following discussion will describe the features of the Islamic State as proposed by Abdul Rauf in his book The Con- cept of Islamic State; with Particular Reference to Treatment of Non-Muslim Citizens (1988). Since his description is not wholly related to the concept of religious freedom advocated by Frank- lin I. Gamwell, I will discuss only four features: sovereignty, shu- ra or the consultative body, human equality and adherence to sharia or Islamic law. Abdul Rauf believes that in the Islamic State, the loyalty and sovereignty belong to Allah. He criticizes modern political ideas, stating that in them, loyalty should be addressed to territorial entity and sovereignty is in the hand of the people. This is differ- 76 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . ent from the Islamic State where the House of Representatives does not have an absolute and independent power, in the sense that it must always seek the revealed guidance, namely the Qu- ran and the Prophetic Tradition (Abdul Rauf 1988, 22). Under the Islamic political system, the rulers should rule on behalf of Allah. This resembles what has been done by the Prophet Muhammad (peace be upon him). The main duty of the rulers is executing what Allah has commanded. If there is a gap between the rulers and the House of Representatives, all have to seek the guidance from the sharia (Abdul Rauf 1988, 22). Since the loyalty and sovereignty belong to Allah, there is no room for nationality in Islam. Nationalism is justifiable only insofar as it is intended to mobilize the people to resist foreign domination (Abdul Rauf 1988, 22). The second feature of the Islamic State which is closely re- lated to the idea of religious freedom is that about Shura or the Consultative body. The Shura institution had long been the practice of the Muslim rulers from the time of the Prophet to the time of Ali, the fourth successor of the Prophet. The Shura terminated when the political system changed from Caliphate to dynasty system (Abdul Rauf 1988, 25–29). The Features of the Islamic State Even though the practice of mutual consultation in the early periods of Islam was conducted in a simple and natural way, this concept has laid down a good foundation for the sake of creating parliamentary as well as check and balance system as practiced in the modern political system. In the decision-making process, the first step to be done is mutual consultation. But if the member of Uli al-Amri (People Assembly) are in disagree- ment, the opinion of the majority should be binding even though this opinion might not in line with the wisdom of the chief ex- ecutive. Yet, to some extent, the chief executive still has the right to veto (Abdul Rauf 1988, 30–31). 77 Walisongo: Jurnal Penelitian Sosial Keagamaan A B U H A P S I N A B U H A P S I N The third feature of the Islamic State which is closely related to the concept of religious freedom is the commitment to sharia. In its broader sense, sharia means the total guidance derived from the Quran and from the model of the Prophet Muham- mad. On this account, the sharia covers regulation of all aspects of a human being either it is formulated in Islamic Theology, Islamic Ethics, Islamic Law or the other branches of Islamic sci- ence. However, in its narrow sense, sharia is always identified with Islamic Law (Abdul Rauf 1988, 38). The fourth feature of the Islamic State is that related to the concept of equality of human being. The Islamic State respects the principle of human equality for all citizens. Both Muslim and non-Muslim have an equal blood price. This is why, as the Quran stipulates, if a Muslim kills a non-Muslim, the Muslim is liable to the death penalty. In brief, in the Islamic State, no class or group of society has the right to special treatment. All of the society “enjoy the equal right and equal opportunity (Abdul Rauf 1988, 36–37).” In legal cases, criminal and civil cases, all citizens, regardless of their religious background, have to be treated equally. The blood and the wealth of the non-Muslim must be protected the same as the blood and the wealth of the Muslim citizen. The non-Muslims have the right to take part in the state services, such as health, education, and economy (Abdul Rauf 1988, 57– 59). In other words, like the Muslim citizen, the non-Muslim citizen has to be trusted to hold public offices and to serve in the military as long as they are qualified. The non-Muslim citi- zen must not suffer from any discrimination, except holding the position with religious nature, such as the Mufti (jurist consult) and the Imam or the leader of a religious congregation (Abdul Rauf 1988, 55; Rahman 1985; Sisk 1992). In the Islamic State the non-Muslims have the right to per- 78 Vol. 25, No.1 (2017) Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . form or to practice their religious convictions as long as doing so does not cause serious harm to others in the community. Walisongo: Jurnal Penelitian Sosial Keagamaan A B U H A P S I N They are not obliged to observe the prohibitions which are peculiar to Muslim, such as consuming fork and wine insofar as this does not invite serious harm to public security. However, the practice of usury, even if the non-Muslim believes it to be lawful, must be forbidden since this practice can elicit a bad influence on the whole society and on the economic condition in general (Abdul Rauf 1988, 50–51). Religious Freedom and Islamic State: Gamwellian Approach Religious Freedom and Islamic State: Gamwellian Approach In the previous section, I have reiterated the features of the Islamic State proposed by Abdul Rauf. I choose only four out of six features promoted by him since the other two features can be explicitly included in those four features. The features of politi- cal authority and the principle of justice can be included in the features of sovereignty and human equality. Actually, the term “Islamic State” itself indicates that the state explicitly supports Islam as the answer to the comprehen- sive question. According to Gamwell, this explicit support is, of course, not justifiable since it will eliminate the free and full public debate or public view (Gamwell 1995, 167, 186). That is why, as long as a state mentions the word “Islamic” explicitly, like the Islamic Republic of Pakistan or The Islamic Republic of Iran, the free and full debate as proposed by Gamwell cannot work out. Consequently, it is impossible to manifest the demo- cratic discourse. Stating the word “Islamic” in the constitution, however, will have a great impact on other decisions. A constitution is a pro- cedure that overrides the other decisions. In the democratic dis- course, it is not justifiable to eliminate the rights of the other adherents of different religious convictions to take part in the 79 A B U H A P S I N public debate. The explicit statement of the Word “Islamic” in the constitution, however, will eliminate the rights of the other religious adherents to express their religious convictions. This is, of course, not in line with the democratic discourse because eliminating the right to express a given religious conviction means eliminating the right to express self-authenticity. In view of this, a question emerges, can a state be named “Islamic State” without mentioning it explicitly in its constitution? If the answer is “no,” it means that, according to Gamwellian approach, there is no religious freedom. A similar question can be posed in relation to the concept of sovereignty. Can an Islamic State put an article in its consti- tution stating that the sovereignty shall be in the hand of the people? An explicit statement about “Allah’s Sovereignty” in the constitution, however, will blur our vision in distinguishing the meaning of political activity and religious activity. Both become indistinct. Religious Freedom and Islamic State: Gamwellian Approach As stated in section two, political activity is addressed to ask and answer explicitly to the question of the state, while religious activity is directed to ask and answer explicitly to the comprehensive question. Thus, if the words “Allah’s sovereign- ty” is written explicitly in a constitution, that constitution no more reflects a political activity. In order for the constitution to reflect the political activity, that constitution must explicitly state that the sovereignty shall be in the hand of the people. Also, the explicit statement about “Allah’s Sovereignty” means that the state affirms and endorse explicitly a given comprehensive conviction, namely the conviction of the people who believe in Allah. In the democratic discourse, the state cannot explicitly sup- port any answer to the comprehensive question. But this does not mean that the state eliminates the role of religion in political life. In the democratic discourse, the religious essential to the 80 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . body politic in the sense that every political decision should im- plicitly represent a valid comprehensive conviction.Whatis not in line with the democratic discourse is the “explicit” endorse- ment of the state to a given comprehensive conviction (Gamwell 1995, 205). This is why, as far as the words “Islamic” and “Al- lah’s Sovereignty” is explicitly stated in the constitution there would be no religious freedom. Suppose the articles which explicitly stated, “Allah’s sov- ereignty” and “Islamic State” in the constitution is the amend- ments produced through an overriding constitutional decision. The question here is, what kind of overriding constitutional de- cision or procedure can be used as a reference to produce these amendments? A democratic constitution, however, must eliminate any ex- plicit supporter explicit affirmation to any comprehensive con- viction (Gamwell 1995, 167, 186). Thus the overriding consti- tutional procedure is possibly formulated: “the state shall not explicitly support or affirm any religious conviction.” If this is the overriding constitutional procedure, then, how can these amendments be produced? If the above overriding constitution- al procedure is affirmed by the body politic, while at the same time there are some decisions, say articles, about “Allah’s sover- eignty” and “Islamic State” in the constitution, the constitution must be self-contradictory. Walisongo: Jurnal Penelitian Sosial Keagamaan Religious Freedom and Islamic State: Gamwellian Approach The other features of the Islamic State that need further dis- cussion are those related to the concept of Shura (Consultative Body) and human equality. As far as I know, Abdul Rauf does not elaborate the criteria for the membership of the Shura. Who can be the member of the Shura and how decision-making pro- cess should be done are not explained. According to the democratic discourse, the member of the Consultative Body must not be limited only to one or some ad- 81 Walisongo: Jurnal Penelitian Sosial Keagamaan A B U H A P S I N herent of religious conviction. This is the logical consequence of the idea that every citizen is free to express his or her own religious conviction which is believed to provide his or her au- thenticity as a human being. That is why there is no reason not to accommodate their aspiration in the Consultative Body. The way in which their aspiration is accommodated could be deferent from one to another country. In a more populated country, one member of the Consultative Body might represent many more constituents than he or she might do in a less popu- lated country. This procedure must be clear and must be stated explicitly in the constitution and must get the affirmation of the body politic. This constitutional procedure must give a room for the whole adherents of different religious convictions. The ques- tion of whether a given religious adherent can utilize that room or not is another thing. The most important thing is that the constitution must fulfill the requirement of what Gamwell call “formal claim about justice,” in the sense that the whole body politic, regardless of their different religious convictions, must affirm the constitutional procedure that overrides any decision (Gamwell 1995, 175). With the above explanation, I would argue that there would not be religious freedom in a country whose Consultative Body member is limited only to the adherents of a given religious conviction. Now, the question is, can an Islamic State provide a constitutional procedure that includes the whole body politic to have the same rights and opportunity to sit in the Shura (Con- sultative Body)? If the answer is “no,” it means that there is no religious freedom. Religious Freedom and Islamic State: Gamwellian Approach Thus, even though Abdul Rauf claims that the non-Muslim citizen has a full religious freedom (Abdul Rauf 1988, 45–51), as far as that procedure is not stated clearly and explicitly in the constitution, according to Gamwell, there is no religious freedom. 82 Vol. 25, No.1 (2017) Walisongo: Jurnal Penelitian Sosial Keagamaan R E L I G I O U S F R E E D O M A N D T H E I D E A . . . As stated in section two, in the democratic discourse, the formal claim about justice which is constituted in the constitu- tional procedure is primary. This is why Gamwell believes that religious freedom as modern political problems is nothing other than a constitutional matter (Gamwell 1995, 37). Because the democratic resolution to the modern political problems empha- sizes primarily on the constitutional procedure, consequently defining religious freedom and its ramifications such as the right to express and to manifest the religious conviction, the rights to sit in the Shura (Consultative Body) and the rights to sit in any political position cannot be dependent only on a given religious conviction. Thus, even though, for instance, the Muslims con- stitute the majority, the concept of religious freedom must be defined by the Muslims and the non-Muslims as well. Also, this concept must be written clearly and explicitly in the constitution and must get the affirmation of the whole adherents of different religious convictions in order to fulfill the formal claim about justice (Fox 2008). Even though in the democratic discourse, the formal claims about justice or the constitutional procedure is primary, this does not mean that the material claim about justice which is con- stituted in every decision can be neglected. Human reason and experience, however, will judge whether the decision produced through the overriding constitutional procedure is just or not. In other words, the human reason and experience shall be the crite- ria in order to measure the material claims about justice. On this account, the treatment of an Islamic State, as well as the concept of religious freedom offered by Abdul Rauf, is, of course, very convincing and beautiful (Abdul Rauf 1988, 45–51). Whoever reads these concepts will come to a conclusion that the Islamic State does fulfill the material claim about justice. However, for Gamwell, no matter how convincing and 83 Walisongo: Jurnal Penelitian Sosial Keagamaan A B U H A P S I N beautiful these concepts are, there would be no religious free- dom insofar as there is no full and free debate. This full and free debate can, of course, be done if only there is a procedural guarantee which is clearly and explicitly stated in a constitu- tional procedure. Thus, the guarantee of material claims about justice as described by Abdul Rauf will be meaningless unless it is accompanied by the procedural guarantee. R E L I G I O U S F R E E D O M A N D T H E I D E A . . . In the democrat- ic discourse, to repeat what I have stated, fulfilling the formal claim about justice is more important than fulfilling the material claim about justice. The last feature is that the Islamic State must have a deep commitment to the dictates of sharia (Abdul Rauf 1988, 38). Again, in the democratic discourse, the state is proscribed to “support the teaching of any religion or answer to the com- prehensive question”(Gamwell 1995, 167, 186). This does not mean that the state prohibits the adherents of Islam from having a deep commitment to the sharia. In the democratic discourse, sharia, which constitutes the answer to the comprehensive ques- tion, is essential to the Muslims. What is not in line with the democratic discourse is the explicit endorsement of the state to the sharia. Thus, the allegiance of the state to a given religious conviction and regards it as the state’s explicit answer to the comprehensive question is unjustifiable (Gamwell 1995, 205). In practice, the state might accept the capital punishment from the sharia, say Islam, but might reject its principle of rela- tive polygamy. Instead, the state might accept the idea of abso- lute monogamy believed by the Christian. Or, after being free- ly and fully debated through a just and affirmed constitutional procedure, the Shariah might become personal and criminal law. But, if the state accepts the sharia as the criminal and personal law, it is simply because the body politic agrees on it, not be- cause of the desire of the state to co-opt Islamic ideas and make 84 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . it as the answer to the comprehensive question. The political ac- tivity, however, is not intended to ask and answer explicitly the comprehensive question. Thus, as far as the criminal and per- sonal law which is derived from the sharia is stipulated through an affirmed constitutional procedure, there is no reason for the other religious adherents not to obey these laws insofar as both laws are not against human reason and experience. In the debate, let us say parliamentary debate, every religious adherent free to refer to their comprehensive understanding or religious convictions. Walisongo: Jurnal Penelitian Sosial Keagamaan R E L I G I O U S F R E E D O M A N D T H E I D E A . . . The Muslims are allowed to refer to their sharia, similarly, the Christians are allowed to refer to their ca- nonical book as a reference, the Buddhists are justifiable to base their argument on Tri Pitaka and the a-theist groups can use their personal truth to buttress their argument. In brief, every re- ligious adherent of a religious conviction is free to use his or her religious conviction as a reference as well as the basis for argu- ment (Gill 2007). This is different from the idea of free-standing justice of John Rawls which requires justification only from the ideas that are implicit in the political culture (Rawls 2005, 10). Hence, no religious conviction is important to politics. Religious conviction should be left behind when we play in the political arena. Even though the sharia has been the main element of the state’s political activities, the state must maintain its neutrality toward the sharia. The state cannot claim the sharia to be its comprehensive understanding of the comprehensive question. When the sharia has been the main element of the state’s politi- cal activity, its function changes. The sharia which was formerly regarded as the answer to the comprehensive question now has been the state’s political activity. Because the political activity is not intended to provide the answer to the comprehensive ques- tion, whatever the decision derived from the sharia must not be 85 A B U H A P S I N addressed to answer the comprehensive question. From the above explanation, its clear that Gamwellian ap- proach on religious freedom requires the neutrality of the state to support explicitly to a given religious conviction, including the sharia. In view of this, the question emerges, can a state be named “Islamic State” without mentioning explicitly about its commitment to the sharia as that proposed by Abdul Rauf? If the answer is “no”, there would be no full and free debate, and, without full and free debate, there would be no democratic dis- course, and, the absence of the democratic discourse means the absence religious freedom. Conclusion Gamwell believes that religious freedom is nothing other than a political discourse that can figure out only through a democratic resolution. According to the democratic resolution, it is justifiable to bring a given religious conviction into the po- litical arena because it is the religious conviction that provides the answer to the comprehensive question, and it is the answer to the comprehensive question that the authenticity of a human being can be found. Because the authenticity of a human being cannot be fragmented, it is necessary that the religious convic- tion is involved in the political arena. As a result, the religious conviction should be ready to be freely and fully debated. This is what Gamwell means with “religion as rational.” Changing paradigm from “religion as non-rational” to “re- ligion as rational” is a necessary condition for entering a public debate. Yet, the sole public debate or public view is not enough to solve the modern political problems. The public debate must be guided by a constitutional procedure affirmed by the body politic so that it fulfills the criteria of formal claim about jus- tice. So important is the affirmed constitutional procedure in 86 86 Vol. 25, No.1 (2017) R E L I G I O U S F R E E D O M A N D T H E I D E A . . . the democratic discourse that Gamwell believes that religious freedom is nothing other than constitutional matter. Because the modern political problem of religious freedom is a constitutional matter, the idea of establishing the Islamic State proposed by Abdul Rauf elicits at least four important questions. First, can a state be named “Islamic State” without mentioning the word “Islamic” in its constitution? Second, can a state be named “Islamic State” without mentioning explicitly the words “sovereignty belongs to Allah” in its constitution? Third, Can a state be named “Islamic State” without mentioning explicitly about its commitment to sharia in its constitution? Fourth, can a state be named “Islamic State” whose constitution states explic- itly that all adherents of different religious convictions have the equal opportunity and rights for any political position, includ- ing the position of the head of the state? If the answer of these four question is “yes”, it means that there is religious freedom. On the contrary, if the answer is “no”, it means that there is no religious freedom. Walisongo: Jurnal Penelitian Sosial Keagamaan Conclusion Another logical consequence of the dictum that “religious freedom is constitutional matter” is that the affirmation of the whole body politic is of a great significance to determine wheth- er or not a given constitutional procedure is just. Since the “affir- mation” of the body politic is a determinant, the formulation of the constitutional procedure related to religious freedom cannot be formulated only by the Muslims, even if they are the major- ity. This formulation requires the involvement of the body poli- tic without regard to their different religious conviction. This is why, even though Abdul Rauf claims that, in an Islamic State, the non-Muslims will have a just treatment and will have the rights to religious freedom, it will be meaningless unless these guarantees are explicitly stated in the constitution. 87 Walisongo: Jurnal Penelitian Sosial Keagamaan 87 Walisongo: Jurnal Penelitian Sosial Keagamaan Bibliography Abdul-Rahman, Muhammad Saed. 2009. Tafsir Ibn Kathir Juz’ 3 (Part 3) : Al-Baqarah 253 To Al-I-’Imran 92. 2nd ed. London: MSA Publication Ltd. Abdul Rauf, Tan Sri Muhammad. 1988. The Concept of Is- lamic State; With Particular Reference to Treatment of Non-Muslim Citizens. Malaysia: Islamic Affairs Division Prime Minister’s Department. Al-ʻUmari, Akram Diyaʼ. 1991. Madīnan Society at the Time of the Prophet Vol I. Virginia: International Institute of Is- lamic Thought. Asad, Muhammad. 1980. The Message of the Quran. Lahore: Maktaba Jawahar ul-Uloom. 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Geneva: World Council of Churches Publications. Koshy, Ninan. 1992. Religious Freedom in a Changing World / Ninan Koshy. - Version Details - Trove. Geneva: World Council of Churches Publications. Mayer, Ann Elizabeth. 1994. “Universal Versus Islamic Human Rights: A Clash of Cultures or a Clash with a Construct?” Michigan Journal of International Law 15 (2). University of Michigan Law School. North, Charles M, and Carl M. Gwin. 2004. “Religious Free- dom and Unintended Consequences of State Religion.” Southern Economic Journal 71 (1): 103–17. Rahman, Fazlur. 1985. “Laws and Ethics in Islam.” In Ethics in Islam, edited by Richard G. Van Hovanism. Malibu: Ude- na Publication. Rawls, John. 2005. Political Liberalism. New York: Columbia University Press. Sisk, Timothy D. 1992. Islam and Democracy; Religion Politics and Power in the Middle East. Washington D.C.: United States Institutes of Peace Press. 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https://openalex.org/W2975195667	https://amt.copernicus.org/articles/12/5137/2019/amt-12-5137-2019.pdf	English	null	A new discrete wavelength backscattered ultraviolet algorithm for consistent volcanic SO&lt;sub&gt;2&lt;/sub&gt; retrievals from multiple satellite missions	Atmospheric measurement techniques	2,019	cc-by	14,954	Correspondence: Bradford L. Fisher (bradford.ﬁsher@ssaihq.com) Our results suggest that the discrete-wavelength MS_SO2 algorithm, al- though less sensitive than hyperspectral PCA algorithm, can be adapted to retrieve volcanic SO2 VCDs from contempo- rary hyperspectral UV instruments, such as OMI and OMPS, to create consistent, multi-satellite, long-term volcanic SO2 climate data records. 1 Introduction Volcanic eruptions are an important natural driver of global climate change, but, unlike other natural climate forcing (e.g., changes in Earth’s orbit, solar irradiance), the magni- tude of volcanic forcing is highly variable and largely un- predictable, and the effects are typically more transient. Of most interest are the episodic, large injections of volcanic sulfur dioxide (SO2) into the Earth’s stratosphere by major explosive volcanic eruptions, the most recent example be- ing the eruption of Pinatubo (Philippines) in June 1991 (e.g., Bluth et al., 1992; Guo et al., 2004). Stratospheric loading of volcanic SO2 by major eruptions leads to the formation of sulfuric acid (or sulfate) aerosols that scatter incoming solar shortwave radiation and absorb outgoing thermal radiation over timescales of months to years, cooling the troposphere and warming the stratosphere (e.g., Robock, 2000). Primary Atmos. Meas. Tech., 12, 5137–5153, 2019 https://doi.org/10.5194/amt-12-5137-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Atmos. Meas. Tech., 12, 5137–5153, 2019 https://doi.org/10.5194/amt-12-5137-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Correspondence: Bradford L. Fisher (bradford.ﬁsher@ssaihq.com) Correspondence: Bradford L. Fisher (bradford.ﬁsher@ssaihq.com) Received: 12 April 2019 – Discussion started: 14 May 2019 Revised: 8 August 2019 – Accepted: 14 August 2019 – Published: 25 September 2019 Received: 12 April 2019 – Discussion started: 14 May 2019 Revised: 8 August 2019 – Accepted: 14 August 2019 – Published: 25 September 2019 Received: 12 April 2019 – Discussion started: 14 May 2019 Received: 12 April 2019 Discussion started: 14 May 2019 Revised: 8 August 2019 – Accepted: 14 August 2019 – Published: 25 September 2019 Abstract. This paper describes a new discrete wavelength algorithm developed for retrieving volcanic sulfur dioxide (SO2) vertical column density (VCD) from UV observ- ing satellites. The Multi-Satellite SO2 algorithm (MS_SO2) simultaneously retrieves column densities of sulfur diox- ide, ozone, and Lambertian effective reﬂectivity (LER) and its spectral dependence. It is used operationally to pro- cess measurements from the heritage Total Ozone Mapping Spectrometer (TOMS) onboard NASA’s Nimbus-7 satellite (N7/TOMS: 1978–1993) and from the current Earth Poly- chromatic Imaging Camera (EPIC) onboard Deep Space Climate Observatory (DSCOVR: 2015–ongoing) from the Earth–Sun Lagrange (L1) orbit. Results from MS_SO2 al- gorithm for several volcanic cases were assessed using the more sensitive principal component analysis (PCA) algo- rithm. The PCA is an operational algorithm used by NASA to retrieve SO2 from hyperspectral UV spectrometers, such as the Ozone Monitoring Instrument (OMI) onboard NASA’s Earth Observing System Aura satellite and Ozone Mapping and Proﬁling Suite (OMPS) onboard NASA–NOAA Suomi National Polar Partnership (SNPP) satellite. For this com- parative study, the PCA algorithm was modiﬁed to use the discrete wavelengths of the Nimbus-7/TOMS instrument, de- scribed in Sect. S1 of the Supplement. Our results demon- strate good agreement between the two retrievals for the largest volcanic eruptions of the satellite era, such as the 1991 Pinatubo eruption. To estimate SO2 retrieval systematic uncertainties, we use radiative transfer simulations explicitly accounting for volcanic sulfate and ash aerosols. Our results suggest that the discrete-wavelength MS_SO2 algorithm, al- though less sensitive than hyperspectral PCA algorithm, can be adapted to retrieve volcanic SO2 VCDs from contempo- rary hyperspectral UV instruments, such as OMI and OMPS, to create consistent, multi-satellite, long-term volcanic SO2 climate data records. uncertainties, we use radiative transfer simulations explicitly accounting for volcanic sulfate and ash aerosols. Bradford L. Fisher1, Nickolay A. Krotkov2, Pawan K. Bhartia2, Can Li2,3, Simon A. Carn4, Eric Hughes5, and Peter J. T. Leonard6 1SSAI, Suite 600, 10210 Greenbelt Rd, Lanham, MD 20706, USA 2Atmospheric Chemistry and Dynamics Laboratory, NASA Goddard Space Flight Center, Greenbelt, MD 20771, USA 3Earth System Science Interdisciplinary Center, University of Maryland, College Park, MD 20742, USA 4Geological and Mining Engineering and Sciences, Michigan Technological University, Houghton, MI 49931, USA 5Miner & Kasch, 8174 Lark Brown Rd, Ofﬁce no. 101, Elkridge, MD 21075, USA 6ADNET Systems, Inc., Suite A100, 7515 Mission Drive, Lanham, MD 20706, USA Published by Copernicus Publications on behalf of the European Geosciences Union. Eruptions (star symbols) are color coded by esti- mated plume altitude and derived from a variety of sources, in- cluding Smithsonian Institution Global Volcanism Program vol- canic activity reports, volcanic ash advisories and satellite data. The annual total explosive volcanic SO2 production (omitting SO2 discharge from effusive eruptions) is shown in black. The or- ange lines above the plot indicate the operational lifetimes of NASA UV satellite instruments: Nimbus-7 (N7), Meteor-3 (M3), ADEOS (AD), Earth Probe (EP) TOMS, OMI (currently opera- tional), and SNPP/OMPS (currently operational), along with the ESA/EU Copernicus S5P/TROPOMI (currently operational). Data shown in this plot are available from the NASA Goddard Earth Sci- ences (GES) Data and Information Services Center (DISC) as a level 4 MEaSUREs (Making Earth System Data Records for Use in Research Environments) data product (Carn, 2019). 2006), the Ozone Mapping and Proﬁler Suite (OMPS, 2012– ongoing; Carn et al., 2015), and EU/ESA Copernicus Sen- tinel 5 precursor (S5P) (Veefkind et al., 2012). This is mani- fested in Fig. 1 as an increased number of detected volcanic eruptions with low SO2 loading (< 10 kt) after 2004 (note that GOME and SCIAMACHY measurements are not shown in Fig. 1), whereas rates of global volcanic activity have not changed signiﬁcantly. UV SO2 retrieval algorithms have also evolved substantially since the 1980s in response to advances from multispectral to hyperspectral sensors, improvements in ozone retrievals, and efforts to account for volcanic ash and aerosol interference (e.g., Krueger et al., 1995, 2000; Krotkov et al., 1997, 2006; Yang et al., 2007, 2010; Li et al., 2013, 2017; Theys et al., 2015). However, to date there has been no attempt to develop a single algorithm that could be used to generate a long-term, consistent SO2 climatology across multiple UV satellite missions. In this paper we de- scribe a new multi-satellite SO2 algorithm (MS_SO2) that is applicable to both multispectral (e.g., TOMS) and hyper- spectral (e.g., OMI) UV measurements. As a ﬁrst step in the generation of a multi-satellite volcanic SO2 record, we apply One of the key challenges in assembling a long-term, con- sistent, satellite-based volcanic SO2 emissions climatology (e.g., Fig. 1) is merging measurements from sensors with different spectral coverage and resolution. This complicates any analysis of “trends” in volcanic SO2 loading (e.g., in the post-2000 period of moderate volcanic eruptions; Fig. 1) or comparisons of eruptions of similar magnitude in different satellite instrumental eras. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. L. Fisher et al.: A new discrete wavelength BUV algorithm Figure 1. Multi-decadal record of SO2 emissions by volcanic eruptions observed by NASA’s ﬂeet of satellites observing TOA UV radiances. Eruptions (star symbols) are color coded by esti- mated plume altitude and derived from a variety of sources, in- cluding Smithsonian Institution Global Volcanism Program vol- canic activity reports, volcanic ash advisories and satellite data. The annual total explosive volcanic SO2 production (omitting SO2 discharge from effusive eruptions) is shown in black. The or- ange lines above the plot indicate the operational lifetimes of NASA UV satellite instruments: Nimbus-7 (N7), Meteor-3 (M3), ADEOS (AD), Earth Probe (EP) TOMS, OMI (currently opera- tional), and SNPP/OMPS (currently operational), along with the ESA/EU Copernicus S5P/TROPOMI (currently operational). Data shown in this plot are available from the NASA Goddard Earth Sci- ences (GES) Data and Information Services Center (DISC) as a level 4 MEaSUREs (Making Earth System Data Records for Use in Research Environments) data product (Carn, 2019). B. L. Fisher et al.: A new discrete wavelength BUV algorithm 5138 volcanic emissions of aerosols such as volcanic ash can also have atmospheric and climatic impacts, but these are typi- cally more short-lived. Volcanic eruptions can also release reactive halogen species into the atmosphere, such as chlo- ride and bromide (Mankin and Coffey, 1984; Bobrowski et al., 2003; Kern et al., 2009). Halogens can impact the to- tal column ozone amount and proﬁle shape if injected into the lower stratosphere (Solomon et al., 1998; Klobas et al., 2017), but sulfate aerosols are also required to catalyze the heterogeneous chemical reactions that can efﬁciently deplete ozone. Hence, to understand the impacts of volcanic erup- tions on climate, and in order to predict possible outcomes in the event of a major eruption, long-term satellite measure- ments of volcanic SO2 emissions are essential. The satellite record of volcanic SO2 emissions by major volcanic eruptions extends back to 1978 and has been de- rived from instruments operating in both the ultraviolet (UV) and infrared (IR) spectral bands (Fig. 1; e.g., Carn et al., 2003, 2016; Carn, 2019; Prata et al., 2003). Published by Copernicus Publications on behalf of the European Geosciences Union. Measurements in the UV band have a longer heritage, since the ﬁrst satel- lite detection of volcanic SO2 was achieved by the UV Total Ozone Mapping Spectrometer (TOMS) in 1982 following the eruption of El Chichón (Mexico; Krueger, 1983; Krueger et al., 2008), and interference from volcanic SO2 must be ac- counted for in order to produce accurate, long-term UV mea- surements of ozone. UV measurements have greater sensitiv- ity to the total atmospheric SO2 column than IR retrievals, and hence the former have been the mainstay of volcanic SO2 monitoring during the satellite era to date. The volcanic SO2 climatology from 1978 to the present (Fig. 1, Carn, 2019) reveals highly variable inter-annual volcanic SO2 forc- ing dominated by two major eruptions (El Chichón in 1982 and Pinatubo in 1991), with the post-2000 period dominated by smaller eruptions. Although none of these smaller erup- tions have, individually, produced measurable climate ef- fects, collectively they have garnered signiﬁcant interest as they may play an important role in sustaining the persistent, background stratospheric aerosol layer, which is an impor- tant factor in global climate forcing (e.g., Solomon et al., 2011; Vernier et al., 2011; Ridley et al., 2014). Figure 1. Multi-decadal record of SO2 emissions by volcanic eruptions observed by NASA’s ﬂeet of satellites observing TOA UV radiances. Eruptions (star symbols) are color coded by esti- mated plume altitude and derived from a variety of sources, in- cluding Smithsonian Institution Global Volcanism Program vol- canic activity reports, volcanic ash advisories and satellite data. The annual total explosive volcanic SO2 production (omitting SO2 discharge from effusive eruptions) is shown in black. The or- ange lines above the plot indicate the operational lifetimes of NASA UV satellite instruments: Nimbus-7 (N7), Meteor-3 (M3), ADEOS (AD), Earth Probe (EP) TOMS, OMI (currently opera- tional), and SNPP/OMPS (currently operational), along with the ESA/EU Copernicus S5P/TROPOMI (currently operational). Data shown in this plot are available from the NASA Goddard Earth Sci- ences (GES) Data and Information Services Center (DISC) as a level 4 MEaSUREs (Making Earth System Data Records for Use in Research Environments) data product (Carn, 2019). Figure 1. Multi-decadal record of SO2 emissions by volcanic eruptions observed by NASA’s ﬂeet of satellites observing TOA UV radiances. 2 Heritage satellite ozone and SO2 algorithms Ozone and SO2 are the two main absorbers in the near UV spectral region between 300 and 340 nm. The relative contri- butions of each gas to the satellite backscattered ultraviolet (BUV) measurements at the three absorbing TOMS channels (317, 331, 340 nm) used in the retrieval, depend on the spec- tral structure of the absorption cross sections, which are mea- sured as functions of wavelength and temperature (Bogumil et al., 2003; Daumont et al., 1992). Figure 2 shows the O3 and SO2 cross sections and the SO2/O3 cross section ratio as a function of wavelength for a spectral UV region span- ning the three absorbing channels of TOMS. At the instru- ment’s spectral resolution (∼1 nm full width at half maxi- mum, FWHM), the SO2 molecule is 2.5 times more absorb- ing than O3 at 317 nm, while O3 is 6 times more absorbing at 331 nm. These differences allow for simultaneous multispec- tral retrievals of O3 and SO2. By explicitly modeling the effect of aerosols using a radia- tive transfer code, Dave (1978) showed that Rs did not vary signiﬁcantly with wavelength for non-absorbing aerosols, hence they produced no ozone error. However, for aerosols that might have strong absorption in the UV, that study pre- dicted that Rs would decrease at shorter wavelengths, pro- ducing an overestimation of ozone. Since aerosol properties in the UV were not known at that time, no correction for aerosol absorption was applied until the mid-1990s when the effect predicted by Dave (1978) was detected in the Nimbus- 7 TOMS data launched in October 1978. Published by Copernicus Publications on behalf of the European Geosciences Union. These proﬁles are then used to esti- mate I, which varies with wavelength (λ), observational ge- ometry, surface pressure and surface reﬂectivity (R). Follow- ing the Dobson convention, log(I) is converted to N value which is deﬁned in Eq. (1) as Figure 2. Spectral dependence of laboratory-measured SO2 (black) and O3 (red) cross sections between 310 and 340 nm at TOMS FWHM ∼1 nm. The SO2/O3 ratio (green) is shown with the scale on the right axis. The nominal locations of the N7/TOMS absorbing bands (317, 331, 340 nm) are shown by vertical blue lines (blue). N = −100log10 I F . (1) (1) F is the extraterrestrial solar irradiance. By linearly inter- polating N between total ozone nodes, one forms the N- curves that are a single valued function of representative of a given latitude band and observational geometry. This ap- proach allows to be estimated by matching the measured N value to the interpolated N values. the MS_SO2 algorithm to the Nimbus-7 TOMS (N7/TOMS) measurements (1978–1993) and present a reanalysis of some of the most signiﬁcant eruptions of the N7/TOMS mission. Over the years several modiﬁcations have been intro- duced to this basic concept. Mateer et al. (1971) proposed a Lambert-equivalent reﬂectivity (LER) concept to estimate the combined contribution of the surface, clouds and aerosols to BUV radiance. In this concept, the scene at the bottom of the atmosphere is assumed to be a Lambertian reﬂector, whose reﬂectivity (Rs) is derived from the measurements at 380 nm where the ozone and SO2 absorption is negligible. The effective pressure of this reﬂecting surface is assumed to vary with Rs, from a surface pressure at Rs < 0.2 to a cloud pressure 0.4 atm at Rs > 0.6, linearly interpolated at interme- diate Rs. The algorithm assumed that Rs, thus derived, did not vary with wavelength. Although in the earlier versions of this algorithm wavelength pairs (313/331, 318/340) were used to derive , Rs was later derived at 331 nm to mini- mize errors due to the spectral dependence of Rs. This made pairing unnecessary (McPeters et al., 1996). Published by Copernicus Publications on behalf of the European Geosciences Union. A step change in SO2 sensitivity occurred when the multispectral, six-channel TOMS instru- ments were superseded by hyperspectral UV sensors, such as the Global Ozone Monitoring Experiment (GOME, 1995– 2003; Khokhar et al., 2005), the Scanning Imaging Absorp- tion Spectrometer for Atmospheric Chartography (SCIA- MACHY, 2002–2012; Lee et al., 2008), the Ozone Mon- itoring Instrument (OMI, 2004–ongoing; Krotkov et al., Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ B. L. Fisher et al.: A new discrete wavelength BUV algorithm B. L. Fisher et al.: A new discrete wavelength BUV algorithm 5139 B. L. Fisher et al.: A new discrete wavelength BUV algorith Figure 2. Spectral dependence of laboratory-measured SO2 (black) and O3 (red) cross sections between 310 and 340 nm at TOMS FWHM ∼1 nm. The SO2/O3 ratio (green) is shown with the scale on the right axis. The nominal locations of the N7/TOMS absorbing bands (317, 331, 340 nm) are shown by vertical blue lines (blue). (TOA) BUV radiances (I) do not follow the Beer–Lambert law. In general, log(I) varies nonlinearly with ozone column amount (), and this relationship is sensitive to the shape of the ozone proﬁle (deﬁned as the ozone density proﬁle nor- malized to total ozone). To account for this effect, Dave and Mateer (1967) proposed constructing a set of lookup tables (LUTs) based on standard ozone proﬁles with different total ozone amounts using ozonesonde and Dobson Umkehr data. Since the shapes of the proﬁles also vary with latitude, they proposed using three sets of proﬁles for low latitudes, midlat- itudes and high latitudes. These proﬁles are then used to esti- mate I, which varies with wavelength (λ), observational ge- ometry, surface pressure and surface reﬂectivity (R). Follow- ing the Dobson convention, log(I) is converted to N value which is deﬁned in Eq. (1) as (TOA) BUV radiances (I) do not follow the Beer–Lambert law. In general, log(I) varies nonlinearly with ozone column amount (), and this relationship is sensitive to the shape of the ozone proﬁle (deﬁned as the ozone density proﬁle nor- malized to total ozone). To account for this effect, Dave and Mateer (1967) proposed constructing a set of lookup tables (LUTs) based on standard ozone proﬁles with different total ozone amounts using ozonesonde and Dobson Umkehr data. Since the shapes of the proﬁles also vary with latitude, they proposed using three sets of proﬁles for low latitudes, midlat- itudes and high latitudes. 2.1 Heritage BUV ozone algorithms Dave and Mateer (1967) ﬁrst proposed a technique to esti- mate total ozone column from nadir backscatter UV mea- surements taken in the Huggins ozone absorption band (310– 340 nm), assuming no SO2 is present. Their algorithm was inspired by the pioneering Dobson spectrophotometer, which measures attenuation of solar irradiance by UV wavelength pairs from which total ozone is derived using the Beer– Lambert law. However, unlike the direct sun technique, radia- tive transfer calculations show that the top-of-the-atmosphere Since the TOMS instrument had three reﬂectivity channels (331, 340, 380 nm), it was possible to compare the reﬂectiv- ities derived from them. This comparison showed that Rs in- www.atmos-meas-tech.net/12/5137/2019/ 2.2 Heritage TOMS SO2 algorithms Krueger (1983) was the ﬁrst to suggest that TOMS could be used to retrieve sulfur dioxide from explosive volcanic events. He correctly interpreted the large positive ozone anomaly observed following the explosive eruption of El Chichón in 1982 as being due to the SO2 released into the atmosphere during the event. To estimate the SO2 inside the plume region, he separated the SO2 and O3 signals by com- puting a residual reﬂectance, estimated as the difference be- tween the interpolated unperturbed background reﬂectances outside the plume and the reﬂectance anomaly inside the plume. This early technique for retrieving SO2 from TOMS ozone estimates became known as the residual method. The residual method, however, failed when the background could not be clearly separated from the ozone anomaly. Krueger subsequently developed the ﬁrst BUV algorithm that sepa- rated the O3 and SO2 radiance contributions, based on an ear- lier methodology developed by Kerr et al. (1980) to retrieve the SO2 column from the ground with a Brewer spectropho- tometer. This method assumed that the BUV radiation was attenuated by the two absorbing species (O3, SO2), leading to an equation describing BUV radiance, I, for a given wave- length, λ, corresponding to the TOMS ﬁeld of view (FoV): Performing online radiative transfer calculations for every satellite ﬁeld-of-view (FoV) can greatly increase the time re- quired to process full orbits of data. To improve the compu- tational efﬁciency of the operational algorithm, N7TOMS- speciﬁc lookup tables (N7TOMS-LUT) were produced of- ﬂine using the inputs listed in Table 1 and convolved with the triangular band pass at each of the six Nimbus-7 TOMS wavelengths (FWHM ∼1 nm). I (λ) = aF (λ)exp −bλ + Sg(τO3 + τSO2) . (2) I (λ) = aF (λ)exp −bλ + Sg(τO3 + τSO2) . (2) The TOMRAD algorithm was conﬁgured to account for two absorbing trace gases: O3 and SO2. The LUTs include 21 total ozone nodes and 12 total SO2 nodes for each of the three assumed SO2 heights. For ozone, the total column amounts and proﬁle shapes vary between latitude bands (see Table 1). For sulfur dioxide, we assumed a Gaussian verti- cal proﬁle shape, which is determined by two parameters: a center of mass altitude (CMA) and a geometrical standard deviation. The CMA represents the altitude of the peak SO2 In Eq. B. L. Fisher et al.: A new discrete wavelength BUV algorithm B. L. Fisher et al.: A new discrete wavelength BUV algorithm 5140 creased signiﬁcantly with wavelength for moderately thick clouds, causing a signiﬁcant underestimation of (up to 3 %). A modiﬁed LER (MLER) concept assuming two Lam- bertian surfaces, one at the surface and the other at the cloud top was applied to minimize this error (Ahmad et al., 2004). umn densities and the dimensionless parameters a and b. This algorithm is generally referred to as the Krueger–Kerr algorithm (Krueger et al., 1995). Krotkov et al. (1997) de- veloped radiative transfer path correction, which explicitly accounted for the Rs, ozone and SO2 vertical proﬁles, replac- ing the geometrical AMF in Eq. (2). The modiﬁed algorithm with empirical background correction has been used ofﬂine on a case-by-case basis for the past 2 decades to retrieve SO2 mass tonnage from medium to large explosive eruptions us- ing TOMS BUV measurements (Krueger et al., 2000; Carn et al., 2003). p pp The most recent version of the TOMS ozone algorithm re- verts back to the LER model, but it assumes that clouds are at the surface, which reduces the Rs wavelength dependence (Ahmad et al., 2004). This simple LER (SLER) model is used in our SO2 algorithm. However, since there are many other reasons for such a dependence, including ocean color, non- Lambertian surfaces, such as ocean glint and fogbow and, most importantly, the absorbing aerosol effect predicted by Dave (1978), Rs is assumed to vary linearly with λ; its slope is derived using 340 and 380 nm radiances. This simple om- nibus approach works well for most cases, except when the UV absorbing aerosols (smoke, dust and volcanic ash) are very thick. Such data are ﬂagged in the TOMS ozone algo- rithm. The new MS_SO2 algorithm is an extension of this algorithm into two dimensions (Sect. 3). 3 New MS_SO2 algorithm The new discrete wavelength SO2 algorithm (MS_SO2) builds on the heritage of the TOMS total ozone algorithm (Sect. 2.1) but adds sulfur dioxide (SO2) as a second ab- sorber. The BUV radiance is simulated with the TOMRAD forward vector radiative transfer (RT) model (Dave, 1964) for a known viewing geometry by assuming a vertically in- homogeneous, pseudo-spherical Rayleigh scattering atmo- sphere with standard ozone proﬁles (Klenk et al., 1983; Bhar- tia, 2002) and a priori SO2 vertical proﬁles (Krueger et al., 1995). The underlying reﬂecting surfaces (land/ocean, clouds and aerosols) are approximated with the simple LER re- ﬂecting surface at terrain height pressure (Sect. 2.1). TOM- RAD accounts for all orders of polarized Rayleigh scatter- ing and for the gaseous absorption (e.g., O3 and SO2), us- ing a priori vertical proﬁles of the gas concentrations and laboratory-measured temperature-dependent gaseous cross sections (Dave and Mateer, 1967; Bogumil et al., 2003; Daumont et al., 1992). Improvements to the TOMRAD model include corrections for molecular anisotropy (Ahmad and Bhartia, 1995), rotational Raman scattering (Joiner et al., 1995) and pseudo-spherical corrections to account for changes to the solar and viewing zenith angles due to the sphericity of the Earth. www.atmos-meas-tech.net/12/5137/2019/ Atmos. Meas. Tech., 12, 5137–5153, 2019 B. L. Fisher et al.: A new discrete wavelength BUV algorithm Table 1. Input parameters used in construction of the Nimbus-7 TOMS LUTs. Table 1. Input parameters used in construction of the Nimbus-7 TOMS LUTs. Table 1. Input parameters used in construction of the Nimbus-7 TOMS LUTs. LUT node Number of nodes Values Surface pressure 2 1013.25 and 500 hPa Wavelength 6 312.5, 317, 331, 340, 360 and 380 Standard ozone proﬁles 21 3 low-latitude, 8 midlatitude and (TOMS version 8) 10 high-latitude bands Gaussian SO2 proﬁles 12 for each CMA 0, 5, 10, 50, 100, 150, 200, (8, 13, 18 km) 250, 350, 450, 550, 650 DU SZA 10 0, 30, 45, 60, 70, 77, 81, 84, 86, 88 VZA 6 0, 15, 30, 45, 60, 70 Table 2. Retrieved state vector. n/a – not applicable. Retrieved parameter Wavelength Symbol First guess∗ (nm) Total column SO2 317 6 60 = 0 Total column O3 331 0 Spectral reﬂectivity 340 dR/dλ dR0/dλ = 0 Dependence Reﬂectivity 380 Rs n/a ∗0 is a climatological value for each of the three latitude bands. Table 2. Retrieved state vector. n/a – not applicable. concentration. LUTs for SO2 are generated for three differ- ent CMAs: 8 km (middle troposphere, TRM), 13 km (upper tropospheric, TRU) and 18 km (lower stratospheric, STL). A constant standard deviation of σ = 2 km is assumed for each SO2 proﬁle. The MS_SO2 algorithm retrieves a four-parameter state vector, x,deﬁned below as x =   6 dRs/dλ Rs  , (3) ∗0 is a climatological value for each of the three latitude bands. (3) The reﬂectivity Rs is computed analytically using the mea- sured BUV radiance at 380 nm (see Supplement, Eq. S4). Note that since the O3 and SO2 cross sections are negligi- ble at 380 nm, the Rs and ∂N380/∂Rs do not change with the iterations (i.e., dRs = 0). where 6 is the retrieved total column sulfur dioxide, is the total column ozone, dR/dλ characterizes the Rs spectral dependence between 340 and 380 nm, and Rs is the LER at 380 nm. The retrieval of sulfur dioxide is carried out in one or two steps described in the next sections, referred to as step 1 and step 2. Equation (4) is solved iteratively by zeroing the residuals, dN = Nm-Nc, and recomputing the Nc and the Jacobians at each iteration step for the four used channels. 3.1 Step 1 retrieval Our step 1 inversion starts with an initial state vector x0, con- sisting of ﬁrst guesses for 60, 0 and dRs0/dλ shown in Ta- ble 2. The ﬁnal state vector, x, is determined iteratively by inverting the Jacobian matrix K at each iteration step: dNk = Nm −Nc,k−1 = Kk−1dxk, (6a) dxk = xk −xk−1 = K−1 k−1dNk. (6b) (6a) (6a) (6b) (6b) Since O3 and SO2 exhibit small absorption at 340 nm, a nonzero R spectral slope (i.e., dR/dλ ̸= 0) accounts for the radiative effects of aerosols and surface reﬂectance (e.g., sun glint). = Kdx, (4) (4) B. L. Fisher et al.: A new discrete wavelength BUV algorithm The state vector is then adjusted after each iteration, xk = xk−1 + dxk, k = 1,2,... until it converges on a solution as described below: dN = Kdx, where dx represents the relative changes in the state vector from the previous iteration and dN represents the residual vector equal to Nm−Nc, computed as the difference between the measured N values, Nm, and the calculated N values, Nc, at the four TOMS channels at 317, 331, 340 and 380 nm. K represents a 4×4 Jacobian matrix computed from the LUTs. These matrix elements are deﬁned as follows: where dx represents the relative changes in the state vector from the previous iteration and dN represents the residual vector equal to Nm−Nc, computed as the difference between the measured N values, Nm, and the calculated N values, Nc, at the four TOMS channels at 317, 331, 340 and 380 nm. K represents a 4×4 Jacobian matrix computed from the LUTs. These matrix elements are deﬁned as follows: As indicated in Table 2, the algorithm initially assumes zero R-λ dependence (i.e., dR/dλ = 0); however, absorbing aerosols (smoke, dust and volcanic ash) cause dR/dλ ̸= 0. The algorithm uses retrieved spectral slope dR/dλ in Eq. (7) below to update the calculated LERs after each it- eration: Ki,j = ∂Nc,i ∂xj , i,j = 1,4, (5) Rj = Rs + ∂R ∂λ × λj −λR j = 1,4, (7) (5) (7) where λj = 312, 317, 331, 340 nm and λR = 380 nm. When SO2 or aerosol loading is high nonlinear R-λ dependence where Nc,i is the forward model calculated N value at wave- length i. 2.2 Heritage TOMS SO2 algorithms (2), F is the incoming solar ﬂux, Sg is the geomet- rical optical path (air mass factor, AMF), and τO3 and τSO2 are the vertical optical thicknesses for O3 and SO2, while the coefﬁcients a and b depend on the satellite viewing ge- ometry, cloud or surface reﬂectance, and volcanic ash and sulfate aerosols (Krueger et al., 1995; Krotkov et al., 1997). Equation (2) can be expressed in matrix form, which is then inverted to obtain estimates for the SO2 and O3 vertical col- Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ 5141 3.2 Step 2 retrieval The MS_SO2 forward model accounts for O3 and SO2 ab- sorption and linear spectral changes in Rs due to the pres- ences of aerosols. The algorithm, however, does not explic- itly characterize the absorption and scattering effects of vol- canic ash (absorbing) and sulfate (non-absorbing) aerosols. The retrieval errors in 6 and caused by volcanic ash dur- ing the ﬁrst few days after an explosive eruption can be sig- niﬁcant in the case of major volcanic eruptions like Pinatubo and El Chichón (Krueger et al., 1995; Krotkov et al., 1997). A step 2 procedure was developed primarily to handle ex- plosive eruptions (Volcanic Explosivity Index, VEI > 3 ), in which large anomalies are identiﬁed to occur in conjunc- tion with high ash concentrations. In step 2, a corrected to- tal ozone cor inside the SO2 cloud is interpolated using the retrieved outside the plume along the orbit for each cross-track position. Even if ozone-destroying chemicals are present, such effects can still be considered negligible over the relatively short time periods that SO2 concentrations are high enough to affect TOMS observations. Figure 3. Aerosol index for the El Chichón eruption on 4 April 1982, computed from retrieved dR/dλ. To illustrate the effects of the step 2 procedure, we con- sider the 1982 explosive eruption of El Chichón, which emit- ted ∼7 Tg SO2 (Krueger et al., 2008) the second largest ob- served in the satellite era (Fig. 1). Figure 3 shows the re- trieved AI map during TOMS overpass of the volcano on 4 April 1982, while it was still erupting. High AI values ex- ceeding a value of 10 correspond to biased high step 1 ozone values (Fig. 4a) and underestimated 6 values (Fig. 4c). Fig- ure 4b shows the step 2 corrected cor, making it consis- tent with the ﬁeld outside of the volcanic cloud. Figure 4d shows the step 2 6, which is much higher than step 1. As can be seen in this particular example, the step 2 correction can signiﬁcantly increase the SO2 mass. In this case, the SO2 mass increased from 2475 (step 1) to 3637 kt (step 2). Peak 6 values increased from 396 to 549 DU in the aerosol affected region. The biases, d and d6, for this case are shown in Fig. S1 in the Supplement. B. L. Fisher et al.: A new discrete wavelength BUV algorithm Figure 3. Aerosol index for the El Chichón eruption on 4 April 1982, computed from retrieved dR/dλ. can cause systematic errors in the retrieval state vector. For this reason, we do not use the shortest 312 nm channel in the retrievals (Eqs. 5–6), but the ﬁnal residual dN312 is used as a diagnostic of the nonlinearity. A step 2 empirical procedure, described in the next section, was developed to correct for the retrieval bias resulting from these errors. www.atmos-meas-tech.net/12/5137/2019/ Atmos. Meas. Tech., 12, 5137–5153, 2019 5142 3.2 Step 2 retrieval Step 2 was developed primarily to handle extreme eruptions (VEI > 3), such as El Chichón and Pinatubo, where large anomalies sometimes occur in conjunction with high ash concentrations. In practice, step 2 corrections tend to be small (or nonexistent) for most of the eruptions detected observed during the observation pe- riod covered by TOMS. In deciding whether to apply step 2, the algorithm consid- ers the retrieved 6, and aerosol index (AI) in Step 1. The AI is estimated from the dR/dλ and the calculated Jacobian dN/dR at 340 nm: AI = ∂N340 ∂R ∂R ∂λ (λ340 −λ380) = −40 · ∂N340 ∂R ∂R ∂λ . (8) (8) Positive AI (dR/dλ > 0) identiﬁes spatial regions affected by absorbing aerosols (dust, smoke and ash). The step 2 se- lection criteria ﬁrst select FoVs where either SO2 > 15 DU (inside the plume) or AI > 6. The additional AI criterion al- lows for the selection of FoVs around the edges of the cloud, where the SO2 can be less than 15 DU due to high aerosol concentrations. In this case, it is assumed that the step 1 SO2 may have been underestimated due to the ozone error caused by high aerosol concentrations (in these cases, the SO2 retrieved in step 2 may still not exceed 15 DU and there- fore would be excluded from the plume in subsequent mass calculations). We describe the methodology for interpolat- ing cor in Eqs. (S5)–(S7). A second retrieval of SO2 and dR/dλ is then performed by inversion using the measured 317 and 340 nm radiances while treating the ozone cor as a constant. This constraint on the ozone bounds the SO2 Jaco- bians computed from the forward model LUTs. The opera- tional MS_SO2 product ﬁles include a step 2 algorithm ﬂag (not applied = 0, applied = 1). Positive AI (dR/dλ > 0) identiﬁes spatial regions affected by absorbing aerosols (dust, smoke and ash). The step 2 se- lection criteria ﬁrst select FoVs where either SO2 > 15 DU (inside the plume) or AI > 6. The additional AI criterion al- lows for the selection of FoVs around the edges of the cloud, where the SO2 can be less than 15 DU due to high aerosol concentrations. www.atmos-meas-tech.net/12/5137/2019/ B. L. Fisher et al.: A new discrete wavelength BUV algorithm Figure 4. MS_SO2 maps showing (a) step 1 total column O3, (b) step 2 total column O3, (c) step 1 total column SO2 and (d) step 2 total column SO2 from the El Chichón eruption on 4 April 1982. Figure 4. MS_SO2 maps showing (a) step 1 total column O3, (b) step 2 total column O3, (c) step 1 total column SO2 and (d) step 2 total column SO2 from the El Chichón eruption on 4 April 1982. to equal zero such that loading causes the reﬂectivity dependence to become non- linear, as the forward model does not explicitly account for volcanic aerosol absorption. This scenario typically lasts for about 1–3 d following a major explosive eruption, during which total retrieved SO2 mass is likely to be underesti- mated and in some cases could even increase with time due to ash and ice fallout and plume dispersion. For such extreme cases we recommend estimating SO2 to sulfate conversion e-folding lifetime using weeks of measurements of the total SO2 cloud daily mass and extrapolating it back in time to estimate total SO2 mass emitted on eruption day. This “day one” time extrapolated SO2 mass is typically larger than re- trieved on days immediately following the eruption (Krotkov et al., 2010). (9) 6true = ⟨6⟩clean = 0. 6true = ⟨6⟩clean = 0. (9) We examined a sample of 90 TOMS orbits in clean regions of the central Paciﬁc Ocean and found a positive bias of about 3 DU (i.e., ⟨6⟩clean ∼3 DU, Fig. 5). A soft calibration procedure was developed for correcting this bias by apply- ing a small constant N340 value adjustment to the measured 340 nm BUV radiances. The details of this procedure are described in Sect. S3.3 in the Supplement. Figure 5 shows probability density functions (PDFs) of the step 1 SO2 be- fore (dashed) and after (solid) applying the correction for 11 November 1981. The mean bias is reduced to < 1 DU after applying the correction. 4.1 Random errors and SO2 detection limit We assume that the background sulfur dioxide is below TOMS detection limit in regions of the atmosphere far away from SO2 sources (e.g., volcanic, anthropogenic). Random errors associated with the retrieval process, however, are nor- mally distributed around zero. We expect that the true vol- canic SO2, 6true and the mean of the distribution, ⟨6⟩clean, The random errors in the MS_SO2 retrieval were estimated from the standard deviation in the SO2 from a large data sam- ple that included 90 central Paciﬁc orbits, spanning a 10-year period between 1980 and 1990. Data were restricted to 6 values between −20 and 20 DU (Fig. 6a). Standard devia- www.atmos-meas-tech.net/12/5137/2019/ Atmos. Meas. Tech., 12, 5137–5153, 2019 3.2 Step 2 retrieval In this case, it is assumed that the step 1 SO2 may have been underestimated due to the ozone error caused by high aerosol concentrations (in these cases, the SO2 retrieved in step 2 may still not exceed 15 DU and there- fore would be excluded from the plume in subsequent mass calculations). We describe the methodology for interpolat- ing cor in Eqs. (S5)–(S7). A second retrieval of SO2 and dR/dλ is then performed by inversion using the measured 317 and 340 nm radiances while treating the ozone cor as a constant. This constraint on the ozone bounds the SO2 Jaco- bians computed from the forward model LUTs. The opera- tional MS_SO2 product ﬁles include a step 2 algorithm ﬂag (not applied = 0, applied = 1). The corrected step 2 values inside the volcanic cloud shown in Fig. 4b appear to be fairly consistent with the re- gional unperturbed ozone ﬁeld, but it should be noted that a few remaining high values in the boundary of the plume still exist, which were not selected for step 2 (Fig. 4b). These pixels were not corrected because the threshold criteria were not met, thus 6 may be underestimated. However, their con- tribution to the total SO2 cloud mass is insigniﬁcant. Step 2 follows a methodology similar to the original resid- ual method developed by Krueger (1983), which separated the O3 and SO2 contributions by subtracting the measured BUV reﬂectance in the unperturbed region from the BUV ra- diance anomaly associated with the SO2 cloud. The MS_SO2 algorithm corrects the overestimated step 1 ozone inside the plume by correcting the positive ozone bias. Our step 2 pro- cedure is typically only applied when the ash and/or SO2 Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ 5143 B. L. Fisher et al.: A new discrete wavelength BUV algorithm B. L. Fisher et al.: A new discrete wavelength BUV algorithm 5144 Figure 5. Probability density function of SO2 background before (dashed) and after (solid) applying N340 value correction. Figure 6b shows that TOMS retrieval noise depends on the swath position, varying from ∼6 DU at nadir to ∼4 DU at higher viewing angles, while OMPS is 2–3 times smaller (∼2 DU) and is relatively independent of the cross-track po- sition. Using the MS_SO2 algorithm, we subsequently es- timate the SO2 detection limit for TOMS and OMPS-NM to be about 15 DU and 6 DU (∼99 % conﬁdence level), re- spectively. We note that when applying the Principal Com- ponent Algorithm (PCA) (Li et al., 2013) to all the 100–200 wavelengths available from OMPS-NM hyperspectral mea- surements, the noise is reduced by an order of magnitude to ∼0.2–0.5 DU, allowing detection of large anthropogenic point sources (emissions more than ∼80 kt yr−1) (Zhang et al., 2017). Figure 5. Probability density function of SO2 background before (dashed) and after (solid) applying N340 value correction. Figure 6. (a) PDF of SO2 background (noise distribution) for TOMS and OMPS based on orbits from clean regions of the central Paciﬁc, and (b) standard deviations of background SO2 for TOMS and the OMPS nadir mapper as a function of the swath position. OMPS noise is more than a factor of 2–3 lower than TOMS and less dependent on cross-track position. 4.2 Systematic errors in volcanic SO2 plumes In this section, we evaluate systematic errors of the MS_SO2 retrievals of volcanic SO2. The two most signiﬁcant errors are caused by volcanic aerosols (ash and sulfate) and in- correct assumptions regarding the SO2 proﬁle, namely the plume height. The radiance tables used by the algorithm ac- count for ozone and SO2 absorption but do not account for the absorption and scattering by aerosols. The ash errors can be signiﬁcant during the ﬁrst couple days after the initial eruption phase (Rose et al., 2003; Guo et al., 2004). The pre- computed radiance tables used by MS_SO2 assume an SO2 column amount and an a priori CMA and standard deviation (Sect. 3). An incorrect CMA assumption can cause signiﬁ- cant SO2 errors that vary with viewing geometry, ozone and SO2 column amounts. We characterize these error sources by applying the MS_SO2 algorithm to synthetic radiances. B. L. Fisher et al.: A new discrete wavelength BUV algorithm (e.g., CMA of 13 km vs. 18 km), lower reﬂectivities (e.g., Rs of 0.05 vs. 0.50) or at high viewing angles relative to the nadir (e.g., VZA of 60◦vs. 0◦). For calculations assuming typical midlatitude and high-latitude conditions, we found similar sensitivities of SO2 Jacobians to CMA. From these calculations, we can estimate the errors in the SO2 Jacobians at 317 nm, assuming that the standard a priori proﬁles used in MS_SO2 retrievals (CMA: 13 and 18 km) have a ±2 km error in CMA. The results for the tropics, midlatitudes and high latitudes are summarized in Tables S1, S2 and S3, re- spectively, in the Supplement. As shown in the tables, for SO2 plumes from relatively moderate eruptions (∼50 DU), the relative errors in SO2 Jacobians due to the error in the CMA are mostly within ±10 %. But for plumes with large SO2 loading (∼200–300 DU) from explosive eruptions such as Pinatubo, the relative error in SO2 Jacobians may reach as high as 30 % for pixels near the edge of the swaths that have low reﬂectivity. Additionally, for pixels with the same reﬂectivity and VZA, the relative errors due to SO2 height are greater for midlatitude and high-latitude eruptions than for tropical eruptions. culations (Tables S4–S7). As shown in the tables, for SO2 plumes with a modest loading (∼50 DU), the relative errors in SO2 column amounts, due to a 2 km error in the a pri- ori proﬁle are typically 10 % or less, whereas the relative er- rors in O3 are within 1 %. For plumes with large SO2 load- ings (200–300 DU), the errors in SO2 amounts due to a 2 km bias in the a priori proﬁle are typically 5 %–15 % but can reach as high as 30 %–40 % for high-latitude plumes with large SZA and VZA. For extreme conditions at high lati- tudes (Table S5, 13 km a priori proﬁle vs. 15 km actual pro- ﬁle, SO2 = 300 DU), the MS_SO2 algorithm failed to con- verge after 20 iterations, due to a signal saturation caused by strong absorption at 317 nm. In these relatively rare cases, it is beneﬁcial to use longer wavelengths (e.g., > 320 nm) for SO2 retrievals (Li et al., 2017; Theys et al., 2015), which are available from the current hyperspectral instruments such as OMI and OMPS but not TOMS. 4.2.1 Uncertainties due to SO2 plume height Figure 6. (a) PDF of SO2 background (noise distribution) for TOMS and OMPS based on orbits from clean regions of the central Paciﬁc, and (b) standard deviations of background SO2 for TOMS and the OMPS nadir mapper as a function of the swath position. OMPS noise is more than a factor of 2–3 lower than TOMS and less dependent on cross-track position. To understand retrieval errors in MS_SO2 algorithm due to assumed a priori SO2 proﬁles, we conducted sensitivity tests using the VLIDORT radiative transfer code for the typical observational conditions in the tropics, midlatitudes and high latitudes. Figure 7 shows column SO2 Jacobians ∂N/∂6 at 317 nm for different SO2 amounts, 6, nadir angles and scene reﬂectance as function of the assumed SO2 height (center of mass altitude, CMA). The Jacobians generally increase with the CMA, meaning that satellite BUV measurements are more sensitive to SO2 at higher altitudes. This means that the MS_SO2 algorithm will overestimate (underestimate) the SO2 column amount if the CMA of the a priori proﬁle is lower (higher) than that of the actual SO2 proﬁle. On the other hand, the sensitivity of SO2 Jacobians with respect to CMA is affected by several other factors, particularly SO2 column amounts, geometry (solar zenith angle and viewing zenith angle), the reﬂectivity of the underlying surface (Rs) and the CMA itself. In general, the sensitivity of SO2 Ja- cobians to CMA is greater for SO2 plumes with large SO2 loading (e.g., 300 DU vs. 50 DU) at relatively low altitudes tions were then computed as a function of the TOMS swath position, as shown in Fig. 6b. Figure 6b can be used to char- acterize the SO2 detection limits for TOMS. In this section, we compare the TOMS error distribution with the UV Ozone Mapping Proﬁle Suite Nadir Mapper (OMPS-NM), a hyper- spectral UV instrument onboard the Suomi National Polar- orbiting Partnership (NPP) and NOAA 20 satellites. For this comparison, we selected 1 month of NPP/OMPS spectral data (central Paciﬁc) and applied the MS_SO2 algorithm us- ing the same four wavelength bands of TOMS (Table 2), which were ﬁrst convolved with the TOMS bandpass func- tion. Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ 5145 B. L. Fisher et al.: A new discrete wavelength BUV algorithm Figure 7. VLIDORT calculated SO2 column Jacobians (∂N/∂6) at 317 nm for typical conditions in the tropics (SZA = 10◦, relative azimuthal angle, RAA = 90◦, O3 = 275 DU) but different SO2 column amounts (50, 100, 200 and 300 DU) and center mass altitudes (11– 20 km). For these calculations, Gaussian SO2 proﬁles with the same standard deviation (2 km) were assumed: (a) VZA = 0 and R = 0.05, (b) VZA = 0 and R = 0.50, (c) VZA = 60 and R = 0.05, and (d) VZA = 60 and R = 0.50. Figure 7. VLIDORT calculated SO2 column Jacobians (∂N/∂6) at 317 nm for typical conditions in the tropics (SZA = 10◦, relative azimuthal angle, RAA = 90◦, O3 = 275 DU) but different SO2 column amounts (50, 100, 200 and 300 DU) and center mass altitudes (11– 20 km). For these calculations, Gaussian SO2 proﬁles with the same standard deviation (2 km) were assumed: (a) VZA = 0 and R = 0.05, (b) VZA = 0 and R = 0.50, (c) VZA = 60 and R = 0.05, and (d) VZA = 60 and R = 0.50. (e.g., CMA of 13 km vs. 18 km), lower reﬂectivities (e.g., Rs of 0.05 vs. 0.50) or at high viewing angles relative to the nadir (e.g., VZA of 60◦vs. 0◦). For calculations assuming typical midlatitude and high-latitude conditions, we found similar sensitivities of SO2 Jacobians to CMA. From these calculations, we can estimate the errors in the SO2 Jacobians at 317 nm, assuming that the standard a priori proﬁles used in MS_SO2 retrievals (CMA: 13 and 18 km) have a ±2 km error in CMA. The results for the tropics, midlatitudes and high latitudes are summarized in Tables S1, S2 and S3, re- spectively, in the Supplement. As shown in the tables, for SO2 plumes from relatively moderate eruptions (∼50 DU), the relative errors in SO2 Jacobians due to the error in the CMA are mostly within ±10 %. But for plumes with large SO2 loading (∼200–300 DU) from explosive eruptions such as Pinatubo, the relative error in SO2 Jacobians may reach as high as 30 % for pixels near the edge of the swaths that have low reﬂectivity. Additionally, for pixels with the same reﬂectivity and VZA, the relative errors due to SO2 height are greater for midlatitude and high-latitude eruptions than for tropical eruptions. www.atmos-meas-tech.net/12/5137/2019/ B. L. Fisher et al.: A new discrete wavelength BUV algorithm 5146 Figure 8. Comparison of the OMPS retrieval SO2 against the GEOS-5 model SO2. The TOA radiances for the OMPS retrieval were generated assuming no aerosol (a), only sulfate aerosols (b), and both ash and sulfate aerosols (c). Figure 8. Comparison of the OMPS retrieval SO2 against the GEOS-5 model SO2. The TOA radiances for the OMPS retrieval were generated assuming no aerosol (a), only sulfate aerosols (b), and both ash and sulfate aerosols (c). inferred from the differences between the model SO2 input and the SO2 retrieved by MS_SO2, using the radiances from the model run. The no aerosol case conﬁrms unbiased SO2 retrievals for SO2 column amounts less than ∼150 DU and small positive bias for larger SO2 amounts. For aerosol cases where sulfates and ash were included in the simulation, we observe a negative bias for SO2 column amounts exceeding ∼100 DU. These negative biases (retrieval saturation) are expected as the MS_SO2 forward model does not explicitly account for volcanic aerosols. This OSSE experiment shows the effects of heavy aerosol loading on the retrieval but also increases conﬁdence in MS_SO2 retrievals between 15 and 100 DU, under nominal conditions, even in the presence of high aerosol concentrations. leads to negative residuals at 312 nm, whereas a negative bias in a priori proﬁle (CMA too low) causes positive residuals. The residuals are generally within 1–2 N value (2 %–5 % er- ror in radiance) for SO2 column amounts of 50–100 DU but can reach 3–7 N value (6 %–15 %) for large SO2 amounts of 200–300 DU. While the 312 nm channel may potentially be used to retrieve SO2 plume height for large volcanic erup- tions, it is strongly affected by volcanic aerosols as demon- strated in the next section. 4.2.2 Ash and sulfate aerosol effects on MS_SO2 retrievals To test the sensitivity of the MS_SO2 algorithm to ash and sulfate aerosols, an Observing System Simulation Ex- periment (OSSE) was conducted. The experiment used the GEOS-5 Earth system model (Molod et al., 2012; Buchard et al., 2017; Colarco et al., 2012), coupled with online God- dard Chemistry Aerosol and Radiation (GOCART) (Chin et al., 2000; Colarco et al., 2010) and Community Aerosol and Radiation Model for Atmospheres (CARMA) (Toon et al., 1988; Colarco et al., 2014). In this experiment, we consid- ered three separate cases for a Pinatubo-like eruption sce- nario: (1) 12 Mt of SO2 and no aerosols; (2) 12 Mt of SO2 and 4 Mt of sulfate aerosols (as reported by Guo et al., 2004); and (3) 12 Mt of SO2, 4 Mt of sulfate aerosols, and 5 Mt of ash uniformly distributed between 18 and 22 km above the location of Pinatubo volcano, on 15 June 1991, from 06:00 to 15:00 UTC. B. L. Fisher et al.: A new discrete wavelength BUV algorithm We also calculated the residual at 312 nm (res312 = Nm − Nc), deﬁned here as the difference between the “measured” synthetic Nm and the Nc at 312 nm using MS_SO2 retrieved ozone and SO2 column amounts. Note that the 312 nm chan- nel was not used in the MS_SO2 algorithm, and the resid- uals at other wavelengths are essentially zero since we are retrieving four parameters from four wavelengths. As shown in Tables S4–S7, a positive bias in the SO2 height (when the is CMA too high when compared with the actual proﬁle) To quantify the retrieval errors due to inaccuracies in the a priori proﬁles, we used the top-of-the-atmosphere syn- thetic radiance data generated by VLIDORT as input to the MS_SO2 algorithm. The retrieved SO2 and O3 column amounts were compared with assumed in VLIDORT cal- www.atmos-meas-tech.net/12/5137/2019/ Atmos. Meas. Tech., 12, 5137–5153, 2019 5 Comparison with PCA SO2 retrievals We directly compared MS_SO2 retrievals with the princi- pal component analysis (PCA) SO2 algorithm adapted to the TOMS 6 spectral channels. In the PCA approach (Li et al., 2013, 2017), a set of principal components (PCs) is ﬁrst ex- tracted from the measured radiances using a PCA technique and ranked in descending order according to the spectral vari- ance they each explain. If derived from SO2-free areas, these PCs represent geophysical processes (e.g., ozone absorption) and measurement details (e.g., wavelength shift) that are un- related to SO2 but may interfere with SO2 retrievals. Next, we ﬁt the ﬁrst nν (non-SO2) PCs and the SO2 Jacobians (∂N/∂6) to the measured radiances (in N value) described in Eq. (10). This allows us to simultaneously estimate the co- efﬁcients of the PCs (ω) and SO2 column amount and helps to minimize the impacts of various interfering processes: The GEOS-5 simulated 4-D proﬁles of ozone, SO2, sulfate aerosols and volcanic ash were used as input to a VLIDORT RT model (Spurr, 2008). The model generated synthetic ra- diances at 317, 331, 340 and 380 nm TOMS bands, using the actual Suomi National Polar Partnership (SNPP)/OMPS- NM viewing geometry, assuming cloud-free conditions. The synthetic radiances produced by the VLIDORT were used as input to the MS_SO2 algorithm to generate “retrieved” columns of ozone and SO2. We note that MS_SO2 algorithm uses LUTs produced using a different TOMRAD RT model. N (ω,6) = Xnv i=1ωivi + ∂N ∂6 6. (10) (10) A more detailed introduction to the PCA SO2 retrieval tech- nique for hyperspectral instruments such as the Ozone Moni- toring Instrument (OMI) and the Ozone Mapping and Proﬁler Suite Nadir Mapper (OMPS-NM) can be found elsewhere (e.g., Li et al., 2013, 2017; Zhang et al., 2017). A more detailed introduction to the PCA SO2 retrieval tech- nique for hyperspectral instruments such as the Ozone Moni- toring Instrument (OMI) and the Ozone Mapping and Proﬁler Suite Nadir Mapper (OMPS-NM) can be found elsewhere (e.g., Li et al., 2013, 2017; Zhang et al., 2017). Figure 8 compares retrieved versus true SO2 column amounts for the three cases considered. The retrieval bias is Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ B. L. Fisher et al.: A new discrete wavelength BUV algorithm B. L. Fisher et al.: A new discrete wavelength BUV algorithm For this comparison we adapt the PCA to the discrete wavelength of N7/TOMS. The Nimbus-7 TOMS PCA SO2 algorithm is similar to the OMI and OMPS-NM version in terms of its overall structure but differs in some implemen- tation details. Speciﬁcally, unlike the OMI/OMPS volcanic SO2 retrievals that use a dynamic spectral ﬁtting window (Li et al., 2017), the TOMS PCA SO2 algorithm uses all six wavelengths available from TOMS in ﬁtting. Also due to the small number of wavelengths, in the TOMS PCA SO2 algorithm, we always use nν = 5 PCs in Eq. (10), lower than the number of PCs used for OMI (nν ≤20) or OMPS (nν ≤15). For OMI and OMPS retrievals, SLER is derived at three wavelengths (342, 354 and 367 nm) and extrapo- lated to other wavelengths using a second-degree polynomial function ﬁtted to these three wavelengths. As for TOMS, SLER is determined at 340 and 380 nm and extrapolated lin- early. Additionally, while the Jacobian lookup tables are con- structed using the VLIDORT radiative transfer code (Spurr, 2008) for both OMI/OMPS and Nimbus-7 TOMS, different, instrument-speciﬁc slit functions are used to band-pass the SO2 Jacobians from the lookup tables. internal SO2 plume structure, as inferred from the SO2 gra- dients across the peak regions of the cloud. Note that for 16– 19 June, part of the observed cloud is missing due to a known mechanical problem with the TOMS instrument. These miss- ing regions can be clearly identiﬁed in the imagery. Figure 11 shows a scatterplot comparing the MS_SO2 and PCA retrievals for the 6 d time series, which included over 7000 matching FoVs. These results show the retrievals are in close quantitative agreement, with a correlation of 0.993 and a slope of 1.00. Since the two algorithms apply funda- mentally different approaches to retrieving SO2, this level of agreement is impressive considered over such a broad range of values. We further compared quantitative estimates of SO2 cloud mass, peak SO2 and plume area. For this comparison, we also considered results from the Krueger–Kerr algorithm (KK), based on the published results of Guo et al. (2004). Table 3 displays daily estimates of the SO2 cloud mass and peak SO2 amounts for the MS_SO2, PCA and KK algorithms for the 6 d period. Guo et al. B. L. Fisher et al.: A new discrete wavelength BUV algorithm (2004) applied a modiﬁed version of the KK algorithm that assumes a radiative transfer air mass factor (AMF), which accounts for the a priori ozone and SO2 absorption proﬁles (Krotkov et al., 1997). The early SO2 mass estimates by Bluth et al. (1992) derived from Pinatubo eruption assumed a geometrical AMF. Also note that Guo et al. (2004) interpolated across the missing data regions of the plume on 16, 18 and 19 June using a punctual kriging statis- tical analysis. Here, we did not correct for the missing data. The three algorithms are in good overall agreement for the period from 17 to 21 June, with the differences within 10 % compared to MS_SO2. The most signiﬁcant differences be- tween the three algorithms are observed on 16 June under conditions of heavy ash loading. KK mass tonnage estimates exceeded MS_SO2 by over 24 % even though MS_SO2 and the PCA differ by just 2 %. Some of the difference between KK and the other two algorithms can be attributed to the fact that the Guo et al. (2004) estimates include contribu- tion from the missing data region at the northern boundary of the plume (compare SO2 and aerosol imagery), but this contribution does not nearly account for the total difference in Table 3. We compared retrievals from the two algorithms for the ﬁrst 6 d of Mount Pinatubo eruption (16–21 June 1991). The Pinatubo case provides a large sample of FoVs spanning a broad range of SO2 amounts from 15 DU (minimum thresh- old) to over 400 DU. In this test of the algorithm, MS_SO2 and PCA retrievals were generated assuming a CMA of 18 km. www.atmos-meas-tech.net/12/5137/2019/ 5147 www.atmos-meas-tech.net/12/5137/2019/ June 1991 eruption of Mount Pinatubo Mount Pinatubo is a large stratovolcano located at 15◦08′ N, 120◦21′ E in western Luzon, Philippines, that erupted explo- sively on 15 June 1991, following weeks of precursory ac- tivity. TOMS SO2 imagery on 15 June shows a narrow, elon- gated SO2 ash plume extending to the west from the loca- tion of the volcano. On the following day TOMS measured a massive SO2 plume to the west of the volcano (Bluth et al., 1992). TOMS continued tracking the daily evolution of the Pinatubo volcanic cloud as it encircled the Earth over a period of about 22 d. Previous estimates of the Pinatubo SO2 height (CMA) range between 18 and 25 km (Self et al., 1996; Guo et al., 2004). The differences can be explained by considering how each algorithm is affected by aerosols. MS_SO2 accounts for ash by retrieving the spectral dependence at 340 nm, which is then adjusted iteratively to correct the reﬂectivity at the two absorbing channels. As explained in Sect. 3.2, absorbing aerosols in the column can cause possible ozone anomalies, which decrease 6. The KK algorithm (Krueger et al., 1995) accounts for ash implicitly by retrieving two linear spectral parameters that adjust calculated Nc to match measured Nm. Like MS_SO2, the KK radiative path LUTs are based on TOMRAD calculations that do not explicitly account for ash (Krotkov et al., 1997). Krueger et al. (1995) estimated that ash aerosols can cause errors in the retrieval up to +30%, depending on the ash size distribution. The PCA algorithm, Figure 9 shows TOMS daily SO2 maps produced with the MS_SO2 and the PCA algorithms for the 6 d period from 16 to 21 June. Corresponding ash index (AI) imagery from MS_SO2 is shown in Fig. 10. SO2 and AI imagery for 16 June show a large SO2 ash cloud propagating to the west. AI values range from 4 to above 12 across the plume. The AI values decreased over the following days due to wind advec- tion and wet deposition (Guo et al., 2004). As the SO2 cloud area continues to expand, total SO2 mass remains high, while SO2 peak values decrease, which is expected from cloud dis- persion. The MS_SO2 and the PCA imagery show excel- lent qualitative agreement in resolving the plume area and Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ B. L. Fisher et al.: A new discrete wavelength BUV algorithm B. L. June 1991 eruption of Mount Pinatubo Fisher et al.: A new discrete wavelength BUV algorithm B. L. Fisher et al.: A new discrete wavelength BUV algorithm Daily SO2 mass and maximal SO2 values for MS_SO2, PCA and KK algorithms for the 6 d following the Pinatubo eruption 15 June 1991. MS_SO2 PCA algorithm Krueger–Kerr Percent algorithm algorithm difference (Guo et al., 2004) (%) Day in SO2 Max SO2 SO2 Max SO2 Max PCA KK June mass SO2 mass SO2 mass SO2 1991 (Mt) (DU) (Mt) (DU) (Mt) (DU) 06/16 9.8 410 10.0 418 12.0∗ 537 −2.0 24.3 06/17 12.1 389 12.1 399 13.0 423 0.0 7.4 06/18 12.0 279 12.4 280 13.1∗ 350 3.3 9.2 06/19 10.9 173 11.6 180 11.4∗ 207 6.2 4.6 06/20 12.6 148 13.2 157 12.2 180 4.7 −4.0 06/21 11.8 125 12.5 130 11.9 137 5.9 0.8 ∗Guo et al. (2004) interpolated values in the missing data region seen in maps for 16, 18 and 19 June. Table 3. Daily SO2 mass and maximal SO2 values for MS_SO2, PCA and KK algorithms for the 6 d following the Pinatubo eruption on 15 June 1991. maximal SO2 values for MS_SO2, PCA and KK algorithms for the 6 d following the Pinatubo eruption on Table 3. Daily SO2 mass and maximal SO2 values for MS_SO2, PCA and KK algorithms for the 6 d follo 15 June 1991. Table 3. Daily SO2 mass and maximal SO2 values for MS_SO2, PCA and KK algorithms for the 6 d following the Pinatubo eruption on 15 June 1991. ∗Guo et al. (2004) interpolated values in the missing data region seen in maps for 16, 18 and 19 June. Figure 11. Scatterplot of retrieved SO2 using PCA and MS_SO2 algorithms for the period 16–21 June 1991. Overall, the PCA retrieved 3 % more total mass tonnage than MS_SO2. These differences are attributed to differences in how the MS_SO2 algorithm handles aerosols and differ- ences in the area of the plume due to differences in the re- trieval near the sensitivity threshold (∼15 DU). Ash, sulfates and high SO2 amounts impact the ozone retrieval, for as was seen in Sect. 3.2, systematic errors in SO2 are anticorrelated with errors in O3 (see Fig. S1). For the case of the KK al- gorithm, the total ozone retrieved inside the SO2 plume can be unrealistically low and even negative in an extreme event like Mount Pinatubo shown in Figs. S4 and S5. Figure S4 compares the KK ozone retrieval with MS_SO2 step 2 ozone retrieval and Fig. B. L. Fisher et al.: A new discrete wavelength BUV algorithm S5 compares scatterplots of SO2 and total ozone for 17 and 18 June. Figure 11. Scatterplot of retrieved SO2 using PCA and MS_SO2 algorithms for the period 16–21 June 1991. Table 4 provides estimates of the plume area for the MS_SO2 and PCA. The area of the plume is most sensi- tive to the minimum detection threshold around the edges of the SO2 cloud. MS_SO2 and the PCA algorithms were directly compared by computing the areal sum of all the pix- els where 6 > 15 DU (Fig. 9). For the 6 d study period, the plume increased in size from about a little over 2 × 106 km2 to ∼9 × 106 km2 The PCA trends observed a larger cloud area for ﬁve of the 6 d, with most of the observed differences within 7 %. On 16 June, shortly after the major eruption of 15 June, the estimated area for the PCA is about 15 % greater than for MS_SO2. The fresh plumes are opaque, which result in underestimating of SO2 mass by all BUV algorithms due to the mixing of aerosols (Krotkov et al., 1997). The PCA appears slightly more sensitive to SO2 near the edges of the cloud, where aerosol loading is high (AI > 1.5). It should be noted that the soft calibration applied to the 340 nm chan- nel, described in Sects. 3.3 and S3.3, may also contribute to the lowering the sensitivity around the edges of the plume. This correction effectively lowered the background by about 3 DU. in contrast, accounts for ash in the separation and ordering of the principal components. The differences between MS_SO2 and KK on 16 and 17 June can be partly ascribed to the ef- fects of aerosols on the retrievals. By 18 June, the ash and SO2 clouds have mostly separated, though, aerosol indices over 4 are still observed in some re- gions of the plume. Pinatubo did not erupt again after the major eruption on 15 June, yet the three algorithms show re- trieved SO2 mass increases on 17 and 20 June (the PCA and KK retrievals also indicate a small increase on 18 June). Guo et al. (2004) attribute these increases to the sequestering of volcanic SO2 by ice–ash mixtures in the plume. They pro- pose the sequestered SO2 was released at a later time through sublimation of ice in the lower stratosphere. B. L. Fisher et al.: A new discrete wavelength BUV algorithm 5148 Figure 9. Daily SO2 imagery for MS_SO2 and the PCA using data from TOMS overpasses of the Pinatubo eruption cloud between 15 and 21 June 1991: (a) MS_SO2 for 16 June and (b) MS_SO2 for 17 June, (c) MS_SO2 for 18 June and (d) PCA for 16 June, (e) PCA for 17 June and (f) PCA for 18 June, (g) MS_SO2 for 19 June and (h) MS_SO2 for 20 June, (i) MS_SO2 for 21 June and (j) PCA for 19 June, and (k) PCA for 20 June and (l) PCA for 21 June. Figure 9. Daily SO2 imagery for MS_SO2 and the PCA using data from TOMS overpasses of the Pinatubo eruption cloud between 15 and 21 June 1991: (a) MS_SO2 for 16 June and (b) MS_SO2 for 17 June, (c) MS_SO2 for 18 June and (d) PCA for 16 June, (e) PCA for 17 June and (f) PCA for 18 June, (g) MS_SO2 for 19 June and (h) MS_SO2 for 20 June, (i) MS_SO2 for 21 June and (j) PCA for 19 June, and (k) PCA for 20 June and (l) PCA for 21 June. Figure 10. Daily AI imagery retrieved using MS_SO2 between 16 and 21 June 1991. Contours show SO2 levels from Fig. 9. Positive AI values over India and the Arabian peninsula are due to dust aerosols, not related to the Pinatubo ash cloud: (a) 16 June, (b) 17 June, (c) 18 June, (d) 19 June, (e) 20 June and (f) 21 June. Figure 10. Daily AI imagery retrieved using MS_SO2 between 16 and 21 June 1991. Contours show SO2 levels from Fig. 9. Positive AI values over India and the Arabian peninsula are due to dust aerosols, not related to the Pinatubo ash cloud: (a) 16 June, (b) 17 June, Figure 10. Daily AI imagery retrieved using MS_SO2 between 16 and 21 June 1991. Contours show SO2 levels from Fig. 9. Positive AI values over India and the Arabian peninsula are due to dust aerosols, not related to the Pinatubo ash cloud: (a) 16 June, (b) 17 June, (c) 18 June, (d) 19 June, (e) 20 June and (f) 21 June. Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ 5149 B. L. Fisher et al.: A new discrete wavelength BUV algorithm Table 3. www.atmos-meas-tech.net/12/5137/2019/ Atmos. Meas. Tech., 12, 5137–5153, 2019 6 Conclusions This paper describes a discrete multi-satellite UV wavelength algorithm (MS_SO2) for retrieving volcanic SO2 that was used operationally to process measurements from the her- itage Nimbus-7 TOMS and the Deep Space Climate Observa- tory Earth Polychromatic Imaging Camera (Carn et al., 2018; Marshak et al., 2018). The MS_SO2 algorithm retrieves four parameters (SO2, O3, dR/dλ and Rs) and can be used to pro- cess data from current hyperspectral UV spectrometers, such as SNPP/OMPS and Aura/OMI, using a convolved, discrete set of wavelengths, offering a viable means for intercompar- ing volcanic SO2 retrievals from different missions. To assess the overall accuracy of the TOMS SO2 retrievals, we compared MS_SO2 and independent PCA algorithms for the ﬁrst 6 d following the 1991 Pinatubo eruption. The daily time series of SO2 retrievals showed high correlation (R2 = 0.986) and excellent agreement between the two re- trievals over a broad SO2 range between 15 and 400 DU. We also compared the SO2 mass, peak SO2 amounts and plume area with the heritage Krueger–Kerr algorithm. This three- way comparison showed the SO2 mass within 10 % for all days, except on 16 June, when the Krueger–Kerr algorithm retrieved 24 % higher SO2 mass. This could be explained by interpolation over a region of missing TOMS measurements on 16 June (Guo et al., 2004). The remaining differences be- tween current MS_SO2 and the PCA algorithms (3 %–7 %) are attributed to the differences in handling of aerosols and different sensitivity thresholds of the algorithms. We estimated random (noise) and systematic errors, re- lated to the effects of volcanic aerosols and uncertainties in SO2 height and partly corrected for absorbing ash, using pos- itive aerosol index (AI) as a proxy for applying a Step 2 cor- rection to the SO2 retrievals. The correction could still under- estimate SO2 mass during the ﬁrst days after extremely large eruptions (VEI > 3) due to BUV saturation. In such cases we recommend estimating e-folding time of the SO2 decay, us- ing later measurements and extrapolating SO2 mass expo- nentially back in time to the eruption day (Krotkov et al., 2010). The reprocessed Nimbus-7 TOMS volcanic SO2 data set (TOMSN7SO2) is now publicly available through the God- dard Earth Sciences Data and Information Services Cen- ter (GES DISC) as part of the NASA’s Making Earth Sys- tem Data Records for Use in Research Environments (MEa- SUREs) program (Krotkov et al., 2019). B. L. Fisher et al.: A new discrete wavelength BUV algorithm Table 4. SO2 plume area and number of ﬁelds of view (NFoVs) where the retrieved SO2 exceeded 15 DU using the MS_SO2 and PCA algorithms for the 6 d following for the Pinatubo eruption on 15 June 1991. Table 4. SO2 plume area and number of ﬁelds of view (NFoVs) where the retrieved SO2 exceeded 15 DU using the MS_SO2 and PCA algorithms for the 6 d following for the Pinatubo eruption on 15 June 1991. Table 4. SO2 plume area and number of ﬁelds of view (NFoVs) where the retrieved SO2 exceeded 15 DU using the MS_SO2 and PCA algorithms for the 6 d following for the Pinatubo eruption on 15 June 1991. MS_SO2 PCA Percent difference (%) Day Area NFovs Area NFovs PCA (×106 km2) (6 > 15 DU) (×106 km2) (6 > 15 DU) 06/16 2.13 442 2.48 519 15.2 06/17 4.19 1006 4.04 971 −3.6 06/18 5.05 1062 5.31 1088 5.0 06/19 5.09 910 5.30 957 4.0 06/20 7.27 1407 7.59 1487 4.3 06/21 8.44 1674 9.02 1805 6.6 6 Conclusions three different SO2 altitudes (8, 13 and 18 km) base their analysis on the altitude that is most a three different SO2 altitudes (8, 13 and 18 km). Users should base their analysis on the altitude that is most appropriate for a particular eruption. B. L. Fisher et al.: A new discrete wavelength BUV algorithm The oxidation of hydrogen sulﬁde offers another mechanism to account for the observed mass increases in the days following the erup- tion. The combined results of the three algorithms support the conclusions of Guo et al. (2004) that the observed mass increases in the temporal evolution of the plume are real. Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ 5150 B. L. Fisher et al.: A new discrete wavelength BUV algorithm Data availability. Our data can be publicly accessed at https://disc.gsfc.nasa.gov/datasets/TOMSN7SO2_3/summary? keywords=TOMSSO2 (Krotkov et al., 2019). Data availability. Our data can be publicly accessed at https://disc.gsfc.nasa.gov/datasets/TOMSN7SO2_3/summary? keywords=TOMSSO2 (Krotkov et al., 2019). Bogumil, K., Orphal, J., Homann, T., Voigt, S., Spietz, P., Fleis- chmann, O. C., Vogel, A., Hartmann, M. Kromminga, H., Bovensmann, H., Frerick, J., Burrows J. P.: Measurements of molecular absorption spectra with the SCIAMACHY pre-ﬂight model: instrument characterization and reference data for at- mospheric remote-sensing in the 230–2380 nm region, J. Pho- toch. Photobio. A, 157, 167–184, https://doi.org/10.1016/S1010- 6030(03)00062-5, 2003. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/amt-12-5137-2019-supplement. Competing interests. The authors declare that they have no conﬂict of interest. Financial support. This work was supported by NASA’s Mak- ing Earth System data records for Use in Research Environments (NNH12ZDA001N-MEASURES) program. Can Li acknowledges support from NASA Earth Science Division for development and analysis of hyperspectral PCA SO2 products for OMPS (grant no. 80NSSC18K0688). Eric Hughes was supported by NASA grant to the University of Maryland no. NNX13AG51G. Simon A. Carn ac- knowledges support from NASA grant NNX13AF50G. Carn, S. A., Yang, K., Prata, A. J., and Krotkov, N. A.: Extending the long-term record of volcanic SO2 emissions with the Ozone Mapping and Proﬁler Suite (OMPS) Nadir Mapper, Geophys. Res. Lett., 42, 925–932, https://doi.org/10.1002/2014GL062437, 2015. Carn, S. A., Clarisse, L., and Prata, A. J.: Multi- decadal satellite measurements of global volcanic degassing, J. Volcanol. Geoth. Res., 311, 99–134, https://doi.org/10.1016/j.jvolgeores.2016.01.002, 2016. Review statement. This paper was edited by Michel Van Roozen- dael and reviewed by two anonymous referees. Carn, S. A., Krotkov, N. A. Fisher, B. L., Li, C., and Prata, A. J.: First observations of volcanic eruption clouds from the L1 Earth- Sun Lagrange point by DSCOVR/EPIC, Geophys. Res. Lett., 45, 11456–11464, https://doi.org/10.1029/2018GL079808, 2018. Chin, M., Rood, R. B., Lin, S., and Muller, J. F.: Atmospheric sul- fur cycle simulated in the global model GOCART: Model de- scription and global properties, J. Geophys. Res.-Atmos., 105, 24671–24688, https://doi.org/10.1029/2000JD900384, 2000. 6 Conclusions We plan to repro- cess all follow-up multispectral UV (TOMS) and hyperspec- tral UV (OMI, OMPS) missions (Fig. 1) with MS_SO2 and PCA algorithms to keep updating our multi-satellite volcanic SO2 mass database archived at GES DISC (Carn, 2019). It is important to continue quantifying SO2 emissions from small explosive eruptions, as they may, collectively, play an im- portant role in sustaining the persistent, background strato- spheric aerosol layer, which is an important factor in global climate forcing. The TOMS Observing System Simulation Experiment simulation, using synthetic radiances, shows unbiased MS_SO2 retrievals of for SO2 < 100–150 DU but low biases for larger SO2 amounts due to the presence of ash and sulfate aerosols. Therefore, operational MS_SO2 retrievals should provide a low boundary constraint on the SO2 mass injected into the atmosphere from large eruptions during ﬁrst days af- ter an eruption. The algorithm can be further improved by explicitly accounting for volcanic ash and sulfate aerosols, which was not feasible in the operational processing. The MS_SO2 retrieval is also sensitive to differences be- tween the a priori and actual SO2 center of mass altitude. Since this key parameter is not retrieved, the TOMS SO2 product provides separate SO2 column amounts assuming Atmos. Meas. Tech., 12, 5137–5153, 2019 www.atmos-meas-tech.net/12/5137/2019/ 5151 Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/amt-12-5137-2019-supplement. Bluth, J. S. G., Doiron, S. D., Schnetzler, C. C., Krueger, A. J., and Walter, L. S.: Global tracking of the SO2 clouds from the June, 1991 Mount Pinatubo eruptions, Geophys. Res. 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https://openalex.org/W2901352217	https://link.springer.com/content/pdf/10.1007/s10570-018-2145-0.pdf	English	null	Comparative moisture and heat sorption properties of fibre and shiv derived from hemp and flax	Cellulose	2,018	cc-by	10,625	Y. Jiang (&) M. Lawrence A. Hussain M. Ansell P. Walker BRE Centre for Innovative Construction Materials, Department of Architecture and Civil Engineering, University of Bath, Bath BA2 7AY, UK e-mail: y.jiang@bath.ac.uk; yunhongjiang@yahoo.com Cellulose (2019) 26:823–843 https://doi.org/10.1007/s10570-018-2145-0 (0123456789().,-volV)(0123456789().,-volV) ORIGINAL RESEARCH Comparative moisture and heat sorption properties of ﬁbre and shiv derived from hemp and ﬂax Graphical abstract Keywords Microstructure Porosity Equilibrium moisture content Sorption isotherm and sorption hysteresis materials have the potential to act as a buil material to moderate humidity levels in an ind environment due to their inherent hygroscopic p erties (Latif et al. 2015; Laborel-Pre´neron et al. 2 Graphical abstract Graphical abstract abstract Keywords Microstructure Porosity Equilibrium moisture content Sorption isotherm and sorption hysteresis materials have the potential to act as a building material to moderate humidity levels in an indoor environment due to their inherent hygroscopic prop- erties (Latif et al. 2015; Laborel-Pre´neron et al. 2016; Hurtado et al. 2016; Bourdot et al. 2017). The components of ﬁbres and shiv of ﬂax and hemp include cellulose, hemicellulose, lignin, pectin, waxes and water soluble substances. The composition of selected ﬁbres and shiv of ﬂax and hemp are shown in Table 1. The major differences in chemical composi- tion between the ﬁbres and shiv were in the amounts of lignin. In addition, ﬁbres had a higher content of cellulose compared with that of shiv. Comparative moisture and heat sorption properties of ﬁbre and shiv derived from hemp and ﬂax Yunhong Jiang . Michael Lawrence . Atif Hussain . Martin Ansell . Pete Walker Received: 2 July 2018 / Accepted: 19 November 2018 / Published online: 23 November 2018 The Author(s) 2018 dependent on the availability of hydroxyl groups for water in the cell wall. The macroscopic surface area and porosity of the specimen on a large scale had little inﬂuence on the availability of hydroxyl groups in the cell wall. The water molecules bound with cell wall molecules through hydrogen bonds over the full range of relative humidities, with a consistent hysteresis difference between the ﬁrst sorption isotherm and subsequent sequential sorption cycles found in the hemp shiv specimens. For both hemp and ﬂax, the isothermic hysteresis of the shiv was much higher than that of the ﬁbre, which was shown to be dependent on the lignin content. The result of heat sorption indicated that some blocked sorption sites become available to water vapour molecules due to the change in molec- ular structure of the cell wall during the adsorption process. This study has improved understanding of the hemp and ﬂax sorption behaviour and is important for optimal application of bio-based insulation materials for construction. Abstract Unlike many mineral-based insulation materials bio-based lignocellulosic ﬁbre and shiv offer a number of beneﬁts including thermal and hygroscopic properties. The microstructure, porosity and chemical compositions of the plant cell walls play a major role in the moisture exchange process. In this paper, the effects of microstructure, chemical compo- sition, porosity and pore size distribution of both ﬁbre and shiv, from hemp and ﬂax plants, on both moisture and heat sorption were studied for the ﬁrst time. The physical and chemical characteristics of the ﬁbre and shiv from hemp and ﬂax were studied by using scanning electron microscopy, mercury intrusion porosimetry and Fourier transform infrared spec- troscopy. Water moisture sorption and heat of sorption were studied using a dynamic vapour sorption appa- ratus and a simultaneous thermal analysis system, combined with a humidity generator and using a copper furnace. Comparative moisture and heat sorption properties of ﬁbre and shiv derived from hemp and ﬂax Results showed that both the ﬁbre and shiv of hemp and ﬂax adsorbed a similar amount of moisture at a given relative humidity, which was 123 123 Cellulose (2019) 26:823–843 824 Graphical abstract Keywords Microstructure Porosity Equilibrium moisture content Sorption isotherm and sorption hysteresis Introduction Agro-industrial lignocellulosic materials such as hemp, ﬂax, corn cob and straw (wheat, rice) are becoming more widely recognised for their excellent hygric and excellent insulating properties (Latif et al. 2014; Walker and Pavı´a 2014; Collet et al. 2017). Hemp and ﬂax are versatile and fast-growing agricul- tural crops, which can be used as part of a crop rotation to condition soil. Acting as a carbon bank, hemp and ﬂax lock in up to two tonnes of CO2 per tonne of ﬁbre, making them a particularly sustainable choice for construction applications (Boutin et al. 2005). In the stem of natural plants, such as hemp and ﬂax, a bark- like layer containing ﬁbres surrounds a woody core (Jiang et al. 2018). These components can be separated either by retting including chemical and microbial actions or mechanically, dividing the stem into bark ﬁbres and woody stem (shiv) fractions. These materials have the potential to act as a building material to moderate humidity levels in an indoor environment due to their inherent hygroscopic prop- erties (Latif et al. 2015; Laborel-Pre´neron et al. 2016; Hurtado et al. 2016; Bourdot et al. 2017). The components of ﬁbres and shiv of ﬂax and hemp include cellulose, hemicellulose, lignin, pectin, waxes and water soluble substances. The composition of selected ﬁbres and shiv of ﬂax and hemp are shown in Table 1. The major differences in chemical composi- tion between the ﬁbres and shiv were in the amounts of lignin. In addition, ﬁbres had a higher content of cellulose compared with that of shiv. Moisture levels in buildings have an inﬂuence on occupant comfort and fabric durability. Many building materials play an important role in inﬂuencing indoor air quality by adsorbing and desorbing moisture with their surroundings (Ferreira et al. 2017). Therefore, understanding hygroscopicity (the ability of a material to respond air moisture levels by either absorbing or releasing water vapour) of building materials is increasingly important. The hygroscopic behaviour of hemp and ﬂax has been studied by a number of researchers (Nilsson et al. 2005; Hill et al. 2009; Collet et al. 2011; Mikhalovska et al. 2012; Ce´lino et al. Introduction However, Thygesen et al. suggested that the moisture condensation in capillaries is insigniﬁcant below 99.5% RH. Engelund et al. further explained this phenomena by using theoretical calculations (En- gelund Emil et al. 2010). Mauze and Stern (1984; Vrentas and Vrentas 1991) reported another potential explanation for this upward curve of the sorption isotherm, which is due to the softening of the amorphous polymers. The sorption hysteresis for wood has been observed over the entire range of relative humidity. There are two main physical explanations for the existence of hysteresis. One explanation refers to narrow connections between large cavities of adjoining pores due to entry pores with small diameter based on the capillary theory of adsorption such as capillary condensation hysteresis; contact angle hysteresis and the ink-bottle effect (Engelund et al. 2013). Second explanation has been suggested that the existence of sorption hysteresis attributes to the process of polymer relaxation in the inter-microﬁbrillar matrix (Hill et al. 2012). 2013). Hill et al. studied the water vapour sorption behaviour of a range of natural ﬁbres (jute, ﬂax, coir, cotton, hemp, sitka spruce). They found that the extent of hysteresis exhibited between the adsorption and desorption isotherms was dependent on the ﬁbre type studied (Hill et al. 2009). Hemp-lime, which is a mixture of hemp shiv, water and lime, has been widely studied as a bio-based building material due to its low thermal conductivity (0.06–0.14 W/m.K) and high moisture buffer value [2.15 g/(m2.%RH)] (Collet et al. 2013). The literature reveals that the low density, complicated microstruc- ture and high porosity of cell wall play a key role to their low thermal conductivity and high moisture buffer value for bio-based plant building materials (Collet et al. 2008; Collet 2014; Bourdot et al. 2017). Collet et al. (2008) studied the porous structure and water vapour sorption of hemp-based materials and showed that lime–hemp render and hemp mortar contain macro-pores, meso-pores and micro-pores. These pores were associated with measured water vapour sorption isotherms classiﬁed as type II. Collet et al. (2013) also reported that the capillary conden- sation occurs in pores of width from 5 nm to 40 nm in the range of relative humidity range 81–97%RH. This range of pore sizes is observed in the cell wall of hemp shiv. Rahim et al. reported on the sorption isotherm curves of the hemp concrete (HLC) and ﬂax concrete (FLC). Introduction Agro-industrial lignocellulosic materials such as hemp, ﬂax, corn cob and straw (wheat, rice) are becoming more widely recognised for their excellent hygric and excellent insulating properties (Latif et al. 2014; Walker and Pavı´a 2014; Collet et al. 2017). Hemp and ﬂax are versatile and fast-growing agricul- tural crops, which can be used as part of a crop rotation to condition soil. Acting as a carbon bank, hemp and ﬂax lock in up to two tonnes of CO2 per tonne of ﬁbre, making them a particularly sustainable choice for construction applications (Boutin et al. 2005). In the stem of natural plants, such as hemp and ﬂax, a bark- like layer containing ﬁbres surrounds a woody core (Jiang et al. 2018). These components can be separated either by retting including chemical and microbial actions or mechanically, dividing the stem into bark ﬁbres and woody stem (shiv) fractions. These Moisture levels in buildings have an inﬂuence on occupant comfort and fabric durability. Many building materials play an important role in inﬂuencing indoor air quality by adsorbing and desorbing moisture with their surroundings (Ferreira et al. 2017). Therefore, understanding hygroscopicity (the ability of a material to respond air moisture levels by either absorbing or releasing water vapour) of building materials is increasingly important. The hygroscopic behaviour of hemp and ﬂax has been studied by a number of researchers (Nilsson et al. 2005; Hill et al. 2009; Collet et al. 2011; Mikhalovska et al. 2012; Ce´lino et al. 12 123 Cellulose (2019) 26:823–843 825 Table 1 Chemical composition of selected ﬁbre and shiv (Garcia et al. 1998; Bledzhi and Gassan 1999; Vignon et al. 1995; Ross and Mazza 2010; Kostic et al. 2008; Kyma¨la¨inen and Sjo¨berg 2008; Viel et al. 2018) Specimen Cellulose (%) Lignin (%) Hemicellulose (%) Pectin (%) Wax (%) Flax ﬁbre 61.0–71.0 1.8–5.7 18.6–20.6 1.8–2.3 0.5–1.7 Hemp ﬁbre 60.0–76.2 3.7–5.7 12.8–22.4 0.9–2.3 0.7–1.7 Flax shiv 39.9–41.0 23.0–30.3 25.2–26.8 – – Hemp shiv 34.0–44.0 22.0–30.0 31.0–37.0 4.0–5.0 0.5–1.4 binding sites, including the hydroxyl (- OH) groups, the carboxyl groups and, if present, the sulfonic acid groups. Most of the absorbed moisture molecules are held in amorphous areas of the cellulose and the hemicellulose and lignin matrix through the hydroxyl groups. Kollmann (1962) reported the sorption isotherm of wood presents an upward bend around 60–70% RH, which is related to sorption of free water in capillaries due to the micropores in the cell wall. Materials and methods The porosity and pore size distribution of ﬁbre and shiv were analyzed by an Autopore Mercury Porosimeter (PASCAL, Thermo Scientiﬁc). This related the radius (r) of pores (assumed to be cylindrical) to the imposed pressure (P) using the Washburn equation (Wang et al. 2016): The ﬁbre and shiv of hemp and ﬂax used in this study were obtained from the CAVAC Company (France), produced by a mechanical de-ﬁbreing process (shown in Fig. 1). The materials and mechanical de-ﬁbering process used are as reported by Viel et al. (2018). The ﬁbres were composed of bundles containing surface defects such as kink band and microcracking, whilst the shiv exhibited a more regular shape with a smoother surface topography. The particle size of hemp shiv and ﬂax shiv was measured using a digital electronic calliper. The test was repeated three times on different orientations for each shiv. The particles of hemp shiv and ﬂax shiv had a mean length of 17.6 ± 2.3 mm and a mean width of 8.4 ± 1.8 mm and a mean length of 13.5 ± 1.6 mm and 3.3 ± 0.6 mm, respectively. The protocol used to dry the samples are as reported by Jiang et al. (2018). The bulk densities of hemp shiv and ﬂax shiv were in the range of 85–90 kg/m3, which is measured based on the followed protocol that was developed by the RILEM Technical Committee 236-BBM (Picandet 2017). The test was repeated three times for each shiv. Th i t t f th ﬁb d hi P ¼ 2c cos h ð Þ=r ð1Þ ð1Þ P ¼ 2c cos h ð Þ=r where c = interfacial energy (surface tension) of mercury, h = contact angle of mercury with the material. The adsorption and desorption of moisture mole- cules by the ﬁbre and shiv specimens were investi- gated using a dynamic vapour sorption apparatus (DVS, Surface Measurement Systems, UK). The methods used here are as reported by Hill et al. (2012). The moisture sorption and heat sorption of bio- aggregates were studied by using a simultaneous thermal analyser (STA-449 F3 Jupiter, NETZSCH, Germany) combined with a humidity generator (Mo- dular Humidity Generator, NETZSCH, Germany) and a copper furnace including coupling adapter. A suitable gas ﬂow rate (100 ml/min) was delivered and controlled by a humidity generator control soft- ware to achieve the desired setpoint for humidity. Introduction HLC reached a lower equilibrium moisture content than FLC because the porosity of ﬂax shiv is smaller than the porosity of hemp shiv, and thus more capillary condensation is promoted, resulting in increased moisture adsorption (Rahim et al. 2015). A better understanding of the moisture sorption mechanism of ﬁbre and shiv of ﬂax and hemp is necessary for developing ﬁbre and shiv-reinforced composite insulation materials. This paper aims to study the effects of the microstructure, chemical composition, pore size distribution and porosity on the hygroscopic behaviour of ﬁbre and shiv. In addition, the moisture sorption properties (sorption The exact mechanisms of moisture sorption in natural plant materials are not yet well understood. Engelund et al. (2013) reported that the moisture molecules can be directly adsorbed by the cell wall polymer of plant materials through the different 12 3 3 Cellulose (2019) 26:823–843 826 isotherms) and the enthalpy of heat sorption were evaluated for both shiv and ﬁbre of ﬂax and hemp during the sorption process. thermal properties of the ﬁbre and shiv specimens were studied using a thermal analyser (STA 449, NETZSCH, Germany) under a pure nitrogen atmo- sphere. The temperature was increased from 25 to 800 C at a constant heating rate of 10 C/min with a nitrogen ﬂow of 20 ml/min. Materials and methods Humidity and temperature values were recorded by means of a humidity sensor which is situated within a heated adapter. To avoid condensation and allow high relative humidity, the experimental temperature set- point was ﬁxed 5 C above the maximum dewpoint. The mass change of the bio-aggregates was measured The microstructure of the ﬁbre and shiv was observed by a scanning electron microscope (JEOL SEM-6480LV, Tokyo, Japan). The sample preparation for SEM measurement has been reported by Jiang et al. (2018). FTIR transmittance spectra were col- lected in an attenuated total reﬂection (ATR) mode with 20 mm-1 resolution and 10 scans were accumu- lated for each spectrum in the range 6–40 m-1. The Fig. 1 Optical photos showing the appearance of studied ﬁbre and shiv of ﬂax and hemp Fig. 1 Optical photos showing the appearance of studied ﬁbre and shiv of ﬂax and hemp 12 3 827 Cellulose (2019) 26:823–843 presence of C–H stretch vibration from polysaccha- rides and CH2 stretch from wax, respectively. The peak at 17.36 m-1 was attributed to C=O unconju- gated stretching of carboxylic acid or ester of the hemicelluloses and the peak at 12.32 m-1 was asso- ciated with C–O stretching vibration of acyl group present in the lignin. The peak at around 10.33 m-1 was assigned to C=O stretch vibration in the hemicel- lulose and pectin and the peak at 8.95 m-1 was due to the presence of C–O–C stretch (Pandey 1999; Rana et al. 2010; Esteves et al. 2013). All above peaks can be observed in all four FTIR spectra. There was no essential difference in the FTIR spectra of the studied ﬁbres or shiv. This indicated that all the specimens had a similar chemical structure. However, certain minor differences were observed between ﬁbre and shiv. For instance, the peak at 16.39 m-1 was assigned to C=C stretching vibration in the lignin, which is only observed in the FTIR spectrum of hemp shiv and ﬂax shiv. The peaks at 15.06 m-1 (C=C aromatic sym- metrical stretching) and 14.25 m-1 (HCH and OCH in plane deformation) were also only observed in the FTIR spectrum of shiv specimens. Meanwhile, the FTIR spectrum of hemp ﬁbre and ﬂax ﬁbre showed a peak at 10.24 m-1 (C–C, C–OH, C–H ring and side group vibration), which does appear in the FTIR spectrum of shiv specimens. TGA analyses of the shiv and the ﬁbres Figure 4 shows the TGA and DTG spectra of ﬁbre and shiv of both hemp and ﬂax at a heating rate of 10 C/ min under a pure nitrogen atmosphere. The peaks in the DTG curves correspond to points of maximum decomposition slope in the original TGA curves. Results showed that all the specimens have a similar degradation process. All the specimens showed two main decomposition peaks on the DTG thermograph. Initial degradation of both shiv and ﬁbre started at around 220 C. Hemicellulose started its exothermal Microstructure Figure 2 shows the microstructure of the shiv and ﬁbre for hemp and ﬂax. The hemp shiv had a foam-like closed cell structure comprised of continuous cells known as tracheids as shown in Fig. 2a. The larger cells which are similar to vessels in hardwoods were approximately 50–80 lm in diameter. Flax shiv (Fig. 2b) had a relatively simple structure compared with hemp shiv. The majority of ﬂax shiv was comprised of long, thin tracheids. The diameter of tracheids was in the range of 20-80 lm. In the centre of the stem, there were relatively large diameter cells with thin walls. Figure 3c–f shows SEM images of hemp ﬁbre and ﬂax ﬁbre. The hemp and ﬂax ﬁbres were in the form of bundles, with elementary ﬁbres joined by middle lamellae. The cross-section of the ﬁbre bundles were irregular and most of them have an ellipsoidal or polygonal shape. The ﬂax ﬁbres (F) were imaged as a partly separated bundle revealing the single ﬁbres. The microstructures of the hemp and ﬂax ﬁbre were similar in terms of their cross-section shape. It was clear that the cell wall microstructure of the hemp and ﬂax shiv was signiﬁcantly different from the microstructure of the hemp and ﬂax ﬁbres. In addition, the microstructure of hemp shiv was different from that of ﬂax shiv with regard to the thickness of cell wall and arrangement of pores. Materials and methods Compared to the FTIR spectra of hemp ﬁbre and ﬂax ﬁbre, both FTIR spectra of hemp shiv and ﬂax shiv showed a strong intensity peaks at 17.36 m-1 and 12.32 m-1. These results indicate that the components of ﬁbres and shiv of ﬂax and hemp include cellulose, hemicellulose, lignin, pectin and waxes. In terms of chemical composition, the shiv had a higher amount of lignin and hemicel- lulose compared with the ﬁbres, which is in a good agreement with literature ﬁndings reported in Table 1. at different relative humidity (RH) levels (33%, 54% and 75%) in both increasing and decreasing steps. Each RH level lasted for 3 h and a microbalance in the STA measured the mass change continuously during the experimentwith a nitrogen ﬂow rate of 20 ml/min at 30 C. At the same time, the heat of sorption, which is related to moisture sorption of the specimens, was also measured by the STA. FTIR of the shiv and ﬁbres Figure 3 shows the FTIR spectra in an ATR mode for shiv and ﬁbre of both hemp and ﬂax. The main peak in the range of 33–35 m-1 was due to the characteristic axial vibration of the hydroxyl group of cellulose (from 2, 3 and 6 carbon of glucose). The adsorption bands at 29.18 and 28.51 m-1 were due to the 12 3 3 Cellulose (2019) 26:823–843 828 decomposition at 220–300 C and the pyrolysis of cellulose took place between 315 and 400 C, whereas that of lignin had a broader temperature range between 220 and 600 C (Stefanidis et al. 2014). The higher decomposition rate of the shiv between 220 and 260 C indicated that the shiv has a much higher content of hemicellulose than the ﬁbres. Both species f ﬁb h d hi h d iti t th hi possessed a higher concentration of cellulose th shiv, because the main decomposition betwee and 400 C correspond to the simultaneous dec sition of cellulose. The data showed that the shi higher mass loss compared to the ﬁbre above 4 as can be seen in Fig. 4a. The mass loss above 4 was assigned to the decomposition of lig i di t d th t b th i f hi h Fig. 2 Scanning electron microscope (SEM) images of cross- sections of hemp shiv (a), ﬂax shiv (b) ﬁbre bundles can be seen in the outer layer of the sample that meant the separation of the two fractions was not perfect, hemp ﬁbre (c) and ﬂ (d) and SEM images of specimens of hemp ﬁbre (e) ﬁbre (f) Fig. 2 Scanning electron microscope (SEM) images of cross- sections of hemp shiv (a), ﬂax shiv (b) ﬁbre bundles can be seen in the outer layer of the sample that meant the separation of the two fractions was not perfect, hemp ﬁbre (c) and ﬂax ﬁbre (d) and SEM images of specimens of hemp ﬁbre (e) and ﬂax ﬁbre (f) decomposition at 220–300 C and the pyrolysis of cellulose took place between 315 and 400 C, whereas that of lignin had a broader temperature range between 220 and 600 C (Stefanidis et al. 2014). The higher decomposition rate of the shiv between 220 and 260 C indicated that the shiv has a much higher content of hemicellulose than the ﬁbres. Porosity and pore size distribution of shiv and ﬁbres hemp and ﬂax had a larger cumulative pore volume (2063–2428 mm3/g) compared to the ﬁbres (365.71–481.41 mm3/g). The increase in Hg uptake was attributed to ﬁlling of the pores. There was an initial steep ﬁlling of the voids in the range of 10–100 lm, followed by a plateau in the range of 0.004–10 lm for both hemp ﬁbre and ﬂax ﬁbre. In contrast, the volumetric data showed a slow ﬁlling of voids between 80 and 100 lm for hemp shiv and ﬂax shiv. Subsequently the ﬂax shiv had a higher increase of Hg uptake around 20 lm and a lower increase of Hg uptake around 0.5 lm compared to the hemp shiv. Figure 5b shows the pore size distribution of speci- mens corresponding to the ﬁlling of Hg under different pressures. Figure 5b shows hemp ﬁbre and ﬂax ﬁbre have a pore size distribution from 5 to 100 lm and both hemp shiv and ﬂax shiv showed a bimodal pore size distribution with two clearly separated peaks. For the hemp shiv, the main pore diameter ranged from 0.03 to 1 lm and the second pore diameter peak was between 20 and 80 lm. On the other hand, ﬂax shiv showed the main pore diameter ranging from 5 lm to 50 lm and the second pore diameter was between 0.03 and 50 lm. Nanoporosity was not observed, and smaller pores of around 3 nm had a lower peak Table 2 summarises the density and porosity of shiv and ﬁbre specimens obtained by MIP. Shiv showed a higher porosity (hemp: 77.93 ± 4.05% and ﬂax: 75.81 ± 3.36%) compared to the hemp ﬁbre (35.71 ± 2.89%) and ﬂax ﬁbre (40.8 ± 2.03%). In addition, the apparent densities of the ﬁbre (hemp: 1519.1 ± 78.9 kg/m3 and ﬂax: 1518.6 ± 78.6 kg/m3) were higher than the apparent densities of the hemp shiv (1453.9 ± 74.1 kg/m3) and ﬂax shiv (1431.8 ± 74.9 kg/m3). Ehrnrooth (1984) reported that the apparent density of the ﬁbres increased with reducing lignin content, and the density of lignin and hemicellulose in situ were measured to be 1397 and 1541 kg/m3 respectively, whereas the density of cellulose was around 1559 kg/m3. Stefanidis et al. (2014) also reported that the true density of a cellulose crystal was between 1582 and 1599 kg/m3, indicating that the ﬁbres have a higher cellulose content than the shiv. By employing Washburn’s Eq. FTIR of the shiv and ﬁbres Both species of ﬁbre showed a higher decomposition rate than shiv between 260 and 400 C. It followed that the ﬁbre possessed a higher concentration of cellulose than the shiv, because the main decomposition between 300 and 400 C correspond to the simultaneous decompo- sition of cellulose. The data showed that the shiv had a higher mass loss compared to the ﬁbre above 400 C as can be seen in Fig. 4a. The mass loss above 400 C was assigned to the decomposition of lignin. It indicated that both species of shiv have a higher concentration of lignin than hemp and ﬂax ﬁbre. 12 3 Cellulose (2019) 26:823–843 829 Fig. 3 Fourier transform infrared spectra for shiv and ﬁbre (Hemp and Flax) Fig. 3 Fourier transform infrared spectra for shiv and ﬁbre (Hemp and Flax) Porosity and pore size distribution of shiv and ﬁbres (1), the pore vol- ume distribution and pore size distribution of shiv and ﬁbre can be derived as shown in Fig. 5. Both shiv of 12 3 3 Cellulose (2019) 26:823–843 830 Fig. 4 TGA (a) and DTG (b) curves of hemp and ﬂax ﬁbre and shiv in a nitrogen atmosphere Table 2 Total intruded volume, Bulk density, Envelope density, Apparent density and Accessible porosity determined by Mercury intrusion porosimetry Specimen Total intruded volume (mm3/g) Bulk density (kg/m3) Envelope density (kg/m3) Apparent density (kg/m3) Accessible porosity (%) Flax shiv 2063.1 ± 108.6 367.4 ± 18.9 367.6 ± 19.2 1431.8 ± 74.9 75.81 ± 3.36 Flax ﬁbre 481.4 ± 25.1 847.6 ± 27.3 850.2 ± 26.8 1519.1 ± 78.9 40.8 ± 2.03 Hemp shiv 2428.8 ± 123.5 320.9 ± 16.5 321.3 ± 17.4 1453.9 ± 74.1 77.93 ± 4.05 Hemp ﬁbre 365.7 ± 21.3 976.4 ± 33.2 992.4 ± 35.7 1518.6 ± 78.6 35.71 ± 2.89 g. 4 TGA (a) and DTG curves of hemp and ﬂax re and shiv in a nitrogen mosphere TGA (a) and DTG rves of hemp and ﬂax nd shiv in a nitrogen phere Table 2 Total intruded volume, Bulk density, Envelope density, Apparent density and Accessible porosity determined by Mercury intrusion porosimetry Table 2 Total intruded volume, Bulk density, Envelope density, Apparent density and Accessible porosity determined by Mercury i t i i t intensity, due to the limit of the sensitivity of MIP technique (pores below 3.7 nm were not measured and pores above 100 nm were excluded). The results showed agreement with the SEM images (Fig. 2). Porosity and pore size distribution of shiv and ﬁbres These sorption curves at the data for different specimens of the same v type were reproducible over most of the RH udied. According to IUPAC classiﬁcation mes et al. 2015), all specimens studied exhib- type II-isotherm curves, which represents the on behaviour of macro-porous and non-porous nts (Brunauer et al. 1938; Thommes et al. A very steep slope especially at the higher RH erved for hemp ﬁbre and hemp shiv as seen in b. Hemp shiv and hemp ﬁbre exhibited higher e content on adsorption compared to ﬂax shiv ﬁbre between 80 and 90%RH. ndividual moisture adsorption and desorption ms over three sorption cycles for the ﬂax ﬁbre Fig. 7. The absolute hysteresis, which was obtained by subtracting the adsorption from desorption isotherm loop moisture content values for the different speci- men over the three cycles, is given in Fig. 8. The sorption isotherms for the ﬂax ﬁbre, hemp ﬁbre and ﬂax shiv specimens were reproducible. For the hemp shiv, the moisture content value of ﬁrst sorption cycle was higher than the second and third cycles. The hysteresis decreased between the ﬁrst and subsequent cycles. The desorption curve of the isotherm remained the same. In addition, results showed the hemp shiv and ﬂax shiv exhibited a higher hysteresis value around 2.5% for second and third cycles at RH 70%. Meanwhile, there was a signiﬁcant decrease in the sorption hysteresis for hemp ﬁbre and ﬂax ﬁbre (approximately 1.2%, average value of three cycles at RH 70%) ore volume n (a) and pore size n (b) of shiv and x and Hemp) (2019) 26:823–843 831 831 Cellulose (2019) 26:823–843 specimens of ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv. The sorption curves for both hemp and ﬂax ﬁbre and shiv are presented in Fig. 6. These sorption curves show that the data for different specimens of the same ﬁbre/shiv type were reproducible over most of the RH range studied. According to IUPAC classiﬁcation (Thommes et al. 2015), all specimens studied exhib- Fig. 7. The absolute hysteresis, which was obtained by subtracting the adsorption from desorption isotherm loop moisture content values for the different speci- men over the three cycles, is given in Fig. 8. The sorption isotherms for the ﬂax ﬁbre, hemp ﬁbre and ﬂax shiv specimens were reproducible. For the hemp shiv, the moisture content value of ﬁrst sorption cycle Fig. Porosity and pore size distribution of shiv and ﬁbres Moisture sorption behaviour of shiv and ﬁbre In view of the variability of natural specimens, preliminary studies were employed here to measure the reproducibility of the sorption process using Table 2 Total intruded volume, Bulk density, Envelope density, Apparent density and Accessible porosity determined by Mercury intrusion porosimetry Specimen Total intruded volume (mm3/g) Bulk density (kg/m3) Envelope density (kg/m3) Apparent density (kg/m3) Accessible porosity (%) Flax shiv 2063.1 ± 108.6 367.4 ± 18.9 367.6 ± 19.2 1431.8 ± 74.9 75.81 ± 3.36 Flax ﬁbre 481.4 ± 25.1 847.6 ± 27.3 850.2 ± 26.8 1519.1 ± 78.9 40.8 ± 2.03 Hemp shiv 2428.8 ± 123.5 320.9 ± 16.5 321.3 ± 17.4 1453.9 ± 74.1 77.93 ± 4.05 Hemp ﬁbre 365.7 ± 21.3 976.4 ± 33.2 992.4 ± 35.7 1518.6 ± 78.6 35.71 ± 2.89 Table 2 Total intruded volume, Bulk density, Envelope density, Apparent density and Accessible porosity determined by Mercury intrusion porosimetry Specimen Total intruded volume (mm3/g) Bulk density (kg/m3) Envelope density (kg/m3) Apparent density (kg/m3) Accessible porosity (%) Flax shiv 2063.1 ± 108.6 367.4 ± 18.9 367.6 ± 19.2 1431.8 ± 74.9 75.81 ± 3.36 Flax ﬁbre 481.4 ± 25.1 847.6 ± 27.3 850.2 ± 26.8 1519.1 ± 78.9 40.8 ± 2.03 Hemp shiv 2428.8 ± 123.5 320.9 ± 16.5 321.3 ± 17.4 1453.9 ± 74.1 77.93 ± 4.05 Hemp ﬁbre 365.7 ± 21.3 976.4 ± 33.2 992.4 ± 35.7 1518.6 ± 78.6 35.71 ± 2.89 Table 2 Total intruded volume, Bulk density, Envelope density, Apparent density and Accessible porosity determined by Mercury intrusion porosimetry l intruded volume, Bulk density, Envelope density, Apparent density and Accessible porosity determined by simetry volume, Bulk density, Envelope density, Apparent density and Accessible porosity determined by Mercury Table 2 Total intruded volume, Bulk density, Envelope density, Apparent density and Accessible porosity intrusion porosimetry Moisture sorption behaviour of shiv and ﬁbre intensity, due to the limit of the sensitivity of MIP technique (pores below 3.7 nm were not measured and pores above 100 nm were excluded). The results showed agreement with the SEM images (Fig. 2). In view of the variability of natural specimens, preliminary studies were employed here to measure the reproducibility of the sorption process using 12 123 123 ns of ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp e sorption curves for both hemp and ﬂax ﬁbre are presented in Fig. 6. Heat sorption behaviour of shiv and ﬁbre The moisture and heat of sorption characteristics of plant material can provide interesting insights into the hygrothermal behaviour of these materials. Figure 12 presents the change of the moisture content and the enthalpy of heat of sorption at various relative humidity levels at 30 C for hemp shiv. Similar results had also been observed for hemp ﬁbre, ﬂax shiv and ﬂax ﬁbre as seen in Table 3. All specimens absorbed higher amounts of moisture content (around 0.24 wt%/ RH%) at high relative humidity between RH 54 and RH 75% compared to moisture content change at low relative humidity (0–33%) and medium relative humidity (33–54%). For the desorption process, the hemp shiv and ﬂax shiv showed a lower mass percentage change between RH 54 and RH 75% and a higher mass percentage change between RH 0 and RH 33% compared to their adsorption process. On the contrary. hemp ﬁbre and ﬂax ﬁbre showed a similar mass change percentage between RH 0 and RH 75%. Thus, the hysteresis of moisture inside shiv was higher than hysteresis inside ﬁbre and the hysteresis effect was more pronounced in high water activity regions, which was in good agreement with DVS results. In addition, the difference in heat of adsorption and desorption was independent of the change in moisture content. The enthalpy of heat of sorption at lower RH (\ 33%) was much higher the enthalpy of heat of sorption at higher RH (54% and 75%) for both adsorption and desorption processes. The enthalpy change over the full range of humidity was greater than the heat of vaporisation of liquid water (2257 kJ/ kg) at 30 C. Fig. 6 Comparison of moisture adsorption behaviour of ﬁbre (a) and shiv (b) (Flax and Hemp) at 23 C. The data for different specimens of the same ﬁbre and shiv were reproducible over most of the RH range studied The differences in hysteresis behaviour are more clearly illustrated when the curves are overlaid as seen in Fig. 9. Hemp ﬁbre and ﬂax ﬁbre showed a similar hysteresis around 1% in the whole part of the hygroscopic range. However, hemp shiv and ﬂax shiv exhibited an increasing hysteresis in the lower part of the hygroscopic range. Hemp shiv and ﬂax shiv exhibited a signiﬁcantly higher sorption hysteresis compared with hemp ﬁbre and ﬂax ﬁbre between 20 and 80%RH. Porosity and pore size distribution of shiv and ﬁbres 5 Pore volume distribution (a) and pore size distribution (b) of shiv and ﬁbre (Flax and Hemp) ( ) size nd specimens of ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv. The sorption curves for both hemp and ﬂax ﬁbre and shiv are presented in Fig. 6. These sorption curves show that the data for different specimens of the same ﬁbre/shiv type were reproducible over most of the RH range studied. According to IUPAC classiﬁcation (Thommes et al. 2015), all specimens studied exhib- ited the type II-isotherm curves, which represents the adsorption behaviour of macro-porous and non-porous adsorbents (Brunauer et al. 1938; Thommes et al. 2015). A very steep slope especially at the higher RH was observed for hemp ﬁbre and hemp shiv as seen in Fig. 6a, b. Hemp shiv and hemp ﬁbre exhibited higher moisture content on adsorption compared to ﬂax shiv and ﬂax ﬁbre between 80 and 90%RH. Fig. 7. The absolute hysteresis, which was obtained by subtracting the adsorption from desorption isotherm loop moisture content values for the different speci- men over the three cycles, is given in Fig. 8. The sorption isotherms for the ﬂax ﬁbre, hemp ﬁbre and ﬂax shiv specimens were reproducible. For the hemp shiv, the moisture content value of ﬁrst sorption cycle was higher than the second and third cycles. The hysteresis decreased between the ﬁrst and subsequent cycles. The desorption curve of the isotherm remained the same. In addition, results showed the hemp shiv and ﬂax shiv exhibited a higher hysteresis value around 2.5% for second and third cycles at RH 70%. Meanwhile, there was a signiﬁcant decrease in the sorption hysteresis for hemp ﬁbre and ﬂax ﬁbre (approximately 1.2%, average value of three cycles at RH 70%). The individual moisture adsorption and desorption isotherms over three sorption cycles for the ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv are plotted in 123 123 Cellulose (2019) 26:823–843 832 Fig. 6 Comparison of moisture adsorption behaviour of ﬁbre (a) and shiv (b) (Flax and Hemp) at 23 C. The data for different specimens of the same ﬁbre and shiv were reproducible over most of the RH range studied the max time limit (360 min). The other three specimens reached equilibrium within the set time limit (360 min) between 81 and 90%RH. Porosity and pore size distribution of shiv and ﬁbres In addition, it was observed that the equilibration time for both ﬁbres were shorter than the equilibration time for both shiv. Heat sorption behaviour of shiv and ﬁbre The mass change of moisture content at varying humidity range for hemp shiv, hemp ﬁbre, ﬂax shiv and ﬂax ﬁbre is shown in Fig. 10. All specimens had a similar mass change of moisture content below 70% RH. The moisture content of hemp shiv and hemp ﬁbre signiﬁcantly increased compared to ﬂax shiv and ﬂax ﬁbre between RH72 and RH90%. 123 Discussion Previous literature studies have reported that water can be found in constitutive, bound or free forms in natural plant material (Berthold et al. 1998; Mokhothu and john 2015). The constitutive water belongs to part of Figure 11 shows that hemp shiv did not reach the equilibrium (dm/dt = 0.002%) between 81 and 90%RH before moving to the next step after reaching 123 123 833 Cellulose (2019) 26:823–843 as difﬁcult to remove. The by hydrogen bonds to the cell liquid water stored inside the netrated in the lumens by n diffused into the cell wall ce. The penetration of water e swelling of the cell wall B h ld l 1994 1998 (FSP) when the sample contained no free water and no bound water had been desorbed from the cell wall materials. The ﬁbre saturation points for different natural plant materials were slightly different depend- ing on structure and chemical compositions of the cell wall and normally ranged from 26 to 32% moisture content. I hi d h l l i b l h Fig. 7 Moisture adsorption and desorption behaviour for ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv at 23 C the cell walls which was difﬁcult to remove. The bound water was linked by hydrogen bonds to the cell wall. Free water was the liquid water stored inside the lumens. The water penetrated in the lumens by capillary forces and then diffused into the cell wall by hydrogen bonding force. The penetration of water molecules induced some swelling of the cell wall (Nakamura et al. 1981; Berthold et al. 1994, 1998; Bouguerra et al. 1999; Hill et al. 2010, 2012; Ce´lino et al. 2013; Engelund et al. 2013; Mokhothu and John 2015). Skaar (1984) deﬁned the ﬁbre saturation point (FSP) when the sample contained no free water and no bound water had been desorbed from the cell wall materials. The ﬁbre saturation points for different natural plant materials were slightly different depend- ing on structure and chemical compositions of the cell wall and normally ranged from 26 to 32% moisture content. In this study, the plant-water relations below the ﬁbre saturation point were investigated. The results from these experiments showed that the moisture in the cell wall of the specimens can be removed without 12 3 3 834 Cellulose (2019) 26:823–843 ans that the water is held ll walls as bound water. Fig. 8 Hysteresis between adsorption and desorption curves (obtained by subtraction of equilibrium moisture contents) for ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv at different values of relative humidity at 23 C Discussion me of the water molecules surface of cell wall by the ome of the water molecules the gas phase due to the face of cell wall. The SEM levels remained more or less similar, regardless of whether the specimen is shiv or ﬁbre. It indicated that the large pores within the tracheids and pith did not affect the adsorption of moisture in the specimens and the available number of hydroxyl groups was more or less independent of the macroscopic surface area of the specimen. Fig. 7 continued levels remained more or less similar, regardless of whether the specimen is shiv or ﬁbre. It indicated that the large pores within the tracheids and pith did not affect the adsorption of moisture in the specimens and the available number of hydroxyl groups was more or less independent of the macroscopic surface area of the specimen. high temperatures. It means that the water is held preferentially in the cell walls as bound water. Figure 7 showed that some of the water molecules had been adsorbed on the surface of cell wall by the hydrogen bonding force. Some of the water molecules were not free to enter the gas phase due to the interactions within the surface of cell wall. The SEM and MIP results showed that the shiv specimens have a larger surface area, wide range of pore distribution and higher porosity compared to the ﬁbre specimens. However, the moisture content at different humidity At molecular/atomic level, the cell wall can be considered as a micro-mesoporous material. Although the sorption isotherms of all specimens are generally similar in shape, there are still considerable variations 12 123 835 Cellulose (2019) 26:823–843 Fig. 8 Hysteresis between adsorption and desorption curves (obtained by subtraction of equilibrium moisture contents) for ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv at different values of relative humidity at 23 C among them with respect to the absolute values of hygroscopicity. Figures 6 and 10 showed the variation was because of difference in the species. The cell wall of a plant is a complex structure, and the chemical composition and microstructure of cell wall vary with the cell type. In previous publications, the single middle lamellae (ML) and primary wall (P) of hemp shiv were found to have thickness of 30–50 nm and 70–110 nm, respectively (Thygesen et al. 2006; Jiang et al. 2018). Discussion The secondary cell wall was found to have a 100–130 nm thick S1 layer and a 3–13 lm thick S2 layer. The major layers were constructed of 100 nm thick lamellae. Whereas, the lamellae of ﬂax varied in thickness from 100 to 200 nm (Jiang et al. 2018). The thickness of the secondary cell wall was a 5.5–8.2 lm (His et al. 2001). The difference of moisture content between hemp and ﬂax at higher relative humidity may be because of the difference in pore size within the cell wall. According to the literature, capillary suction begins at higher relative humidity due to the presence of nanopores in the cell wall (Skaar 1984). Details of the formation, arrangement and at 23 C layer. The major layers were constructed of 100 nm thick lamellae. Whereas, the lamellae of ﬂax varied in thickness from 100 to 200 nm (Jiang et al. 2018). The thickness of the secondary cell wall was a 5.5–8.2 lm (His et al. 2001). The difference of moisture content between hemp and ﬂax at higher relative humidity may be because of the difference in pore size within the cell wall. According to the literature, capillary suction begins at higher relative humidity due to the presence of nanopores in the cell wall (Skaar 1984). Details of the formation, arrangement and among them with respect to the absolute values of hygroscopicity. Figures 6 and 10 showed the variation was because of difference in the species. The cell wall of a plant is a complex structure, and the chemical composition and microstructure of cell wall vary with the cell type. In previous publications, the single middle lamellae (ML) and primary wall (P) of hemp shiv were found to have thickness of 30–50 nm and 70–110 nm, respectively (Thygesen et al. 2006; Jiang et al. 2018). The secondary cell wall was found to have a 100–130 nm thick S1 layer and a 3–13 lm thick S2 12 3 3 Cellulose (2019) 26:823–843 836 Fig. 8 continued composition of the layers need to be determined in order to understand their inﬂuence on water sorption. cellulose. This indicated that the moisture content would increase as the relative proportion of hemicel- luloses increases, and would decrease as the relative proportion of lignin increases. 123 Discussion However, it was observed in this work that both ﬁbre and shiv from both species had a similar moisture content at a particular relative humidity, even though they had a totally different content of cellulose, hemicellulose and lignin because the shiv had a higher proportion of lignin and hemicellulose compared to the ﬁbres, they had sorption capacities either side of the value for cellulose. The accurate sorption capacity of lignin, The chemical components of the cell wall play a big role on their capacity to bind water. Siau (1984), Skaar (1984) explained that the cell wall of plant contains cellulose, hemicelluloses and lignin. All of these polymers have hydroxyl groups, which can adsorb water molecules by hydrogen-bonding force. The cellulose and hemicellulose have more hydroxyl groups to bind water molecules than lignin, but hydroxyl groups of cellulose are inaccessible to water molecules due to the crystalline regions of the 123 123 hemicellulose and cellulose need to be further studied d th t lli i d h i f hysteresis. One hypothesis was attributed to the ill th f d ti d t th i Fig. 9 Graph comparing the hysteresis (cycle 2) between adsorption and desorption curves for ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv at 23 C Fig. 10 Graph comparing the mass changes (cycle 2-average value of three specimens of the same ﬁbre and shiv) among the different relative humidity ranges for ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv at 23 C Cellulose (2019) 26:823–843 837 837 Cellulose (2019) 26:823–843 Fig. 9 Graph comparing the hysteresis (cycle 2) between adsorption and desorption curves for ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv at 23 C Fig. 10 Graph comparing the mass changes (cycle 2-average value of three specimens of the same ﬁbre and shiv) among the different relative humidity ranges for ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv at 23 C Fig. 10 Graph comparing the mass changes (cycle 2-average value of three specimens of the same ﬁbre and shiv) among the different relative humidity ranges for ﬂax ﬁbre, hemp ﬁbre, ﬂax shiv and hemp shiv at 23 C hemicellulose and cellulose need to be further studied and the crystalline regions and amorphous regions of cellulose also need to be further investigated. hysteresis. Discussion One hypothesis was attributed to the capillary theory of adsorption due to the micropore in the cell wall and another hypothesis was due to polymer relaxation in the cell wall matrix. In our study, Figs. 6 and 10 showed that hemp shiv and hemp ﬁbre had a similar sorption behaviour, which was slightly different to the sorption behaviour of ﬂax shiv The hysteresis for all specimens has been observed to span over the full range of measured relative vapour pressures. As discussed in the introduction, there is a difference of opinion about the mechanism of 12 3 Cellulose (2019) 26:823–843 838 Fig. 11 Graph comparing the time changes between 81% RH and 90% RH for ﬂax ﬁbre, hemp ﬁbre, and ﬂax shiv and hemp shiv at 23 C Fig. 11 Graph comparing the time changes between 81% RH and 90% RH for ﬂax ﬁbre, hemp ﬁbre, and ﬂax shiv and hemp shiv at 23 C Fig. 11 Graph comparing the time changes between 81% RH and 90% RH for ﬂax ﬁbre, hemp ﬁbre, and ﬂax shiv and hemp shiv at 23 C 12 Cellulose (2019) 26:823–843 839 Fig. 11 continued Fig. 11 continued Fig. 11 continued 3 Cellulose (2019) 26:823–843 840 Fig. 12 Mass change percentage of moisture content for hemp shiv at different RH (33%, 54%, 75%) and enthalpy of heat sorption for hemp shiv at different RH (33%, 54%, 75%) under 30 C Fig. 12 Mass change percentage of moisture content for hemp shiv at different RH (33%, 54%, 75%) and enthalpy of heat sorption for hemp shiv at different RH (33%, 54%, 75%) under 30 C Table 3 Mass change percentage of moisture content and enthalpy of heat sorption for hemp shiv, hemp ﬁbre, ﬂax shiv and ﬂax ﬁbre at different RH levels (33%, 54%, 75%) under 30 C Name Humidity (%) Mass change (wt%/RH %) Heat sorption (kJ/kg) Adsorption Desorption Adsorption Desorption Hemp shiv 33 0.15 0.19 3533.7 3243.9 54 0.12 0.15 2862.7 2673.7 75 0.24 0.16 2396.9 2615.8 Hemp ﬁbre 33 0.13 0.14 3045.3 2884.3 54 0.13 0.14 2962.5 2644.8 75 0.24 0.22 2654.3 2628.5 Flax shiv 33 0.15 0.2 3464.9 3163.2 54 0.16 0.16 3123.9 2760 75 0.26 0.17 2747.2 2693.8 Flax ﬁbre 33 0.13 0.13 3505.7 3040.1 54 0.11 0.12 3125.3 2739.1 75 0.22 0.20 2854.1 2710.7 and ﬂax ﬁbre due to the thickness which vary with the cell type. Table 3 Mass change percentage of moisture content and enthalpy of heat sorption for hemp shiv, hemp ﬁbre, ﬂax shiv and ﬂax ﬁbre at different RH levels (33%, 54%, 75%) under 30 C Discussion It meant that the micropores in the cell wall for hemp and ﬂax were different. Figure 9 showed that both hemp shiv and ﬂax shiv had a higher hysteresis than hemp ﬁbre and ﬂax ﬁbre. It can be concluded that the micropores in the cell walls of hemp and ﬂax do not signiﬁcantly affect the hysteresis of sorption. Furthermore, the hemp shiv and ﬂax shiv had similar proportions of chemical composition (proportion of cellulose, hemicellulose and lignin), which were considerably different from that of hemp ﬁbre and ﬂax ﬁbre. Both hemp shiv and ﬂax shiv had higher lignin contents compared to hemp ﬁbre and ﬂax ﬁbre. It indicated that the hysteresis is dependent on the lignin content and independent of the micropores in the cell wall. This is the ﬁrst time that difference in the proportions of chemical composition (proportion of cellulose, hemicellulose and lignin), which were considerably different from that of hemp ﬁbre and ﬂax ﬁbre. Both hemp shiv and ﬂax shiv had higher lignin contents compared to hemp ﬁbre and ﬂax ﬁbre. It indicated that the hysteresis is dependent on the lignin content and independent of the micropores in the cell wall. This is the ﬁrst time that difference in the 12 123 841 Cellulose (2019) 26:823–843 Fig. 13 a Minimal 3D model of water vapour molecules sorption process in bio-based plant materials. b Cross sectional view of biomass matrix: Cellulose crystals (black) are separated by hemicellulose and lignin bundles dry (L0), during hydration, the separation between the cellulose/matrix polymer bundles surface increases (L3 [ L2 [ L1 [ L0). With the increase in this soften space, water vapour molecules can penetrate into the blocked space at a lower relative humidity. c Schematic representation of water vapour adsorption regime on cell wall of bio-based plant materials involving Monolayer sorption—I, Multilayer sorption—II and formation of water cluster (Capil- lary condensation)—III Fig. 13 a Minimal 3D model of water vapour molecules sorption process in bio-based plant materials. b Cross sectional view of biomass matrix: Cellulose crystals (black) are separated by hemicellulose and lignin bundles dry (L0), during hydration, the separation between the cellulose/matrix polymer bundles surface increases (L3 [ L2 [ L1 [ L0). With the increase in this soften space, water vapour molecules can penetrate into the blocked space at a lower relative humidity. Conclusions Berthold J, Olsson RJO, Salme´n L (1998) Water sorption to hydroxyl and carboxylic acid groups in carboxymethyl- cellulose (CMC) studied with NIR-spectroscopy. Cellulose 5(4):281–298 It is the ﬁrst time to compare the moisture and heat sorption characteristics of hemp shiv, hemp ﬁbre, ﬂax shiv and ﬂax ﬁbre based on their physical and chemical properties. Although the shiv and ﬁbre for both ﬂax and hemp had difference in microstructure, porosity and portion of chemical composition, all specimens had a similar moisture adsorption content at a particular relative humidity. This was dependent on the available hydroxyl groups for water molecules in the specimens, which was more or less independent of the macroscopic surface area and porosity of the specimen at large scale ([ 50 nm). The isotherm hysteresis of the shiv were much higher than that of the ﬁbre in the range of 9–90% relative humidity. The extent of hysteresis exhibited was dependent on the content of lignin. As a result, for the ﬁrst time the woody hemp shiv has been reported to have a differentsorption isotherm between the ﬁrst and sub- sequent sorption cycles. The enthalpy of heat sorption was stronger for the initial layer of water molecules and then progressively decreased over successive layers. The total enthalpy change across the full range of humidity was greater than the heat of vaporisation of liquid water. This indicated that some blocked sorption sites become available to water vapour molecules due to the change in molecular structure of the cell wall during the adsorption process. This work offers a developed understanding of the moisture sorption mechanism for bio-based plant materials. Bledzhi AK, Gassan J (1999) Composites reinforced with cel- lulose based ﬁnbres. Prog Polym Sci 24(2):221–274 Bouguerra A, Salle´e H, de Barquin F, Dheilly RM, Que´neudec M (1999) Isothermal moisture properties of wood-cemen- titious composites. Cem Concr Res 29(3):339–347 Bourdot A, Moussa T, Gacoin A, Maalouf C, Vazquez P, Thomachot-Schneider C, Bliard C, Merabtine A, Lachi M, Douzane O, Karaky H, Polidori G (2017) Characterization of a hemp-based agro-material: inﬂuence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties. Energy Build 153(Supplement C):501–512 Boutin M-P, Flamin C, Quinton S, Gosse G (2005) Analyse du cycle de vie de mur en be´ton chanvre banche´ sur ossature en bois (life cycle analysis of a cast hemp-lime timber- framed wall). References Berthold J, Desbrie`res J, Rinaudo M, Salme´n L (1994) Types of adsorbed water in relation to the ionic groups and their counter-ions for some cellulose derivatives. Polymer 35(26):5729–5736 Discussion c Schematic representation of water vapour adsorption regime on cell wall of bio-based plant materials involving Monolayer sorption—I, Multilayer sorption—II and formation of water cluster (Capil- lary condensation)—III sorption isotherm between the ﬁrst and subsequent sorption cycles for the hemp shiv has been reported. Popescu and Hill reported that a difference was found in the sorption isotherm for the ﬁrst and second sorption cycles for naturally aged Tilia cordata Mill wood between 60 and 80%RH. They assumed a possible annealing effect associated with plasticisa- tion of the cell wall polymetric matrix occurring at high moisture contents (Popescu and Hill 2013). This is not in good agreement with this study from the results that had been observed. The potential for the variation between ﬁrst and subsequent sorption cycles for hemp shiv is associated with possible difference in ﬁne cell wall microstructure such as the structure of bordered pits or extractive content. molecules occupy the spaces between the microﬁbils in the cell wall resulting in expansion of the cell wall. These changes most likely affect the degree of available hydroxyl groups due to changes in molecular structure of cell wall. The water molecules form a monolayer of water molecules on the surface of cell wall by hydrogen bonds with the available hydroxyl groups at low relative humidity (monomolecular adsorption—Stage I). However, there are still many sorption sites for water vapour adsorption which are blocked due to the complex arrangement of cellulose, hemicellulose and lignin matrix as visualized in Fig. 13a. For relative humidity between 30 and 50%, the water vapour molecules build up a multilayer (Multilayer adsorption—II) and some blocked hydro- xyl groups are available to water vapour molecules due to the change in molecular structure of the cell wall. Multilayer adsorption continues above RH 50%, but water sorption by capillary condensation becomes dominant. The pore size distribution of the specimens is associated to the beginning of capillary condensa- tion. Finally, there is a small portion of blocked According to the ﬁndings in this study, the binding force was stronger for the initial layer of water molecules and then reduced for the successive layers. The total enthalpy change over the full range of humidity was greater than the heat of vaporisation of liquid water. The water vapour molecules sorption process in bio-based cellulose materials has been shown in Fig. 13. 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https://openalex.org/W2969332135	https://cardiab.biomedcentral.com/track/pdf/10.1186/s12933-019-0913-2	English	null	Temporal relationship between inflammation and insulin resistance and their joint effect on hyperglycemia: the Bogalusa Heart Study	Cardiovascular diabetology	2,019	cc-by	8,591	© The Author(s) 2019. 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. Yan et al. Cardiovasc Diabetol (2019) 18:109 https://doi.org/10.1186/s12933-019-0913-2 Yan et al. Cardiovasc Diabetol (2019) 18:109 https://doi.org/10.1186/s12933-019-0913-2 Cardiovascular Diabetology Abstract Background: Inflammation and insulin resistance play crucial roles in the development of type 2 diabetes mellitus (T2DM). We aim to examine the temporal relationship between high-sensitivity C-reactive protein (hsCRP) and insulin resistance in non-diabetic adults and their joint effect on the development of hyperglycemia. Methods: The longitudinal cohort from the Bogalusa Heart Study consisted of 509 non-diabetic adults (360 whites and 149 blacks, mean age = 42.8 years at follow-up) who had hsCRP, fasting glucose and insulin measured twice at baseline and follow-up over 6.8 years. Cross-lagged panel model was used to examine the temporal relationship between hsCRP and homeostasis model assessment for insulin resistance (HOMA-IR). Information on incident T2DM was collected in a survey in 6.1 years after the follow-up survey. Results: After adjusting for race, sex, age, body mass index, smoking, alcohol drinking and follow-up years, the path coefficient from baseline hsCRP to follow-up HOMA-IR (β2 = 0.105, p = 0.009) was significant and greater than the path from baseline HOMA-IR to follow-up hsCRP (β1 = 0.005, p = 0.903), with p = 0.011 for the difference between β1 and β2. This one-directional path from baseline hsCRP to follow-up HOMA-IR was significant in the hyperglycemia group but not in the normoglycemia group. In addition, participants with high levels of baseline hsCRP and follow-up HOMA-IR had greater risks of T2DM (odds ratio, OR = 2.38, p = 0.035), pre-T2DM (OR = 2.27, p = 0.006) and hyperglyce- mia (OR = 2.18, p = 0.003) than those with low–low levels. Conclusions: These findings suggest that elevated hsCRP is associated with future insulin resistance in non-diabetic adults, and their joint effect is predictive of the development of T2DM. Keywords: Inflammation, C-reactive protein, Insulin resistance, Diabetes shown that multiple inflammation markers including hsCRP are correlated with insulin resistance in diabetic and nondiabetic individuals [12, 13]. It is generally con- sidered that hsCRP, insulin resistance, and T2DM are linked through obesity [14]. To date, there have been no studies focusing on the joint effect of increased hsCRP and insulin resistance on the development of T2DM independent of obesity.h Backgroundl Chronic inflammation has been shown to play a cru- cial role in the development of type 2 diabetes (T2DM) [1–4] and cardiovascular disease [5–9]. High-sensitivity C-reactive protein (hsCRP) is a sensitive marker of sub- clinical inflammation and strongly predicts increased risks of T2DM [10, 11]. Cross-sectional studies have The strong association between hsCRP and insulin resistance suggests that chronic inflammation and insu- lin resistance can influence each other based on patho- physiological and metabolic mechanisms [3, 15–17]. Correspondence: wchen1@tulane.edu Correspondence: wchen1@tulane.edu 3 Department of Epidemiology, Tulane University School of Public Health and Tropical Medicine, 1440 Canal Street, Room 1504G, New Orleans, LA, USA Full list of author information is available at the end of the article Full list of author information is available at the end of the article Measurements Insulin, hsCRP, and glucose were measured at the same time at respective baseline and follow-up surveys. Standardized protocols were used by trained examin- ers across all surveys. Subjects were instructed to fast for 12 h before screening. Replicate measurements of height and weight were made, and the mean values were used for analysis. Body mass index (BMI, weight in kilo- grams divided by height in meters squared) was used as a measure of overall adiposity. Information on medica- tion history, smoking and alcohol drinking was obtained in a questionnaire survey. Current smoking and alcohol drinking were defined as smoking at least one cigarette per day and consuming alcohol every day, respectively, during the prior 12 months. Utilizing longitudinal data from the Bogalusa Heart Study, the current study aims to examine the temporal relationship between inflammation measured by hsCRP and homeostasis model assessment for insulin resistance (HOMA-IR) in non-diabetic adults and their joint effect on the development of T2DM. Plasma hsCRP was measured by latex particle- enhanced immunoturbidimetric assay on Hitachi 902 Automatic Analyzer. Plasma glucose levels were meas- ured as part of a multiple chemistry profile (SMA20; Laboratory Corporation of America, Burlington, NC). A commercial radioimmunoassay kit was used for meas- uring plasma immunoreactive insulin levels (Phadebas; Pharmacia Diagnostics, Piscataway, New Jersey). The intraclass correlation coefficients between blind duplicate values ranged from 0.86 to 0.98 for insulin, hsCRP, and glucose. HOMA-IR was calculated using the formula as described by Matthews [25]: HOMA-IR = fasting plasma insulin (μU/mL) × fasting plasma glucose (mmol/L)/22.5. Diagnosis of T2DM and pre‑T2DMi Diagnosis of T2DM and pre‑T2DM T2DM was defined as fasting glucose ≥ 126 mg/dL or taking anti-diabetic medications. Prediabetes (pre- T2DM) was defined as fasting glucose of 100–125 mg/ dL. Hyperglycemia was defined as being T2DM and/or pre-T2DM. © The Author(s) 2019. 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. Yan et al. Cardiovasc Diabetol (2019) 18:109 Page 2 of 10 Yan et al. Cardiovasc Diabetol (2019) 18:109 Review Board of the Tulane University Health Sciences Center. Review Board of the Tulane University Health Sciences Center. Inflammation may activate the innate immune system and interact with adipose tissue-specific macrophages [15, 17]. Although their relationship is likely bidirectional based on the existing literature in this regard, convincing evidence is lacking from the general population regard- ing the temporal relationship (causal sequence) between hsCRP and insulin resistance. Several longitudinal stud- ies have demonstrated that higher baseline hsCRP and greater change in hsCRP are associated with the devel- opment of insulin resistance [18–21]; however, Mende- lian randomization studies failed to demonstrate a causal relationship between hsCRP levels and insulin resistance/ T2DM [22, 23]. Understanding the potential causal rela- tionship between inflammation and insulin resistance may help provide new insights into the underlying mech- anisms and yield novel approaches to prevent T2DM and cardiovascular diseases by focusing on interventions tar- geting at the causal factor. Inflammation may activate the innate immune system and interact with adipose tissue-specific macrophages [15, 17]. Although their relationship is likely bidirectional based on the existing literature in this regard, convincing evidence is lacking from the general population regard- ing the temporal relationship (causal sequence) between hsCRP and insulin resistance. Several longitudinal stud- ies have demonstrated that higher baseline hsCRP and greater change in hsCRP are associated with the devel- opment of insulin resistance [18–21]; however, Mende- lian randomization studies failed to demonstrate a causal relationship between hsCRP levels and insulin resistance/ T2DM [22, 23]. Understanding the potential causal rela- tionship between inflammation and insulin resistance may help provide new insights into the underlying mech- anisms and yield novel approaches to prevent T2DM and cardiovascular diseases by focusing on interventions tar- geting at the causal factor. Study populationh y The Bogalusa Heart Study is a series of long-term epide- miologic studies in a semi-rural, biracial (65% white and 35% black) community in Bogalusa, Louisiana founded by Dr. Gerald Berenson in 1973. This study focuses on the early natural history of cardiovascular disease from childhood [24]. Between 2000 and 2016, three cross- sectional surveys (baseline, follow-up and outcome sur- veys) of adults aged 24 to 58 years were conducted in Bogalusa, Louisiana. By linking the first two surveys (baseline in 2000–2002 and follow-up in 2006–2010), 755 non-diabetic adult participants were identified who had cardiovascular risk factors, including hsCRP, fasting plasma glucose and insulin, measured twice in both base- line and follow-up surveys, with an average follow-up period of 6.8 years. Among these 755 non-diabetic par- ticipants, 509 adults (360 Whites and 149 Blacks; 34.6% males, mean age = 49.3 years) were enrolled in the last survey (the outcome survey) in 2013–2016. These 509 participants who had incident T2DM and anti-diabetic medication information in the last survey formed the longitudinal cohort for the current study, with an aver- age follow-up period of 6.1 years (range = 3.5–10.0 years) from the second (follow-up) survey. Statistical analysis In more recent years, structural equation modeling has been used to estimate the path coefficients [27]. A simplified and conceptual version of the model is presented in Fig. 1. The path with β1 describes the effect of baseline HOMA-IR on subse- quent hsCRP, and the path with β2 describes the effect of baseline hsCRP on subsequent HOMA-IR. Prior to cross-lagged path analysis, the baseline and follow-up values of log-transformed hsCRP and log-transformed HOMA-IR were adjusted for age, BMI, smoking, and alcohol drinking by regression residual analyses and then standardized by Z-transformation (mean = 0, SD = 1) in race-sex groups. The four variables, i.e. hsCRP and HOMA-IR at baseline and follow-up, gen- erated six pair-wise observed correlations, and five correlations in Fig. 1 were used to estimate β1 and β2 because the connecting path through hsCRP-HOMA- IR correlation at follow-up is illegal according to the path analysis rules. Pearson correlation coefficients of the covariate-adjusted and Z-transformed variables of hsCRP and HOMA-IR at baseline and follow-up were calculated; correlation coefficients between baseline and follow-up values were calculated with additional adjustment for follow-up years. The cross-lagged path coefficients (β1 and β2) in the path diagram in Fig. 1 were estimated simultaneously based on the correla- tion matrix using the maximum likelihood method by the program LISREL 8.52. The validity of model fit- ting was indicated by root mean square residual (RMR) and comparative fit index (CFI) [28]. A significant path coefficient (β1 or β2) suggests the directionality, and a significant difference between β1 and β2 provides stronger evidence for the directional path between the two variables measured over time.i Stratified analyses were performed to examine the dif- ference in cross-lagged path parameters between groups classified by glycemic status, race, sex, and follow-up period. The difference between β1 and β2 derived from the standardized variables (Z-scores) was tested using Fisher’s Z-test. Multivariable logistic regression models were used to examine the joint effect of baseline hsCRP and follow-up HOMA-IR on T2DM, pre-T2DM, and hyperglycemia. Low and high levels of baseline hsCRP and follow-up HOMA-IR were defined by their medians. Statistical analysis Values for hsCRP, insulin, and HOMA-IR were log- transformed before subsequent analyses because of their skewed distributions. Analyses of covariance (general- ized linear models) were performed to test differences in continuous study variables between race and sex groups.h The study design of longitudinal changes of hsCRP, glucose, and insulin measured at two time points in the baseline and follow-up surveys was typically a cross-lagged panel design [26, 27]. The cross-lagged panel analysis is a form of path analysis that simulta- neously examines reciprocal and longitudinal relation- ships among a set of intercorrelated variables. Kenny et al. first proposed the cross-lagged analysis model by All subjects gave informed consent for each survey. Study protocols were approved by the Institutional Yan et al. Cardiovasc Diabetol (2019) 18:109 Page 3 of 10 calculating partial correlation coefficients to measure the direct paths [26]. In more recent years, structural equation modeling has been used to estimate the path coefficients [27]. A simplified and conceptual version of the model is presented in Fig. 1. The path with β1 describes the effect of baseline HOMA-IR on subse- quent hsCRP, and the path with β2 describes the effect of baseline hsCRP on subsequent HOMA-IR. Prior to cross-lagged path analysis, the baseline and follow-up values of log-transformed hsCRP and log-transformed HOMA-IR were adjusted for age, BMI, smoking, and alcohol drinking by regression residual analyses and then standardized by Z-transformation (mean = 0, SD = 1) in race-sex groups. The four variables, i.e. hsCRP and HOMA-IR at baseline and follow-up, gen- erated six pair-wise observed correlations, and five correlations in Fig. 1 were used to estimate β1 and β2 because the connecting path through hsCRP-HOMA- IR correlation at follow-up is illegal according to the path analysis rules. Pearson correlation coefficients of the covariate-adjusted and Z-transformed variables of hsCRP and HOMA-IR at baseline and follow-up were calculated; correlation coefficients between baseline and follow-up values were calculated with additional adjustment for follow-up years. The cross-lagged path coefficients (β1 and β2) in the path diagram in Fig. 1 were estimated simultaneously based on the correla- tion matrix using the maximum likelihood method by the program LISREL 8.52. The validity of model fit- ting was indicated by root mean square residual (RMR) and comparative fit index (CFI) [28]. A significant path coefficient (β1 or β2) suggests the directionality, and calculating partial correlation coefficients to measure the direct paths [26]. Characteristics of participantsh The mean levels of continuous variables were compared between race and sex groups, adjusting for age (except age itself). Although log-transformed hsCRP, insu- lin, and HOMA-IR were used for subsequent analyses, the medians (interquartile ranges) of their original val- ues were presented in Table 1. Race differences in BMI, insulin, and HOMA-IR (blacks > whites) in females and sex differences in glucose (males > females), insu- lin (males > females), HOMA-IR (males > females) and hsCRP (males < females) in whites were observed in both baseline and follow-up surveys. In the outcome survey, race differences in BMI (blacks > whites) in females and incidence of T2DM (blacks > whites) in males and sex differences in hyperglycemia (males > females) and pre- T2DM (males > females) in blacks and whites were found. r1=0.102, p=0.021 r2=0.494, p<0.001 r3=0.437, p<0.001 β1=0.005, p=0.903 β2=0.105, p=0.009a Baseline hsCRP Baseline HOMA-IR Follow-up HOMA-IR (R2=0.186) Follow-up hsCRP (R2=0.224) Fig. 1 Cross-lagged path analysis models for hsCRP and HOMA-IR hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model assessment for insulin resistance; β, standardized regression coefficient; r1, synchronous correlation; r2 and r3, tracking correlations. Goodness-of-fit: root mean square residual = 0.037 and comparative fit index = 0.960. a, p = 0.011 for the difference between β1 and β2 Follow-up hsCRP (R2=0.224) Follow-up HOMA-IR (R2=0.186) Baseline HOMA-IR 3 p Fig. 1 Cross-lagged path analysis models for hsCRP and HOMA-IR hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model assessment for insulin resistance; β, standardized regression coefficient; r1, synchronous correlation; r2 and r3, tracking correlations. Goodness-of-fit: root mean square residual = 0.037 and comparative fit index = 0.960. a, p = 0.011 for the difference between β1 and β2 Yan et al. Characteristics of participantsh Cardiovasc Diabetol (2019) 18:109 Page 4 of 10 Table 1 Characteristics of study participants in baseline, follow-up and outcome surveys by race and sex Continuous variables are presented as means (SD) or medians (interquartile range) BMI, body mass index; hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model assessment for insulin resistance; T2DM, type 2 diabetes mellitus White Black p for race differencea Male (n = 138) Female (n = 222) Male (n = 38) Female (n = 111) Male Female Baseline survey Age (years) 36.4 (4.5) 36.2 (4.2) 36.6 (4.2) 35.0 (4.5) 0.975 0.041 BMI (kg/m2) 28.1 (4.9) 27.2 (6.2) 29.4 (6.7) 31.3 (7.8) 0.286 < 0.001 Smoking, n (%) 35 (25.4) 41 (18.5) 15 (39.5) 33 (29.7) 0.088 0.020 Alcohol drinking, n (%) 54 (39.1) 98 (44.1) 8 (21.1) 42 (37.8) 0.039 0.272 Glucose (mg/dL) 83.5 (9.4)* 79.6 (8.1) 83.2 (8.9) 81.2 (9.3) 0.861 0.121 Insulin (μU/mL)b 10.0 (7.0–14.0)* 8.0 (6.0–13.0) 9.0 (5.0–17.0) 11.0 (7.0–17.0) 0.782 0.001 HOMA-IRb 2.04 (1.45–2.82)* 1.65 (1.11–2.60) 1.89 (1.06–3.66) 2.27 (1.43–3.46) 0.786 0.001 hsCRP (mg/L)b 0.93 (0.37–1.93) 1.65 (0.71–4.15) 1.78 (0.60–3.09) 2.07 (0.58–4.80) 0.131 0.830 Pre-diabetes, n (%) 5 (3.6) 3 (1.4) 2 (5.3) 2 (1.8) 0.155 0.255 Follow-up survey Age (years) 43.2 (4.7) 43.0 (4.5) 43.6 (4.5) 41.9 (4.7) 0.674 0.043 Follow-up period (years) 6.8 (0.9) 6.8 (0.9) 7.0 (0.8) 6.9 (0.9) 0.222 0.123 BMI (kg/m2) 29.6 (5.6) 28.6 (6.7) 31.8 (8.2) 33.0 (8.2) 0.131 < 0.001 Smoking, n (%) 32 (23.2) 50 (22.5) 15 (39.5) 33 (29.7) 0.045 0.152 Alcohol drinking, n (%) 52 (37.7) 91 (41.0) 7 (18.4) 38 (34.2) 0.026 0.233 Glucose (mg/dL) 90.6 (10.7) 86.7 (8.8) 92.5 (12.2)** 88.8 (8.6) 0.345 0.036 Insulin (μU/mL)b 10.1 (6.3–16.2)* 8.2 (5.1–13.0) 13.6 (6.7–24.0) 12.8 (6.96–18.9) 0.052 < 0.001 HOMA-IRb 2.26 (1.35–3.63)* 1.74 (1.04–3.00) 3.33 (1.20–5.78) 2.82 (1.43–4.56) 0.065 < 0.001 hsCRP (mg/L)b 0.93 (0.41–1.97)** 1.35 (0.52–3.14) 1.09 (0.56–2.94) 2.07 (0.58–4.80) 0.186 0.062 Pre-diabetes, n (%) 22 (15.9)* 19 (8.6) 11 (29.0)** 10 (9.0) 0.069 0.891 Outcome survey Age (years) 49.6 (4.5) 49.5 (4.2) 50.1 (4.2)* 48.0 (4.7) 0.547 0.003 Follow-up period (year)c 6.0 (1.1) 6.1 (1.1) 6.2 (1.1) 5.9 (1.1) 0.386 0.067 BMI (kg/m2) 30.3 (5.4) 29.5 (6.7) 32.3 (8.6) 34.9 (8.5) 0.085 < 0.001 Smoking, n (%) 33 (23.9) 45 (20.3) 20 (52.6) 27 (24.3) < 0.001 0.397 Alcohol drinking, n (%) 71 (51.5) 88 (55.4) 18 (47.4) 18 (16.2) 0.656 < 0.001 Hyperglycemia, n (%) 76 (55.1)** 78 (35.1) 27 (71.1)* 50 (45.1) 0.077 0.080 Pre-diabetes, n (%) 61 (44.2)** 51 (22.9) 17 (44.8)* 34 (30.7) 0.288 0.091 T2DM, n (%) 15 (10.9) 27 (12.2) 10 (26.3) 16 (14.4) 0.016 0.563 Table 1 Characteristics of study participants in baseline, follow-up and outcome surveys by race and sex c Follow-up period from the time point of the follow-up survey the difference in coefficients of follow-up log-hsCRP and log-HOMA-IR between races (0.248 vs 0.043) (Table 2). Correlations between hsCRP and HOMA‑IR at baseline and follow‑up After adjusting for race, sex, age, BMI, smoking and alco- hol use, with additional adjustment for follow-up years for baseline-follow-up correlations, most correlation coefficients between baseline and follow-up values of log- hsCRP and log-HOMA-IR were significant in the total sample and subgroups based on the cut-off values for the significance level (p < 0.05) listed below the table. All the coefficients did not differ between subgroups except for Continuous variables are presented as means (SD) or medians (interquartile range) Cross‑lagged path analysis of hsCRP and HOMA‑IR Cardiovasc Diabetol (2019) 18:109 Page 5 of 10 Table 2 Pearson correlation coefficients between log-transformed hsCRP and HOMA-IR at baseline and follow-up in the total cohort and subgroups, adjusted for covariates Covariates included in the models were race, sex, age, BMI, smoking and alcohol use for the total sample and Normo/Hyper groups, with additional adjustment for follow-up years for baseline-follow-up correlations hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model assessment for insulin resistance; Normo, normoglycemia in the outcome survey; Hyper, hyperglycemia in the outcome survey a Correlation coefficients greater than 0.087 are significant (p < 0.05) b Correlation coefficients greater than 0.103 for whites and 0.161 for blacks are significant (p < 0.05) c Correlation coefficients greater than 0.148 for males, and 0.108 for females are significant (p < 0.05) d Correlation coefficients greater than 0.118 for normoglycemia, and 0.129 for hyperglycemia are significant (p < 0.05) * p < 0.05 for race difference Group Variable Baseline hsCRP Baseline HOMA-IR Follow-up hsCRP Totala Baseline HOMA-IR 0.102 Follow-up hsCRP 0.473 0.053 Follow-up HOMA-IR 0.147 0.418 0.187 White/blackb Baseline HOMA-IR 0.108/0.090 Follow-up hsCRP 0.494/0.416 0.082/− 0.014 Follow-up HOMA-IR 0.154/0.152 0.437/0.369 0.248/0.043* Male/femalec Baseline HOMA-IR 0.036/0.139 Follow-up hsCRP 0.444/0.495 0.029/0.068 Follow-up HOMA-IR 0.060/0.190 0.460/0.396 0.161/0.201 Normo/hyperd Baseline HOMA-IR 0.146/0.043 Follow-up hsCRP 0.483/0.455 0.034/0.064 Follow-up HOMA-IR 0.129/0.175 0.340/0.437 0.121/0.268 Table 2 Pearson correlation coefficients between log-transformed hsCRP and HOMA-IR at baseline and follow-up in the total cohort and subgroups, adjusted for covariates C i t i l d d i th d l BMI ki d l h l f th t t l l d N /H ith dditi l dj t t f Group Variable Baseline hsCRP Baseline HOMA-IR Follow-up hsCRP Totala Baseline HOMA-IR 0.102 Follow-up hsCRP 0.473 0.053 Follow-up HOMA-IR 0.147 0.418 0.187 White/blackb Baseline HOMA-IR 0.108/0.090 Follow-up hsCRP 0.494/0.416 0.082/− 0.014 Follow-up HOMA-IR 0.154/0.152 0.437/0.369 0.248/0.043* Male/femalec Baseline HOMA-IR 0.036/0.139 Follow-up hsCRP 0.444/0.495 0.029/0.068 Follow-up HOMA-IR 0.060/0.190 0.460/0.396 0.161/0.201 Normo/hyperd Baseline HOMA-IR 0.146/0.043 Follow-up hsCRP 0.483/0.455 0.034/0.064 Follow-up HOMA-IR 0.129/0.175 0.340/0.437 0.121/0.268 Table 2 Pearson correlation coefficients between log-transformed hsCRP and HOMA-IR at baseline and follow-up in the total cohort and subgroups, adjusted for covariates and β2. The tracking correlations of hsCRP and HOMA- IR from baseline to follow-up and the synchronous cor- relation between hsCRP and HOMA-IR at baseline were significant. The variance (R2) of follow-up hsCRP (0.224) and follow-up HOMA-IR (0.186) explained by baseline predictors was significant. Cross‑lagged path analyses by subgroupshfi The path coefficients (β1) of baseline HOMA-IR → fol- low-up hsCRP in all subgroups were nonsignifi- cant (p > 0.05). The path coefficients (β2) of baseline hsCRP → follow-up HOMA-IR in subgroups of hyper- glycemia (β2 = 0.156, p = 0.008), pre-T2DM (β2 = 0.177, p = 0.012), whites (β2 = 0.108, p = 0.023), females (β2 = 0.138, p = 0.006) and follow-up years above median (β2 = 0.199, p < 0.001) were significant. Signifi- cant differences in the path coefficients (β1 and β2) were noted only between normoglycemia and T2DM groups and between follow-up period groups. The model fit- ting parameters of RMR ranged from 0.002 to 0.148 and CFI from 0.862 to 1.000, indicating a relatively good fit to the observed data (Table 3). The path coefficient of baseline hsCRP → follow-up insulin (β2 = 0.107, p = 0.008) was greater than the path of baseline Cross‑lagged path analysis of hsCRP and HOMA‑IR RMR and CFI were 0.037 and 0.960, respectively, indicating a good fit to the observed data according to the criteria of RMR < 0.05 and CFI > 0.90 (Fig. 1). insulin → follow-up hsCRP (β1 = − 0.001, p = 0.998), with p = 0.004 for the difference between β1 and β2 (Additional file 1: Fig. S1). In contrast, neither the path coefficient of hsCRP → glucose (β2 = 0.040, p = 0.339) nor the path of glucose → hsCRP (β1 = − 0.025, p = 0.520) was significant. In addition, the path coef- ficient of hsCRP → insulin (β2 = 0.152, p = 0.009) was significant in the hyperglycemia group, but not signifi- cant in the normoglycemia group (β2 = 0.088, p = 0.121) (Additional file 1: Fig. S2). Cross‑lagged path analysis of hsCRP and HOMA‑IR gg p y After adjusting for race, sex, age, BMI, smoking, alcohol drinking, and follow-up years, the path coefficient from baseline hsCRP to follow-up HOMA-IR (β2 = 0.105, p = 0.009) was greater than the path coefficient from baseline HOMA-IR to follow-up hsCRP (β1 = 0.005, p = 0.903), with p = 0.011 for the difference between β1 Yan et al. Principal findingsh This community-based longitudinal cohort study of black and white adults examined the temporal relation- ship between inflammation measured by hsCRP and insulin resistance estimated by HOMA-IR in a com- munity-based cohort using a cross-lagged path analy- sis model, a statistical approach to dissecting a causal relationship between inter-correlated variables [26, 27]. We found that higher hsCRP preceded HOMA-IR rather than vice versa in non-diabetic adults, and this one-directional relationship was found to be significant in pre-T2DM and hyperglycemia groups but not in the normoglycemia group; high levels of baseline hsCRP and follow-up HOMA-IR were significantly associ- ated with increased risks of T2DM, pre-T2DM and Joint effect of inflammation and insulin resistance on hyperglycemiah The percentages of T2DM (p = 0.010), pre-T2DM (p = 0.006) and hyperglycemia (p = 0.001) in the outcome survey were significantly greater among participants with high hsCRP at baseline and high HOMA-IR at follow- up (high–high group) than among participants with low hsCRP at baseline and low HOMA-IR at follow-up (low– low group) (Fig. 2). After adjusting for race, sex, age, BMI, smoking, and alcohol drinking, participants in the high– high group had greater risks of T2DM (OR = 2.38, 95% CI 1.06–5.53), pre-T2DM (OR = 2.27, 95% CI 1.27–4.05) and hyperglycemia in the outcome survey (OR = 2.18, 95% CI 1.30–3.64) than those in the low–low group (Table 4). Yan et al. Joint effect of inflammation and insulin resistance on hyperglycemiah Cardiovasc Diabetol (2019) 18:109 Page 6 of 10 Table 3 Cross-lagged path coefficients between log-transformed hsCRP and HOMA-IR by glycemia status, race, sex, and follow-up period β standardized regression coefficient; RMR root mean square residual; CFI comparative fit index; hsCRP high sensitivity C reactive protein; HOMA IR homeostasis Path coefficients Goodness-of-fit HOMA-IR → hsCRP hsCRP → HOMA-IR pa RMR CFI β1 p β2 p Glycemia status Normoglycemia (n = 278) − 0.037 0.483 0.081 0.155 0.032 0.022 0.990 Hyperglycemia (n = 231) 0.045 0.449 0.156 0.008 0.059 0.054 0.914 T2DM (n = 68) 0.131 0.206 0.143 0.199 0.911 0.031 0.994 Pre-T2DM (n = 163) 0.006 0.938 0.177 0.012 0.015 0.061 0.884 pb 0.141 0.194 pc 0.011 0.380 pd 0.475 0.121 Race White (n = 360) 0.029 0.530 0.108 0.023 0.111 0.097 0.932 Black (n = 149) − 0.052 0.490 0.120 0.117 0.022 0.148 1.000 pe 0.148 0.843 Sex Male (n = 176) 0.013 0.848 0.044 0.518 0.645 0.104 0.928 Female (n = 333) − 0.001 0.986 0.138 0.006 0.005 0.111 0.962 pf 0.801 0.095 Follow-up period Below median (n = 245) − 0.018 0.756 0.035 0.560 0.352 0.067 0.862 Above median (n = 264) 0.026 0.627 0.199 < 0.001 < 0.001 0.002 0.981 pg 0.885 0.003 Table 3 Cross-lagged path coefficients between log-transformed hsCRP and HOMA-IR by glycemia status, race, sex, and follow-up period path coefficients between log-transformed hsCRP and HOMA-IR by glycemia status, race, sex, β, standardized regression coefficient; RMR, root mean square residual; CFI, comparative fit index; hsCRP, high-sensitivity C-reactive model assessment for insulin resistance; T2DM, type 2 diabetes mellitus a p value for difference between β1 and β2 f 1 2 b p value for difference in β1 and β2 between hyperglycemia and normoglycemia groups c p value for difference in β1 and β2 between T2DM and normoglycemia groups d p value for difference in β1 and β2 between pre-T2DM and normoglycemia groups e p value for difference in β1 and β2 between whites and blacks f p value for difference in β1 and β2 between males and females g p value for difference in β1 and β2 between follow-up period groups (median = 6.7 years) hyperglycemia. The findings suggest that hsCRP levels are associated with subsequent HOMA-IR in non-dia- betic adults, and their joint effect predicts the develop- ment of T2DM. Temporal relationship between inflammation and insulin resistance Inflammation and insulin resistance are two well-known mechanisms linking obesity, T2DM and cardiovascular disease [29, 30]. Multiple inflammation markers includ- ing hsCRP are correlated with hyperglycemia and insulin resistance in majority of previous studies [13, 21, 31–36]. An important question raised by these observations is whether adiposity-induced inflammation reaction pre- cedes insulin resistance or vice versa, or whether this relationship is bidirectional, especially in the evolution of T2DM. Cross-sectional study design limits the inference Yan et al. Cardiovasc Diabetol (2019) 18:109 Page 7 of 10 0 10 20 30 40 50 60 1 2 3 ) % ( e g a t n e c r e P p=0.010 p=0.006 p=0.001 T2DM Pre-T2DM Hyperglycemia Baseline hsCRP: High Low High Low High Low Follow-up HOMA-IR: High Low High Low High Low N: 88 104 117 127 141 140 Fig. 2 Percentage of T2DM, Pre-T2DM and hyperglycemia in the outcome survey between high–high and low–low groups of baseline hsCRP and follow-up HOMA-IR. hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model assessment for insulin resistance; T2DM, type 2 diabetes mellitus. High and low levels of hsCRP and HOMA-IR were defined by their medians Fig. 2 Percentage of T2DM, Pre-T2DM and hyperglycemia in the outcome survey between high–high and low–low groups of baseline hsCRP and follow-up HOMA-IR. hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model assessment for insulin resistance; T2DM, type 2 diabetes mellitus. Temporal relationship between inflammation and insulin resistance High and low levels of hsCRP and HOMA-IR were defined by their medians Table 4 ORs for T2DM, Pre-T2DM, and hyperglycemia in the outcome survey associated with the high–high group with the low–low group as reference, adjusting for covariates Table 4 ORs for T2DM, Pre-T2DM, and hyperglycemia in the outcome survey associated with the high–high group with the low–low group as reference, adjusting for covariates Table 4 ORs for T2DM, Pre-T2DM, and hyperglycemia in the outcome survey associated with the high–high group with the low–low group as reference, adjusting for covariates High and low levels of baseline hsCRP and follow-up HOMA-IR were defined by their medians OR, odds ratio; CI, confidence interval; T2DM, type 2 diabetes mellitus; BMI, body mass index; hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model assessment for insulin resistance a Coding: high–high group = 1; low–low group = 0 Independent variable Outcome T2DM (37 vs 155) Pre-T2DM (89 vs 155) Hyperglycemia (126 vs 155) OR (95% CI) p OR (95% CI) p OR (95% CI) p Black race 1.14 (0.50–2.63) 0.758 0.98 (0.51–1.90) 0.953 1.07 (0.60–1.91) 0.823 Female sex 0.72 (0.30–1.73) 0.467 0.39 (0.21–0.72) 0.003 0.49 (0.28–0.84) 0.010 Age 1.05 (0.96–1.16) 0.286 1.10 (1.02–1.17) 0.010 1.08 (1.02–1.15) 0.011 BMI 1.07 (1.01–1.12) 0.014 1.09 (1.05–1.14) < 0.001 1.08 (1.04–1.12) < 0.001 Smoking 1.74 (0.71–4.25) 0.224 1.17 (0.59–2.32) 0.647 1.28 (0.70–2.34) 0.419 Alcohol drinking 0.30 (0.11–0.86) 0.025 1.06 (0.57–1.96) 0.855 0.77 (0.44–1.36) 0.367 Groupa 2.38 (1.06–5.33) 0.035 2.27 (1.27–4.05) 0.006 2.18 (1.30–3.64) 0.003 High and low levels of baseline hsCRP and follow-up HOMA-IR were defined by their medians OR, odds ratio; CI, confidence interval; T2DM, type 2 diabetes mellitus; BMI, body mass index; hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model assessment for insulin resistance a Coding: high–high group = 1; low–low group = 0 examined the bidirectional relationship between inflam- mation and insulin resistance and showed that base- line levels of hsCRP and interleukin-6 were positively associated with subsequent increases in fasting insulin, HOMA-IR, and beta-cell function. In the reverse analy- sis, baseline HOMA-IR was not associated with change in inflammation biomarkers [19]. However, these tra- ditional longitudinal analysis models cannot address the causal relationship. The present study confirmed and extended previous findings by showing that higher hsCRP preceded insulin resistance among non-diabetic adults using a cross-lagged path analysis model. This regarding the temporal relationship between hsCRP and insulin resistance. Joint effect of inflammation and insulin resistance on T2DM Joint effect of inflammation and insulin resistance on T2DM Joint effect of inflammation and insulin resistance on T2DM one-directional temporal relationship was observed only in participants who had hyperglycemia but not in nor- moglycemic adults. In addition to HOMA-IR, we found that hsCRP elevation preceded increased fasting insulin but not for fasting glucose, suggesting that insulin levels contribute more than glucose to the HOMA-IR index to measure insulin resistance, and fasting insulin alone may be a simple and effective surrogate measure of insulin resistance [37]. The causal relationship between hsCRP and insulin resistance may account for the observation that anti-inflammatory treatments can improve glycemia and insulin resistance among diabetic patients [38, 39].hl Previous longitudinal studies have demonstrated inde- pendent effects of hsCRP and HOMA-IR in predicting the risk of T2DM [47, 48]; however, their joint effect on the development of T2DM has not been reported. In the present study, two approaches were applied to test the joint effect of hsCRP and HOMA-IR on T2DM, pre- T2DM and hyperglycemia, i.e. estimating the effect of baseline hsCRP on follow-up HOMA-IR in glycemic groups and assessing the risk of hyperglycemia of the group with high levels of baseline hsCRP and follow- up HOMA-IR with the low–low group as reference. We found that the path coefficients of baseline hsCRP → fol- low-up HOMA-IR were significant in the pre-T2DM and hyperglycemia groups; individuals with high lev- els of baseline hsCRP and follow-up HOMA-IR had significantly increased risks of T2DM, pre-T2DM and hyperglycemia compared with those who had low–low levels. The results suggest that inflammation and insu- lin resistance have a joint effect on the pathogenesis of hyperglycemia. g p The mechanisms linking inflammation to insulin resist- ance are incompletely elucidated. Existing data sup- port the concept that the temporal relationship between chronic inflammation and insulin resistance is bidi- rectional like a “two-way street”, i.e. they are mutually influenced based on the underlying metabolic and physi- ologic mechanisms [3, 15]. Previous studies suggest that inflammation may promote insulin resistance through the production of proinflammatory cytokines such as interleukin-1β, interacting with adipose tissue-specific macrophages and activation of innate immune system [3, 15, 40]. In addition, inhibition of inflammatory signaling by knockout of key pathways in obese mice can directly prevent the development of insulin resistance [41, 42]. Strengths and limitations Th b d l This community-based longitudinal cohort study pro- vides a unique opportunity to examine the temporal relationship between increased hsCRP and insulin resist- ance and their joint effect on hyperglycemia. However, several limitations should be noted. First, the relatively small sample size of subgroups, especially the num- ber of T2DM patients, had a limited statistical power to detect a weak-to-moderate association. Second, we assessed chronic inflammation state using hsCRP, which is a downstream inflammatory marker. Further studies are required involving a wider spectrum of inflammatory biomarkers such as fibrinogen, interleukin 6, and tumor necrosis factor-α. Third, T2DM was diagnosed based on fasting blood glucose and anti-diabetic medication his- tory in this study without 2 h blood glucose data, which may lead to missed diagnosis. Several studies have reported significant sex and black- white differences in the cross-sectional association between hsCRP and HOMA-IR [44–46]. In the present study, however, we did not find significant sex and race differences in the cross-sectional correlations (except for the correlation at follow-up) and longitudinal directional path parameters between hsCRP and HOMA-IR. These inconsistencies may be due to limited sample size and different age periods, and thus future studies with larger sample sizes are needed to assess the sex- and race-spe- cific association, particularly in the temporal relationship analysis. Joint effect of inflammation and insulin resistance on T2DM On the other hand, experimental studies in mice have suggested that insulin resistance may cause inflamma- tion through the production of the chemokine monocyte protein 1 [16]. It is well-known that inflammation and insulin resistance are linked through obesity [14]. In the present observational population study, the results from the cross-lagged analyses indicated that elevated hsCRP occurred prior to increased HOMA-IR independent of BMI in non-diabetic participants, but the reverse was not true. The finding of this one-directional time sequence from our study provided a necessary first step in estab- lishing causation between systemic inflammation and insulin resistance. Further large and well-controlled pro- spective clinical trials targeting inflammatory pathways for its treatment are warranted [43]. Temporal relationship between inflammation and insulin resistance Several longitudinal studies have attempted to demonstrate the temporal relationship between inflammation, insulin resistance, and T2DM [10, 11, 18, 19]. Multiple inflammatory markers, including hsCRP, interleukin-6, and plasminogen activator inhibi- tor-1, have been shown to independently predict incident T2DM [10, 11]. The Coronary Artery Risk Development in Young Adults study showed that elevated hsCRP was associated with concurrent and future insulin resist- ance after accounting for adiposity and oxidative stress markers [18]. To date, only one longitudinal study has Yan et al. Cardiovasc Diabetol (2019) 18:109 Page 8 of 10 Conclusionsh This study demonstrates that elevated CRP is associated with future insulin resistance in non-diabetic adults, and their joint effect is predictive of the development of T2DM. The findings will improve our understanding of the pathobiology, mechanisms, and natural history of T2DM. Interventional studies are warranted regarding whether anti-inflammatory therapy is an effective strat- egy for reducing the risk of insulin resistance and T2DM. Page 9 of 10 Yan et al. Cardiovasc Diabetol (2019) 18:109 Yan et al. Cardiovasc Diabetol Author details 1 Department of Epidemiology, Capital Institute of Pediatrics, Beijing, China. 2 Department of Non‑communicable Disease Management, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China. 3 Department of Epidemiology, Tulane University School of Public Health and Tropical Medicine, 1440 Canal Street, Room 1504G, New Orleans, LA, USA. 4 Children’s Minnesota Research Institute, Children’s Hospitals and Clinics of Minnesota, Minneapolis, MN, USA. 5 Department of Cardiology, The First Affiliated Hospital of Soochow University, Suzhou, China. 17. Savage DB, Petersen KF, Shulman GI. Mechanisms of insulin resist- ance in humans and possible links with inflammation. Hypertension. 2005;45(5):828–33. 17. Savage DB, Petersen KF, Shulman GI. Mechanisms of insulin resist- ance in humans and possible links with inflammation. Hypertension. 2005;45(5):828–33. 18. Park K, Steffes M, Lee DH, Himes JH, Jacobs DR. Association of inflam- mation with worsening HOMA-insulin resistance. Diabetologia. 2009;52(11):2337–44. 18. Park K, Steffes M, Lee DH, Himes JH, Jacobs DR. Association of inflam- mation with worsening HOMA-insulin resistance. Diabetologia. 2009;52(11):2337–44. 19. Herder C, Færch K, Carstensen-Kirberg M, Lowe GD, Haapakoski R, Witte DR, Brunner EJ, Roden M, Tabák AG, Kivimäki M, et al. Biomarkers of sub- clinical inflammation and increases in glycaemia, insulin resistance and beta-cell function in non-diabetic individuals: the Whitehall II study. Eur J Endocrinol. 2016;175(5):367–77. Received: 4 May 2019 Accepted: 12 August 2019 20. Uemura H, Katsuura-Kamano S, Yamaguchi M, Bahari T, Ishizu M, Fujioka M, Arisawa K. Relationships of serum high-sensitivity C-reactive protein and body size with insulin resistance in a Japanese cohort. PLoS ONE. 2017;12(6):e0178672. Abbreviations 5. Kaptoge S, Di Angelantonio E, Lowe G, Pepys MB, Thompson SG, Collins R, Danesh J. C-reactive protein concentration and risk of coronary heart disease, stroke, and mortality: an individual participant meta-analysis. Lancet. 2010;375(9709):132–40. 5. Kaptoge S, Di Angelantonio E, Lowe G, Pepys MB, Thompson SG, Collins R, Danesh J. C-reactive protein concentration and risk of coronary heart disease, stroke, and mortality: an individual participant meta-analysis. Lancet. 2010;375(9709):132–40. T2DM: type 2 diabetes; hsCRP: high-sensitivity C-reactive protein; HOMA-IR: homeostatic model assessment for insulin resistance; RMR: root mean square residual; CFI: comparative fit index; OR: odds ratio; CI: confidence interval. 6. Bao X, Borne Y, Johnson L, Muhammad IF, Persson M, Niu K, Engstrom G. Comparing the inflammatory profiles for incidence of diabetes mellitus and cardiovascular diseases: a prospective study exploring the ‘common soil’ hypothesis. Cardiovasc Diabetol. 2018;17(1):87. 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https://openalex.org/W2590939961	https://hts.org.za/index.php/hts/article/download/4344/9253	English	null	The role of spirituality as a coping mechanism for South African traffic officers	HTS teologiese studies	2,017	cc-by	5,716	HTS Teologiese Studies/Theological Studies ISSN: (Online) 2072-8050, (Print) 0259-9422 HTS Teologiese Studies/Theological Studies ISSN: (Online) 2072-8050, (Print) 0259-9422 Page 1 of 6 Original Research Page 1 of 6 Original Research Original Research Page 1 of 6 Dates: How to cite this article: Jacobs, R. & Van Niekerk, A., 2017, ‘The role of spirituality as a coping mechanism for South African traffic officers’, HTS Teologiese Studies/ Theological Studies 73(3), a4344. https://doi.org/ 10.4102/hts.v73i3.4344 Copyright: © 2017. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Introduction The work of traffic officers is very important yet quite stressful, thus the need for emotional stability, self-awareness and stress management skills in their field (Pienaar 2007:3). Interestingly, people in general respond differently even when they are confronted with identical stressors (Louw & Viviers 2010:1), and so do traffic officers. There might be various reasons for these different responses. One such reason might be situated in the concept of spirituality and how a person applies spirituality to cope more effectively in the place of work (Jacobs 2013:160; Moos 2002:67). The role of spirituality as a coping mechanism for South African traffic officers Read online: Scan this QR code with your smart phone or mobile device to read online. Authors: Rochelle Jacobs1 Annelize van Niekerk1 Affiliations: 1Department of Industrial and Organisational Psychology, University of South Africa, South Africa Corresponding author: Annelize van Niekerk, vnieka2@unisa.ac.za Dates: Received: 14 Oct. 2016 Accepted: 24 Nov. 2016 Published: 22 Feb. 2017 How to cite this article: Jacobs, R. & Van Niekerk, A., 2017, ‘The role of spirituality as a coping mechanism for South African traffic officers’, HTS Teologiese Studies/ Theological Studies 73(3), a4344. https://doi.org/ 10.4102/hts.v73i3.4344 Copyright: © 2017. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Traffic officers are faced with many stressful situations, yet each traffic officer might cope differently with these stressors. Spirituality is regarded as an essential defence in stressful situations. Therefore, this article provides a basic framework guiding traffic officers and practitioners, on how spirituality can be used as a coping mechanism when faced with various work-related stressors. An interpretative, qualitative study was conducted utilising purposive sampling in which 10 traffic officers participated in in-depth interviews. In line with the interpretive paradigm, data were analysed using content analysis. The research findings indicate when utilising spirituality to various degrees in their workplace, traffic officers displayed adaptive coping capabilities. Traffic officers associated less spirituality or a lack thereof with weaker coping capability. Furthermore, spirituality in traffic officers is informed by their spiritual or religious foundation, their purpose in work and life, their connection to a spiritual source, and the fruits of spirituality. The coping ability of traffic officers is influenced by their upbringing and background, by stressors in their work environment and by their coping mechanisms. The role of spirituality in the coping of traffic officers culminated in their ability to interpret the meaning of spirituality, and then implementing spirituality as a coping mechanism. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Copyright: Copyright: © 2017. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. The context within which the South African traffic officer functions stems firstly from their appointment as prescribed by the National Road Traffic Act, No. 93 (1996). Accordingly, their duties include inspecting any vehicle for compliance within the provisions of this Act, temporarily forbidding a person from driving a vehicle should that person seem incapable, controlling and regulating traffic on any public road and requiring any person to furnish any particulars needed for identification. Actual law enforcement is done on all the public roads within the boundaries of a particular local municipal or provincial area (National Road Traffic Act 1996). The work environment of the traffic officer also includes the Law Enforcement Section within Traffic Services, where the officer carries out administrative duties. Secondly, traffic officers are appointed as peace officers in terms of section 334(1) (a) of the Criminal Procedure Act, No. 51 (1977), as published on 02 September 2011 (Gazette No. 34583, Regulation 707). These duties include all powers bestowed upon peace officers and all powers awarded to police officials, with the exclusion of specified sections and subject to certain provisions. Given the established overlapping nature of the duties of police officers and traffic officers, it is useful to consider findings from police research which are relevant to the current study. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Studies on policing and stress indicated that police work is indeed stressful (Anderson, Litzenberger & Plecas 2002:399). Police tasks contain a spiritual element because in their job they pursue what is good, face threats to their lives and find their worth being ignored by the community (Smith & Charles 2010:320). Similarly, the road environment places growing demands on traffic officers (Pienaar 2007:3), as they may be attacked by disgruntled motorists, targeted for their fire arms and thrown off motor cycles (Mushwana 1998:108). This may be detrimental to their well-being and affect the individual’s perspective on life (Pienaar 2007:93; Smith & Charles 2010:320). Nonetheless, research indicated that people can affect their own well-being through Read online: Scan this QR code with your smart phone or mobile device to read online. How to cite this article: Jacobs, R. & Van Niekerk, A., 2017, ‘The role of spirituality as a coping mechanism for South African traffic officers’, HTS Teologiese Studies/ Theological Studies 73(3), a4344. https://doi.org/ 10.4102/hts.v73i3.4344 Spirituality Existing research related to the spiritual experiences of traffic officers is either fairly old (Mushwana 1998; Van Heerden 1990) or explores spirituality from a police officer’s perspective (Collett 2011; Joubert & Grobler 2013; Karakas 2010; Kinjerski & Skrypnek 2008; Pienaar & Rothmann 2003). As ‘spirit at work’ begins with the individual and requires a holistic consideration of existence (Gnanaprakash 2013:383; Kourie 2009:151; Krok 2008:643), more attention needs to be given to the human element of traffic officers and not just to the law enforcement aspects of their profession (Pienaar 2007:5). Human elements include the values that drive them (Pienaar 2007:5) and how to cope with stress presented during the performance of their daily duties (Pienaar 2007:5). Research specific to the concept of coping of traffic officers seems to be limited (Pancheri et al. 2002; Pienaar 2007; Van Heerden 1990). Defining the complex concept of spirituality seems challenging (Jacobs 2013:1; Naidoo 2014:4) because of its subjective nature (Karakas 2010:26). Among the various definitions of spirituality, some or all of the following elements seem to be included: meaning and purpose, ethical values and beliefs, relationships or connectedness, and transcendence (Jacobs 2013:159). Other definitions describe a personal experience (Lombaard 2011:77; Waaijman 2007:5) searching for a persistent understanding of the existential self, connecting with the sacred (Karakas 2010:8), believing in a higher power and understanding life through lived experience (Jacobs 2013:160; Welzen 2011:38). Spirituality relates to the place of work as ‘spirit at work’ and entails becoming aware of your higher self, within which one seeks to be purposeful in life and work (Kinjerski & Skrypnek 2004:319; Schreuder & Coetzee 2011:11). It is also concerned with cherished relationships with both a higher force and other people (Mohan & Uys 2006:58). Connectedness with people is part of the need for self-actualisation in Maslow’s hierarchy of needs, which motivates some people (Theron 2009:132). Motivation involves spiritual purpose which includes being driven by a need for spiritual wholeness, deeper meaning and the search for creative self-expression through work and relationships (Coetzee & Roythorne- Jacobs 2007:162). ‘Spirit at work’ comprises interpersonal, physical, emotional, mental and spiritual characteristics, and this definite state entails physiological stimulation and positive affect (Kinjerski & Skrypnek 2004:26). However, Naidoo (2014:3) cites Ali and Gibbs (1998) and shows that the work ethics of believers may also bring about disadvantages such as discrimination because of spiritual beliefs (Karakas 2010:26). Copyright: Read online: Scan this QR code with your smart phone or mobile device to read online. http://www.hts.org.za Page 2 of 6 Page 2 of 6 Original Research Original Research practices of forgiveness, spirituality and religion (Boehm & Lyubomirsky 2009:8; Schreuder & Coetzee 2011:275). The differences between, and the overlapping aspects related to, spirituality and religion are acknowledged (Collett 2011:50; Jacobs 2013:164; Karakas 2010:8; Kourie 2009:153; Welzen 2011:37) and often applied interchangeably and complimentary to each other (Kourie 2009:153). 2006:58). Perhaps, the issue is more about whether there is a common definition understood by all, as there may be too many people defining spirituality differently. For the purpose of this study, spirituality is defined as individuals’ experiences of the Divine (Lombaard 2011:77) on their journey of meaningful authentic self-discovery (Karakas 2010:8) and entails how people express these experiences through actions and attitudes (Lombaard 2011:77). Spirituality Notwithstanding, adopting spirituality on a personal level also prompts people to consider ethical aspects, which leads to individual and societal transformation (Kourie 2009:168; Naidoo 2014:1). Hence, the benefits of spirituality in the workplace seem to outweigh the disadvantages (Van Tonder & Ramdass 2009:2), with its motivating energy facilitating service regardless of challenges (Kinjerski & Skrypnek 2004:28), which increases commitment and productivity (Naidoo 2014:1). Spirituality A foundation of spirituality or religion plays a fundamental role in the development and formation of the traffic officer’s spirituality. Participants indicated that the influence of a higher power was important to their spirituality (Kinjerski & Skrypnek 2008:319). In the challenging high-risk traffic environment (Mushwana 1998:134), traffic officers rely on this higher power for the protection of the self, family and different road users who are all at risk of accidents and dangerous incidents. Upbringing within spirituality or religion by parents and others seems to play a fundamental role in the formation of participants’ foundation and subsequent views on spirituality. Participants further indicated that spirituality provided values and morals (Jacobs 2013:159), helping them to distinguish between right and wrong and to explore ethical aspects (Kourie 2009:168). Coping to Pienaar, Rothmann and Van De Vijver (2007:14), people can manage the effects of negative work experiences by confronting the stressors and looking for lessons in such experiences from a religious perspective. Coping Generally, coping is a positive psychological construct (Coetzee & Viviers 2007:485), which refers to the perceptual, mental or behavioural efforts that people employ to deal with situations deemed potentially difficult and stressful (Schreuder & Coetzee 2011:383). Coping ability may be viewed by some as the presence or absence of coping skills (Ryan, Rapley & Dziurawiec 2014:1069), but may differ in the extent to which contextual factors, such as social factors as opposed to individual factors, are considered (Ryan, Rapley & Dziurawiec 2014:1069). For the purpose of this study, coping is viewed as the efforts employed by individuals to reduce the negative influences of stress on personal well-being, which may involve individual resources as well as their perceptions of challenges (Cheng, Mauno & Lee 2014:73; Edwards 1988:243). Lack of coping skills may negatively affect employees and organisations, through poor service to society, increased employee absenteeism and turnover (Dewe, O’Driscoll & Cooper 2010). Pienaar (2007:93) propose the solution to be increased traffic officer training at departmental level focused at assisting them to cope better with stressors. Generally, internal organisational stressors include red tape, internal politics and role uncertainty (Luthans 2008:249). External organisational stressors include technology, changes in the economy, globalisation, the effects of family on the community, and race and gender considerations (Luthans 2011:282). Traffic officers might be faced with internal organisational stressors such as rotating work shifts, tight controls and demanding work conditions, as well as external organisational stressors such as those listed above. According The post-modern approach recognises all these different views and truths on spirituality (Jacobs 2013:164) and emphasises the interconnectedness of living things (Kourie 2009:152). Even though literature indicates that there is no clear definition of spirituality (Jacobs 2013:1), it can be contested that the issue may be less about clarity, as commonalities in perspectives are evident (Mohan & Uys http://www.hts.org.za Open Access Page 3 of 6 Page 3 of 6 Original Research Data were collected with semi-structured, in-depth interviews. Interviews were primarily conducted away from the work environment to avoid interruptions, ensure privacy and create a relaxed environment (Kelly 2006a:298; Terre Blanche, Kelly & Durrheim 2006:276). Permission was obtained from the participants to audio record the interviews and to make verbatim transcriptions (Koekemoer & Mostert 2010:3; Wassenaar 2009:75). Conversing with traffic officers on two or more occasions further contributed towards ensuring that findings about their perceptions were correct representations of their views (Kelly 2006b:380). Theoretical relationship The theoretical relationship between spirituality and coping in the workplace has been explored and is depicted in Figure 1. Trends in the literature indicate the influence of spirituality on coping (Gnanaprakash 2013:383; Krok 2008:643) and that people may respond differently given the same challenges (Louw & Viviers 2010:1). The same stressor may be posed to different people. Yet, higher and lower levels of spirituality result in the application of positive and negative thoughts, respectively. Therefore, according to Rowe and Allen (2003:63) and Bryant-Davis et al. (2012), positive thoughts are associated with high spirituality and appear to result in stronger coping styles, while the application of negative thoughts is associated with lower spirituality, resulting in weak coping styles. Conventional content analysis was applied to analyse the data as it assisted with reducing textual data of subjective experiences into meaningful categories (McMillan & Schumacher 2010:367; Muchinsky, Kriek & Schreuder 2009:31) through inductive reasoning (Yin 2010:94). Findings The data analysis yielded various themes and subthemes that relate to spirituality, coping and the role of spirituality in the coping of traffic officers. Subsequently, this study aimed to explore how traffic officers experience the role of spirituality in coping within the South African work context and to provide a framework that can assist towards better understanding this phenomenon. Methodological approach (Boehm & Lyubomirsky 2009:1), by making conscious decisions informed by prayer and spiritual beliefs. They take responsibility for what manifests in their lives, thus acknowledging their free will (Bergh 2009:317). The traffic work environment presents stressors (Mushwana 1998:134), the effects of which may manifest as aggression and frustration, adversely affecting relationships with others (Pienaar 2007:93). One such stressor is work conditions. Brutal incidents, crime and possibly being involved in accidents seem to pose a threat to work conditions. These may affect individuals emotionally and psychologically (Edwards 1992:245). Further, exhausting shift work or odd working hours may have a negative influence on physical well-being. A second stressor seems to be treatment from others. Traffic officers may face negative attitudes from road users (Pienaar 2007:63), poor treatment by colleagues and unfounded accusations. To cope participants may alter their perceptions around this treatment (Edwards 1992:243), alter distressed relationships between themselves and others, and regulate the emotional distress experienced (Boehmer, Luszczynska & Schwarzer 2007:63). A further stressor appears to be inadequate coping mechanisms. Participants indicated that there are inadequate coping mechanisms and counselling services available at departmental level to assist in dealing with these stressors, and subsequently issues are just left without being attended to. Participants apply various strategies to connect to their higher power or spiritual source (Mohan & Uys 2006:58). One such strategy is through involvement in church. One traffic officer thought it not necessary to go to church to experience this connection, thus viewing spirituality as independent from denomination (Karakas 2010:8). Traffic officers who value involvement in church use it as a coping mechanism, by drawing support and learning spiritual lessons from the social connections. Another strategy is through prayer. When connecting to their spiritual source (Kinjerski & Skrypnek 2008:319), traffic officers use prayer to talk to God and ask for help in various situations (Mohan & Uys 2006:58). Lastly, to sustain the connection with their higher power (Karakas 2010:8), the traffic officers use various spiritual resources, including listening to spiritual music, conversing with family and friends, or reading spiritual material. Participants describe that spirituality manifests in their expression (Lombaard 2011:77) of values in their work and private lives. Individuals may be recognised by their actions or by the fruits of spirituality, which emerged primarily as calmness, positive attitudes, forgiveness, inner strength and awareness. Participants treasure the ability to become calm and exert self-control when faced with challenges. Methodological approach They must deal with the public’s negative attitudes towards them (Pienaar 2007:63), thus the importance of dealing responsibly with emotions. A positive attitude is associated with spirituality, and the two cannot be separated (Rowe & Allen 2003:64). Spirituality facilitates coping by inspiring positive attitudes, as participants are able to tap into their best, despite negative situations. Spirituality further fosters forgiveness, which apparently helps to overcome disappointment and moving forward (Schreuder & Coetzee 2011:275). Forgiveness facilitates coping through helping traffic officers to voluntarily change how they feel about, and respond to, negative occurrences. Inner strength is important to participants as they view their higher power as a source of strength (Mohan & Uys 2006:58), which makes it possible to withstand the demands of the job, and thus cope. Lastly, spirituality leads to an increased sense of awareness (Kinjerski & Skrypnek 2004:32), including awareness of the self, of God and others. The awareness of negative consequences also emerged (Chopko 2007:89), involving the effects of one’s actions or words on others. Personal coping mechanisms comprise positive attitudes, thankfulness and recreation, involving strategies employed to deal with stressors, as illustrated below. Participants also found it useful to surround themselves with positive people and to employ positive ways of thinking to assist them in coping with their work environment (Boehm & Lyubomirsky 2009:1). Coping is also made possible by being thankful to God and people close to one in the midst of challenges. Traffic officers furthermore use physical activities and socialising to combat stress. The effects of stressors also manifest on physical levels (Edwards 1992:245), and recreational activities affect physical well-being and thus coping. Methodological approach Exploring the question ‘how do traffic officers experience the role of spirituality in coping?’ required a methodology of the heart with characteristics of truth (Denzin, Lincoln & Giardina 2006:770). Therefore, the researcher considered the nature of the reality being studied (Ponterotto 2005:130), in selecting a qualitative study – specifically an interpretive approach – to explore participants’ related lived experience in their specific context (Terre Blanche, Kelly & Durrheim 2006:276). A non-probability purposive sample of 10 traffic officers was used (Durrheim & Painter 2006:139), with a biographical composition consisting of one black, one white and eight mixed-race participants. Their gender distribution consisted of 6 males and four females of which 4 were between the ages of 26 and 35, 3 between the ages of 36 and 45, and 3 between the ages of 46 and 55 years. Participants suppose that spirituality informed the construction of their reality (Ponterotto 2005:130), and subsequently spirituality or religion dictates their lives through their actions and reflecting on the truth that they strive towards (Leedy & Ormrod 2010:135). Furthermore, participants feel that spirituality gives purpose and meaning to life. Participants described that the guidance obtained from spirituality added meaning to work and life (McLeod 2003:142; Mohan & Uys 2006:58), creating a feeling that work has purpose (Karakas 2010:20). This entails a desire to be of service and staying the course even in difficult times. Through spirituality, participants are able to be passionate about their work and view their career as a calling (Coetzee & Roythorne- Jacobs 2012:220; Hall & Chandler 2005:1; Karakas 2010:2). Spirituality, furthermore, seems to enable decision-making. Participants believe that they have the power to choose what they want to happen in their lives through mindful actions The research was located within the South African traffic law enforcement environment, comprising a Law Enforcement Section within the particular Traffic Service Centre where they perform administrative duties, as well as the public roads according to the area of jurisdiction, where actual law enforcement takes place. Same stressor Higher spirituality Lower spirituality Coping style apparently stronger Coping style not so strong Posive thoughts Unhealthy thoughts FIGURE 1: Theoretical relationship between spirituality and coping. FIGURE 1: Theoretical relationship between spirituality and coping. Open Access Open Access http://www.hts.org.za http://www.hts.org.za Page 4 of 6 Page 4 of 6 Original Research instilled during childhood. Most participants appear to have been taught adaptive coping mechanisms at a young age. The role of spirituality in the coping of traffic officers Participant spirituality entails an interpretation of spirituality, which may be taught during childhood or by conversing with others on scriptures. Interpreting the meaning of spirituality deepens their understanding of the positive fruits of spirituality, and positive thoughts contribute significantly to effective coping ability (Rowe & Allen 2003:63). The ability to understand others and their views, and to respond to them in caring ways, is linked to the ability to cope effectively (Krishnakumar et al. 2015:21). The interpretation of spirituality facilitates better coping by enhancing social connections. Discussion Research findings revealed that traffic officers in the sample all utilised spirituality to various degrees and exhibited adaptive coping abilities when doing so. Traffic officers associated less spirituality or a lack thereof with weaker coping abilities. The following framework was derived (Figure 2) from the contributions of the participants and reflects their experiences regarding the role of spirituality in their coping as traffic officers: • Stressors occur: It is sometimes expected of the traffic officers to still remain humane and unaffected, at the end of a shift, when something stressful has happened to them. Coping Human context is vital to understanding coping (Moos 2002:67), thus background and upbringing emerged to include communities plagued by gangsterism, crime, violence, alcohol, drugs, poverty and peer pressure. Traffic officers revealed that childhood circumstances developed their coping abilities, because beliefs and spiritual values were Life is understood through lived experiences (Welzen 2011:38), as reflected by traffic officers’ implementation of spirituality. They apply what spirituality means to them within the context of work and life, which enables them to cope better. They treasure viewing their work in a spiritual Open Access http://www.hts.org.za Page 5 of 6 Original Research Stressor Connecon to spiritual source Interpretaon of spirituality Extract fruits of spirituality Implementaon of spirituality Coping Spirituality FIGURE 2: Framework towards understanding the role of spirituality in the coping of traffic officers. Original Research Page 5 of 6 FIGURE 2: Framework towards understanding the role of spirituality in the coping of traffic officers. (Jacobs 2013:144), as well as comparison with traffic officers who do not utilise spirituality at all in their opinion. way in order to persist and remain peaceful amidst challenges. It is within the context of their external work environment that one participant introduced the concept ‘spirituality on the road’ – a concept complementing the experiences of some other participants as well. Acknowledgements Competing interests • Connection to spiritual source: In challenging times, individuals need to go to that emotional place where they can tap into their spiritual source or higher power. The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. • Interpretation of spirituality: The connection to the spiritual source enables the individual to become aware of what their spiritual source means to them, and how they are being served spiritually by this higher power. Authors’ contributions A.v.N. and R.J. were co-responsible for the experimental and project design. R.J. collected and analysed the data, with A.v.N. quality assuring the data collection and data analyses process. R.J. and A.v.N. co-wrote the manuscript. • Extracting the fruits of spirituality: Individuals then take this spiritual meaning and extract what they need at the time, for example, inner strength, self-confidence or perseverance to deal with the given stressor. • Implementation of spirituality: The spiritual resources received are then utilised in work and in life. Conclusion This article concludes that the utilisation of spirituality enables traffic officers to cope more effectively with the daily demands they are faced with. Spirituality in traffic officers is further informed by their foundation of spirituality or religion, purpose to work and life, the connection to their spiritual source and the fruits of spirituality. 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Terre Blance, K. Durrheim & D. Painter (eds.), Research in practice: Applied methods for the social sciences, pp. 60–79, UCT Press, Cape Town. References • Coping made possible: With the implementation of what spirituality means to the individual, or by applying the fruits of spirituality to the stressful situation, adaptive coping is made possible. Anderson, G.S., Litzenberger, R. & Plecas, D., 2002, ‘Physical evidence of police officer stress’, Policing: An International Journal of Police Strategies & Management 25(2), 399–420. http://dx.doi.org/10.1108/136395510210429437 Bergh, Z., 2009, ‘Personality through self-experience’, in Z. Bergh & A. Theron (eds.), Psychology in the work context, pp. 315–330, Oxford University Press, Cape Town. Boehm, J.K. & Lyubomirsky, S., 2009, ‘The promise of sustainable happiness’, in Handbook of positive psychology, pp. 667–677, viewed 13 March 2014, from http://sonjalyubomirsky.com/wpcontent/themes/sonjalyubomirsky/papers/ BLinpressa.pdf Future research may draw upon these findings and replicate the study with respondents from a wider variety of South African Traffic Services Centres. Research on the role of spirituality in the coping of traffic officers can also be expanded upon by exploring the influences of such factors as race, age and gender. Future research may also consider the latest relevance of spirituality in different religious contexts Boehmer, S., Luszczynska, A. & Schwarzer, R., 2007, ‘Coping and quality of life after tumor surgery: Personal and social resources promote different domains of quality of life’, Anxiety, Stress, and Coping 20(1), 61–75. http://dx.doi. org/10.1080/10615800701195439 Bryant-Davis, T., Ellis, M.U., Burke-Maynard, E., Moon, N., Counts, P.A. & Anderson, G., 2012, ‘Religiosity, spirituality and trauma recovery in the lives of children and adolescents’, Professional Psychology: Research and Practice 43(4), 306–314. http://dx.doi.org/10.1037/a0029282 Open Access http://www.hts.org.za Original Research Page 6 of 6 Lombaard, C., 2011, ‘Biblical spirituality and human rights’, Old Testament Essays 24(1), 74–93, viewed 10 September 2014, from http://0-reference.sabinet.co.za.- oasis.unisa.ac.za/sa_epublications Cheng, T., Mauno, S. & Lee, C., 2014, ‘The buffering effect of coping strategies in the relationship between job insecurity and employee well-being’, Economic and Industrial Democracy 35(1), 71–94. http://dx.doi.org/10.1177/0143831X12463170 Louw, G.J. & Viviers, A., 2010, ‘An evaluation of a psychosocial stress and coping model in the police work context’, SA Journal of Industrial Psychology 36(1), 1–11. http:// dx.doi.org/10.4102/sajip.v35i1.442 Chopko, B.A., 2007, ‘The relationship between mindfulness and posttraumatic growth in law enforcement officers’, PhD dissertation, University of Akron. Coetzee, M. & Roythorne-Jacobs, H., 2007, Career counselling and guidance in the workplace: A manual for career practitioners, Juta, Cape Town. Luthans, F., 2008, Organizational behaviour, 11th edn., McGraw Hill, Boston, MA. Coetzee, M. References Krok, D., 2008, ‘The role of spirituality in coping: Examining the relationships between spiritual dimensions and coping styles’, Mental Health, Religion and Culture 11(7), 643–653, viewed 6 April 2014, http://psycnet.apa.org/psycinfo/2008-14449-001 Welzen, H., 2011, ‘Contours of biblical spirituality as a discipline’, Acta Theologica 31, 37–60. http://dx.doi.org/10.4314/actat.v31i1S.4 Leedy, P.D. & Ormrod, J.E., 2010, Practical research: Planning and design, 11th edn., Prentice Hall, Upper Saddle River, NJ. Yin, R.K., 2010, Qualitative research from start to finish, Guilford Press, New York. http://www.hts.org.za Open Access
https://openalex.org/W3211947624	https://bmjopenquality.bmj.com/content/bmjqir/10/4/e001604.full.pdf	English	null	Patient safety during joint replacement surgery: experiences of operating room nurses	BMJ open quality	2,021	cc-by	6,898	To cite: Nyberg A, Olofsson B, Otten V, et al. Patient safety during joint replacement surgery: experiences of operating room nurses. BMJ Open Quality 2021;10:e001604. doi:10.1136/ bmjoq-2021-001604 Original research Original research Open access copyright. o http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from copyright. on October 23, 2024 by guest. P http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from INTRODUCTION Perioperative and postoperative complica- tions result in much patient morbidity and mortality,1 2 and some of these are prevent- able.3 4 Often, well-founded treatment recommendations are available to help to prevent complications. Implementation and maintenance of evidence-based practice is a challenge, including in perioperative practice where there are complex organ- isational challenges. Good technical and 23, 2024 by guest. Protected by copyright. on October 23, 2024 by guest. Protected by http://bmjopenquality.bmj.com/ Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from Anette Nyberg ‍ ‍ ,1,2 Birgitta Olofsson,1,3 Volker Otten,3 Michael Haney,4 Ann-Mari Fagerdahl5 ABSTRACT 1Department of Nursing, Faculty of Medicine, Umeå University, Umeå, Sweden 2Department of Surgical and Perioperative Sciences, Faculty of Medicine, Umeå University, Umeå, Sweden 3Department of Surgical and Perioperative Sciences, Orthopedics, Faculty of Medicine, Umeå University, Umeå, Sweden 4Department of Surgical and Perioperative Sciences, Anesthesiology, Faculty of Medicine, Umeå University, Umeå, Sweden 5Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, Stockholm, Sweden Correspondence to Anette Nyberg; anette.nyberg@umu.se © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. Conclusion The conditions to support patient safety, or limit complication risk, during joint replacement surgery continue to be at times inconsistent, and require steady performance attention. Operating room nurses make adjustments to help solve problems as they arise, where there are obvious risks for patient complications. The organisational patient safety management process still seems to allow deviation from established practice standards at times, and relies on individual-based corrective measures at the ‘bedside’ at times for good results. on October 23, 2024 by guest. Protected by bmj.com/ This study aimed to explore current experi- ence of ORNs focused on their routine work- place reflections concerning patient safety factors that they can influence. The specific aims were to present and analyse current experience for ORNs in their work to prevent perioperative mishaps and SSI risk for a very frequently done operation, where individual, team and organisation factors are relevant. INTRODUCTION ABSTRACT non-technical skills are needed in order to reduce and manage threats to patient safety.5 One example of this is the WHO Surgical Safety Checklist (SSC),6 which was launched in 2008. Employing the SSC is an example of consolidating individual, team, and organisa- tional planning and control steps designed to reduce the risk of avoidable complications for patients (enhance patient safety). Background Avoidable complications for surgical patients still occur despite efforts to improve patient safety processes in operating rooms. Analysis of experiences of operating room nurses can contribute to better understanding of perioperative processes and flow, and why avoidable complications still occur. Aim To explore aspects of patient safety practice during joint replacement surgery through assessment of operating room nurse experiences. Method A qualitative design using semistructured interviews with 21 operating room nurses currently involved in joint replacement surgery in Sweden. Inductive qualitative content analysis was used. Results The operating room nurses described experiences with patient safety hazards on an organisational, team and individual level. Uncertainties concerning a reliable plan for the procedure and functional reporting, as well as documentation practices, were identified as important. Teamwork and collaboration were described as crucial at the team level, including being respected as valuable, having shared goals and common expectations. On the individual level, professional knowledge, skills and experience were needed to make corrective steps. Received 5 July 2021 Accepted 25 October 2021 Joint replacement surgery is a complex but largely standardised operation where surgical complications can be devastating for patients. Besides optimising operative logis- tics, all available steps to minimise risk for surgical site infection (SSI) or periprosthetic joint infection (PJI) are incorporated into current arthroplasty operations, given that PJIs are recognised as devastating for patients where they occur.7 Operating room nurses (ORNs) are constantly managing risks and preventing harm with the aim of zero periop- erative mishaps and zero postoperative infec- tions.8 Nevertheless, they occur with alarming frequency.4 9 Deviations from widely accepted best practice occur, but it is unclear to what extent that these will increase risk for compli- cations. Still, to work to minimise risk means to find the practical steps in practice where perioperative staff experience that commonly accepted or established good practice10–12 is difficult follow. 1Department of Nursing, Faculty of Medicine, Umeå University, Umeå, Sweden 2Department of Surgical and Perioperative Sciences, Faculty of Medicine, Umeå University, Umeå, Sweden 3Department of Surgical and Perioperative Sciences, Orthopedics, Faculty of Medicine, Umeå University, Umeå, Sweden 4Department of Surgical and Perioperative Sciences, Anesthesiology, Faculty of Medicine, Umeå University, Umeå, Sweden 5Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, Stockholm, Sweden Correspondence to Anette Nyberg; anette.nyberg@umu.se © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. Open access Open access The reporting complies with Consolidated Criteria for Reporting Qualitative Research.13 Reliable procedural plan The ORNs stated their need for a reliable preopera- tive plan to ensure a safe procedure. By planning and preparing well for the procedure, they attempted to reduce the time for surgical procedure. Before preparing for the procedure, they often needed to confirm the information from a computerised surgical planning system with the orthopaedic surgeon, due to occurrence of failure in updating the plan. This need to confirm the plan was perceived as unsatisfying and experienced as time-consuming. Though instead of addressing the main problem, the participants resorted to adapting to the incomplete planning. Participants Important preconditions on the organisational level that were highlighted included a reliable plan for the procedure, and reporting and documentation practices enabling a functional exchange of information. Inclusion criteria were: certified and active ORNs, with at least 1 year of experience of joint replacement surgery. Purposive sampling was performed. Nursing department leaders identified ORNs who met inclusion criteria. Twenty-one ORNs were interviewed, median age 43 years (range 27–64), 2 men and 19 women. Median experience as ORN was 7 years (range 2–41). Setting and Swedish context The interviews were conducted at three different hospi- tals in Sweden: one university hospital, one public general hospital and one private orthopaedic hospital. There are international differences concerning areas of responsibility in the OR. In Sweden, ORNs are respon- sible for preparing instruments and implants for the operation, patient positioning and OR asepsis.14 The ORN training in Sweden starts as a registered nurse, then a 1-year ORN specialist programme, with certification and a master’s degree.14–16 Data collection A semistructured interview guide consisted of three ques- tions: ‘What does patient safety in perioperative care during arthroplasties include for you?’, ‘What do you consider most important in securing patient safety?’ and ‘Do you detect any weak areas in patient safety?’. Probing questions were asked when needed. Data were collected from April 2020 to August 2020. All interviews were conducted by the first author. Sixteen of the 21 interviews took place in person, and 5 by telephone. The interviews lasted median 31 min (range 20–45) and were digitally recorded and transcribed verbatim. But it requires extra work when something does not seem to be right, that is when you start to suspect something, that someone has to call and talk to those who are going to operate and so extra time and energy goes into something that should not be needed. (IP16) METHOD Design Correspondence to Anette Nyberg; anette.nyberg@umu.se This was a qualitative study analysing data collected through semistructured interviews. Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604 1 Table 1 Categories and subcategories Categories Subcategories Organisational level Reliable procedural plan Functional reporting and documentation Team level Interprofessional and interdisciplinary collaboration Protocol, checklist and standardisation implementation Aseptic principles compliance Individual level Professional knowledge, skills and experiences Personal engagement Table 1 Categories and subcategories Categories Subcategories Organisational level Reliable procedural plan Functional reporting and documentation Team level Interprofessional and interdisciplinary collaboration Protocol, checklist and standardisation implementation Aseptic principles compliance Individual level Professional knowledge, skills and experiences Personal engagement copyright. on October 23, 2024 by guest. Prote http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604 Data analysis The responses were analysed inductively using quali- tative content analysis, which is used to describe simi- larities and differences in the manifest content and to interpret the latent content in the phenomenon under exploration.17–19 We performed a manifest analysis. The transcripts were imported into MAXQDA 2020 (VERBI Software, Germany). First, the recorded interviews were reviewed and transcripts read through to get a sense of the whole. Second, the text was divided into meaning units relevant to the aim of the study. Third, the meaning units were carefully condensed to avoid losing content, and each meaning unit was coded. The codes were abstracted into subcategories and categories. The anal- ysis was discussed among coauthors until consensus was reached. Ensuring the availability of instruments and implants for the planned procedure was considered a challenge. With procedural planning not being updated, essential instru- ments or implants for the procedure could be missing, and the ORN needed to start preparing for another surgical system instead. This was considered a disturbance in workflow in the OR, and made it difficult to accom- plish the list of operations scheduled for the day. The participants needed to prepare fast-paced, and expressed concern for missing crucial items when doing so. on October 23, 2024 by guest. Protected by com/ r 23, 2024 by guest. Protected by For some participants, the main source of patient- related information was derived from the surgical planning system and the anaesthesia preoperative assess- ment. They did not find time to get information from the main health records, and were thereby not routinely accessing information which included notifications from orthopaedic ward nurses, such as already-identified patient risk for pressure ulcers and nutrition status. FINDINGS From the analysis, three main categories and seven subcat- egories emerged (table 1). 2 Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604 Open access copyright. on October 23, 2024 by gues http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from Therefore, the possibility to prepare for a safe proce- dure was reduced, and they experienced important patient information in different computerised systems in parallel a risk. Team level Collaboration, established safety controls and compliance with aseptic principles were stated as important aspects for safety practice within the team. Compliance with aseptic principles was considered to vary among different professions within the team. copyright. on October 23, 2024 by guest. Protected by http://bmjopenquality.bmj.com/ en Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from Functional reporting and documentation There were concerns that the reporting and docu- mentation practices threatened continuity of patient care. Some participants explained that they were docu- menting their perioperative care both in the comput- erised system and also in paper form. This practice was considered both time-consuming and a risk for patient safety. The documentation on paper was deemed neces- sary as ORNs rarely reported in person to colleagues on the postoperative ward. The nurses on the postoperative and orthopaedic ward were working in the main health record system, not in the planning system where ORNs documented their care, and thereby could not consult the information the ORNs had documented there. The participants suggested that the planning system should be seen merely as a planning system and not as a tool for documentation. Interprofessional and interdisciplinary collaboration Interprofessional and interdisciplinary collaboration The importance of teamwork and collaboration for patient safety was mentioned by all participants. The team shared a goal to do the best for each patient, and every professional’s expertise was a valuable contribu- tion. The ORNs were expecting all team members to perform responsibly, and when this was not the case, it became a strain in the workplace for them. In this collab- oration the ORNs felt that their professional knowledge was respected. Steady communication with other OR personnel was seen important. A lot of teamwork, because I mean, without my anesthesiologist, without my anesthesia nurse, without my assistant nurse, without my surgeon, without me there will be no surgery and without the patient there neither be any surgery. So, there are many people and it gives a sense of security in fact that everyone tries to think; Avoid injuries, not to cause harm - how will it be best. (IP1) A big problem (documentation) and above all, you are not used to each other’s journal system so the nurses at the postoperative wards rarely go in and read. So, if we write: See Orbit (the planning system), they don’t care about it because they think it is hard to get into it because it is not a system they usually work within. So, all that is important, you have to either say it or write it on the paper and then it becomes double documentation, which feels unnecessary time consuming when we now have so little time on everything as it is. (IP7) The work required that the ORNs constantly develop their skills, and they tried to improve their work steadily. Being alone in their profession as ORN in the OR put limits on opportunities to ask colleagues for advice and support. In situations where two ORNs collaborated during surgery, they had opportunities to support and learn from each other, and thereby improve their work. Reporting to a colleague and at the same time keeping the surgery on course was perceived as a risk. ORNs tried to avoid personnel breaks or shift changes during surgery, but sometimes it was seen as inevitable, especially during revision joint replacement surgery lasting the whole day. It was considered a challenge to remember to report everything, and some recalled the need to call the OR on their way home to fill in missing parts in their report. Open access Open access copyright. on October 23, 2024 by guest. Protected by http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from copyright. on October 23, 2024 by gue http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from … that I know that I have the right patient to begin with, that planning is consistent with the x-rays or when we do ‘Sign in’ that it is consistent with the patient records and the ID bracelet and side, that you operate on the correct side (IP11) Professional knowledge, skills and experience The ORNs knew what was expected of them to preserve patient safety. They used professional knowledge and skills to protect patients from risks. This included positioning the patient safely on the operating table among others. They had a responsibility for what they saw during the procedure by being a part of the operating team. If a situ- ation occurred putting the patient at risk, the ORNs had to speak up. They experienced that notifying the ortho- paedic surgeon of near misses required confidence, and this was gained through experience. Unexpected things happen during surgery, and the participants needed to be prepared to adjust their plan. Individual level The ORNs felt a personal responsibility to the patients, and felt guilty when failing to protect the patient from harm. They protected patients by using their professional knowledge and skills, and by having the confidence to speak up if the patient was put at risk in the process. copyright. on October 23, 2024 by guest. Protected by http://bmjopenquality.bmj.com/ : first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from The ORNs considered established safety protocols neces- sary. For example, using a protocol to ensure availability for the correct implant was named as important. This step is performed by both the orthopaedic surgeon and the ORN, reading on the implant package before it was opened and delivered to the sterile field. Some partici- pants considered standardisation of procedures as a way to ensure safety. They explained that they were constantly assessing what was best for each patient, emanating from existing routines and standardisation. Personal engagement O f l ORNs felt a great responsibility for the patients’ sense of security and comfort in perioperative care. They had the ambition to greet the patient before starting preparation and wanted patients to know that everyone in the OR would do their best for them in a dignified way. The participants perceived a feeling of guilt when situ- ations occurred and they failed to protect the patient from coming to harm. Some described situations occur- ring where they would not have wanted to be the patient. Although the ORNs were well aware that many factors could have led to an infection, they felt accountable for it if a patient acquired an SSI or a PJI. The ORNs also emphasised a need to improve staff behaviour regarding adherence to the aseptic princi- ples. When they insisted on observing aseptic protocols, it sometimes was considered a disturbance of the flow, affecting both the surgical procedure and the whole day operation schedule. Such protocols could be handling of specimens and urinary tract catheters. When notifying others on breaks of aseptic principles, some participants perceived that they were seen as annoying. on October 23, 2024 by guest. Protected by .bmj.com/ Then you are as an OR nurse, there must be something that we have in us, as soon as there is an infection then you have to go straight into the old medical record and see if you were there during the previous operation. Because you think that you, yourself carry the responsibility, and of course I understand that it is not how it is, there are many factors that come into play, but I think many of us feel responsible; have I done something wrong, is it my mistake now that led to this patient becoming infected? (IP8) ober 23, 2024 by guest. Protected by So it is, we are herd animals, we do as our colleagues do and it is uncomfortable if someone gives a reprimand or is a hygiene-witch… Even if people might think you're irritating, I think you still get some kind of respect in that you have competence and can see that this is important. Even if you are considered awkward, you are trusted as the person who also is competent and good, good for the group and for the patient. Aseptic principles compliance The ORNs stated being responsible for sterility and infection control was within the domain of their main concern. They kept guarding sterility throughout the entire surgical procedure by keeping an eye on the activ- ities of other team members, which sometimes could be challenging. If you see something in the surgical field, I feel that you have a responsibility when you see something, then of course it depends on experience, how much experience you have and what you know and what you have seen before, but it is also a part of patient safety that you are involved in the operation and are taking part in the operation. (IP17) Participants noted that the prerequisites for work in an aseptic environment were present. National guidelines for preventing PJIs were established, and there was most often compliance with these. One example given was a guideline to control the traffic and avoid disturbance of the ventilation by opening the doors and trying to mini- mise the number of persons in the OR. Still, some partic- ipants experienced that compliance with guidelines varied within the team. For example, some orthopaedic surgeons followed the guidelines more strictly than others. With an interesting surgical case, minimising the amount of personnel in the OR was disregarded by some surgeons. Protocol, checklist and standardisation implementation The participants explained that they were responsible for several established safety controls, including to ensure that all instruments and other sterile material needed were available and functioning—surgical count. The first surgical count was performed before the patient entered the OR. Another surgical count was performed during the surgical procedure before closing the wound to ensure that no surgical items were accidentally left in the operative site. on October 23, 2024 by guest. Protected by ty.bmj.com/ The participants in this study were not convinced that reporting incidents led to any actual improvements in patient safety. OR management sent information about new routines and incidents by email, and these were sometimes perceived to not reach the appropriate OR personnel. The ORNs voiced a desire to receive feed- back on the treatment results for the patients. They wanted to know if there had been injuries for patient were there had been intraoperative challenges. If the orthopaedic surgeons were asked for the results for one specific patient, they shared the result with the ORNs, but there was no systematic feedback on results or complica- tions. For example, some participants emphasised that they wanted to know the infection rates for their specific department. Another important responsibility for the ORNs was to verify the patient identity when entering the OR, as well as confirming the indicated side on the patient with the planning and the x-rays. Through the timeout in the SSC, the participants confirmed the preparation with the orthopaedic surgeon. However, they experienced that the SSC was not always implemented as designed. The checklist was often performed while the ORNs were busy with final preparations, and not participating with whole attention. How the timeout was performed depended on the orthopaedic surgeon’s interest in the checklist, and differed from one surgeon to another. ober 23, 2024 by guest. Protected by 3 Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604 DISCUSSION These findings show that ORNs identify several meas- ures at different levels (organisation, team and indi- vidual) where there can be safety improvements in the (IP10) Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604 4 Open access copyright. on Octo http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from copyright. on October 23, 2024 by http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from OR during joint replacement surgery. These occurrences cause disruptions in the workflow and sometimes even threatening patient safety. These findings also show how upcoming problems that occur were solved in everyday work, to get on with the job and to maintain safety. system miscommunication. This development of work- arounds can involve taking a risk as well as limiting the possibility for a change.21 The risk involved documenting in two places and continuing working in diverse comput- erised systems alleviated pressure on the hospital manage- ment for process improvement. Documentation practices may be directed by traditions and everyday conditions but to ensure safe care the documentation tool should align with the actual practice.27 Some suggested that the planning system should be only a tool for planning, not for documentation, and that documentation should be managed in one patient record system. To ensure a safe surgical procedure, the ORNs iden- tified a need for a reliable preoperative plan, which confirms previous findings.20 This is in line with resilience engineering, where predictive information is needed to anticipate for decision making.21 Some participants iden- tified problems when confirming the plan between the computerised planning system and the surgeon, which was considered time-consuming and unsafe. The purpose of computerised planning systems is to organise a reli- able plan22 but achieving this requires an engagement of all stakeholders involved in the planning.23 Without an updated plan there is a risk for disruptions which can both compromise patient safety and affect productivity of the organisation, similar findings are presented in previous reports.22 24 Participants gave priority to fixing the imme- diate problem in order to maintain the workflow in OR, instead of addressing the main issue, that the plan was not adequately updated. This can be seen as resilience by the frontline workers,25 where the ORNs adapted in order to accomplish a good outcome even when their working conditions were disadvantageous. DISCUSSION Although the intention of the ORNs adaptability is good, it can create a disconnection between the OR management’s work- as-imagined and the ORNs’ work-as-done. This type of disconnection can constrain the possibility for necessary change. Work-as-imagined means practising by following protocols and standards for safe performance,25 but the staff in OR know that a certain performance variability is expected, as is practice safely. In this study, the underlying problem with low engagement in planning documenta- tion can continue as long as the OR managers do not recognise the problem or do not have the opportunity to solve it. The issue with the planning documentation not being updated might be considered as an annoying repetitive problem by ORNs. In the long term, repetitive annoying problems that lead to frustration, cynicism and turnover among the front-line staff impact the resilience in the system.21 copyright. on October 23, 2024 by guest. Protected by http://bmjopenquality.bmj.com/ as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from g p y It was experienced that teamwork and collaboration are crucial in preventing adverse events, which supports findings of previous studies.28 29 Expertise from every profession was considered a valuable contribution to the process and every team members need to feel confi- dent to speak up when alert is needed, as reported previ- ously.30 The ORNs felt respected for their professional knowledge in collaboration with other team members, although previous research suggests that the collabora- tion between nurses and physicians needs improvement, both in OR31 and in other context within the hospital.32 Their professional skills and experience informed their patient safety practice. A combination of technical skills and care for the patient were identified as important, similar findings have been presented earlier.20 33 A sense of personal engagement among the ORNs was prominent. Guilty feelings were named if they failed to protect a patient from harm. They also expected all team members to accept their professional responsibilities and emphasised need for a shared goal. An open dialogue within the team, with confirmed expectations were noted as important, which confirms a previous report.34 The ORNs identified primary responsibility in assuring compliance with aseptic principles. They experienced variable compliance with this within the team, as has been reported previously.35 Guidelines for preventing infec- tions were established, but the experience was that the levels of interest to comply varied among team members. DISCUSSION For best effect, teams should share goals and accept workgroup hierarchy, which includes mutual respect and good leadership within the team.36 Where some everyday performance variability is expected, things should still go right. Individual performance variability should not be understood as necessarily dangerous, though a team not complying with existing safety protocols is beyond expected performance variability.37 Patient safety cannot be improved solely by introducing safety policies—they need to be implemented.38 copyright. on October 23, 2024 by g http://bmjopenquality.bmj.com/ November 2021. Downloaded from on October 23, 2024 by guest. Protected by ality.bmj.com/ y Diverse computerised systems were seen obstructing the transfer of information, with a potential to affect the care of patients, which confirms previous findings on documentation.26 The ORNs did not find time to access information from the main health record. A technology allowing fast access to relevant information should be provided by the organisations.24 In this study, the ORNs did not report directly, or in person, to the postoperative ward, and this to save time. To avoid gaps in the continuity of care which could result, some reported developing a workaround in their documentation practice. This work- around necessitated duplication of documentation in separate systems to ensure that the nurses on postoper- ative ward received relevant information. Participants adapted their routines, demonstrating resilience to avoid October 23, 2024 by guest. Protected by Safety controls, such as checklists, were identified as crucial for preserving safety during surgery, as presented in an earlier report.39 Control steps were seen as guid- ance for safe practice, and specifically SSC as a useful tool to maintain a high level of safety awareness. In other reported findings SSC was noted to increase communica- tion and teamwork in OR40 41 and to recognise potential risks.42 In this study, it was noted that SSC was not always 5 Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604 Open access used as designed mostly dependent on the surgeon. Perceptions of the SSC as nonessential complicated the implementation of SSC.43 Previous reporting suggests that the implementation of SSC might be more successful where physicians engaged with leading it.44 Standard- isation of procedures was considered a way to improve the perioperative process. DISCUSSION ORCID iD Anette Nyberg http://orcid.org/0000-0002-2057-4410 Strengths and limitations p q [ y ] 7 Moore AJ, Blom AW, Whitehouse MR, et al. Deep prosthetic joint infection: a qualitative study of the impact on patients and their experiences of revision surgery. BMJ Open 2015;5:e009495. p q [ y ] 7 Moore AJ, Blom AW, Whitehouse MR, et al. Deep prosthetic joint infection: a qualitative study of the impact on patients and their experiences of revision surgery. BMJ Open 2015;5:e009495. Strengths and limitations 1 WHO. Patient safety, 2019. Available: https://www.who.int/news- room/fact-sheets/detail/patient-safety [Accessed Jun 2021]. 1 WHO. Patient safety, 2019. Available: https://www.who.int/news- room/fact-sheets/detail/patient-safety [Accessed Jun 2021]. During the analysis, there were interactive discussions within the research group which increases credibility for this study. Direct quotations with descriptions are presented for credibility. The diversity of professionals involved in the analysis also strengthens this study. The degree of transferability must be up to the reader.18 One limitation is that all interviews were conducted by the first author, who is an ORN and professionally known to some of the participants. Dual roles for interviewer can potentially influence interviewee reporting.47 This dual interviewer role may have helped create a safe environ- ment, and the shared understanding can deepen the reporting.48 2 Slawomirski L, Auraaen A, Klazinga NS. The economics of patient safety: strengthening a value-based approach to reducing patient harm at national level, 2017. Available: https://www.oecd-ilibrary.org/ content/paper/5a9858cd-en [Accessed Jun 2021]. 2 Slawomirski L, Auraaen A, Klazinga NS. The economics of patient safety: strengthening a value-based approach to reducing patient harm at national level, 2017. Available: https://www.oecd-ilibrary.org/ content/paper/5a9858cd-en [Accessed Jun 2021]. p p [ ] 3 de Vries EN, Ramrattan MA, Smorenburg SM, et al. The incidence and nature of in-hospital adverse events: a systematic review. Qual Saf Health Care 2008;17:216–23. 3 de Vries EN, Ramrattan MA, Smorenburg SM, et al. The incidence and nature of in-hospital adverse events: a systematic review. Qual Saf Health Care 2008;17:216–23. 4 The National Board of Health and Welfare. Serious injuries and care injuries - In-depth analysis of injuries and care injuries in somatic care of adults at emergency hospitals, 2019. Available: https://www. socialstyrelsen.se/globalassets/sharepoint-dokument/artikelkatalog/ ovrigt/2019-4-3.pdf [Accessed May 2021]. 4 The National Board of Health and Welfare. Serious injuries and care injuries - In-depth analysis of injuries and care injuries in somatic care of adults at emergency hospitals, 2019. Available: https://www. socialstyrelsen.se/globalassets/sharepoint-dokument/artikelkatalog/ ovrigt/2019-4-3.pdf [Accessed May 2021]. 5 Mitchell L, Flin R, Yule S, et al. Thinking ahead of the surgeon. An interview study to identify scrub nurses' non-technical skills. Int J Nurs Stud 2011;48:818–28. 5 Mitchell L, Flin R, Yule S, et al. Thinking ahead of the surgeon. An interview study to identify scrub nurses' non-technical skills. Int J Nurs Stud 2011;48:818–28. 6 WHO. Surgical safety checklist, 2009. Available: https://apps.who.int/ iris/bitstream/handle/10665/44186/9789241598590_eng_Checklist. pdf?sequence=2 [Accessed May 2021]. 6 WHO. Surgical safety checklist, 2009. Available: https://apps.who.int/ iris/bitstream/handle/10665/44186/9789241598590_eng_Checklist. pdf?sequence=2 [Accessed May 2021]. DISCUSSION Both appropriate standardi- sation and certain flexibility are needed for an organisa- tion to succeed.45 While standardisation protects against predictable and preventable errors, flexibility supports resilience in unpredictable situations, where balance between these is needed.46 This is in line with another report that suggests that to manage the complexity in OR and maintain safe care necessitates the ability to respond to both the expected and the unexpected.39 Our find- ings show that resilience exists within the organisations, but it is not only used in unpredictable situations. The ORNs demonstrated resilience in managing the everyday work. This shows ORNs ability to make adjustments and maintain them. However, where there is every day need for resilience, capacity to respond to new challenges may be restricted.21 Resilience should be needed for resolving unplanned situations rather than everyday occurrences. Safety threats in everyday work should be recognised and managed as the organisation improves. approved the final manuscript version. BO is responsible for the overall content and conduct of the study as a guarantor. approved the final manuscript version. BO is responsible for the overall content and conduct of the study as a guarantor. Funding This study was supported by grants from the Strategic Research Area Health Care Science (SFO-V), grant number 350033022 SFO-V, and the JC Kempe’s foundation, and funding from Umeå University, no grant numbers, all in Sweden. Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. Patient consent for publication Not applicable. Patient consent for publication Not applicable. Ethics approval This study was jugded to be acceptable by the Swedish Ethical Review Authority (reg. no. 2020-00111). Before the interviews, the ORNs gave their written informed consent to participate, participating was voluntarily and they could interrupt their interview at any time. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data can be made available on reasonable request. Data availability statement Data can be made available on reasonable request. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. copyright. on October 23, 2024 b http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from 15 Swedish Ministry of Education and Research. The higher education ordinance, SFS 1993:100 (up to and including amendments to 2014:1096), 1993. Available: https://www.uhr.se/en/start/laws-and- regulations/Laws-and-regulations/The-Higher-Education-Ordinance/ Annex-2/ [Accessed cited 2021 June]. 31 Makary MA, Sexton JB, Freischlag JA, et al. Operating room teamwork among physicians and nurses: teamwork in the eye of the beholder. J Am Coll Surg 2006;202:746–52. g ; 32 Filizli G, Önler E. Nurse-physician collaboration in surgical units: a questionnaire study. J Interprof Educ Pract 2020;21:100386. 33 Alfredsdottir H, Bjornsdottir K. Nursing and patient safety in the operating room. J Adv Nurs 2008;61:29–37. [ ] 16 Crafoord M-T, Fagerdahl A-M. Clinical supervision in perioperative nursing education in Sweden - A questionnaire study. Nurse Educ Pract 2017;24:29–33. 34 Gillespie BM, Gwinner K, Chaboyer W, et al. Team communications in surgery - creating a culture of safety. J Interprof Care 2013;27:387–93 https://doi.org/10.3109/13561820.2013.784243 17 Graneheim UH, Lindgren B-M, Lundman B. Methodological challenges in qualitative content analysis: a discussion paper. Nurse Educ Today 2017;56:29–34. 35 Megeus V, Nilsson K, Karlsson J, et al. Hand contamination, cross- transmission, and risk-associated behaviors: an observational study of team members in ORs. Aorn J 2015;102:645.e1–645.e12. 18 Graneheim UH, Lundman B. Qualitative content analysis in nursing research: concepts, procedures and measures to achieve trustworthiness. Nurse Educ Today 2004;24:105–12. 36 Gillespie BM. Setting a quality improvement and research agenda for patient safety in a tertiary hospital in Australia. Aorn J 2016;103:632–5. y 19 Lindgren B-M, Lundman B, Graneheim UH. Abstraction and interpretation during the qualitative content analysis process. Int J Nurs Stud 2020;108:103632. Å 37 Hollnagel E, Wears RL, Braithwaite J. From Safety-I to Safety-II: a white paper. The resilient health care net. Australia: University of Southern Denmark, University of Florida, USA, and Macquarie University, 2015. 20 Sandelin A, Kalman S, Gustafsson Birgitta Åkesdotter. Prerequisites for safe intraoperative nursing care and teamwork-Operating theatre nurses' perspectives: a qualitative interview study. J Clin Nurs 2019;28:2635–43. y 38 Leape LL. Errors in medicine. Clin Chim Acta 2009;404:2–5. 39 Göras C, Nilsson U, Ekstedt M, et al. Managing complexity in the operating room: a group interview study. BMC Health Serv Res 2020;20:440. 21 Hollnagel E, Braithwaite J, Wears RL. Resilient health care. Ashgate: Farnham, 2013. 22 Guerriero F, Guido R. Operational research in the management of the operating theatre: a survey. Health Care Manag Sci 2011;14:89–114.i 40 Nordström A, Wihlborg M. A phenomenographic study of Swedish nurse Anesthetists' and or nurses' work experiences. CONCLUSION Protected b http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from Open access Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604 CONCLUSION p g y p 8 Ingvarsdottir E, Halldorsdottir S. Enhancing patient safety in the operating theatre: from the perspective of experienced operating theatre nurses. Scand J Caring Sci 2018;32:951–60. 8 Ingvarsdottir E, Halldorsdottir S. Enhancing patient safety in the operating theatre: from the perspective of experienced operating theatre nurses. Scand J Caring Sci 2018;32:951–60. The conditions to support patient safety, or limit compli- cation risk, during joint replacement surgery continue to be at times inconsistent, and require steady perfor- mance attention. ORNs make adjustments to help solve problems as they arise, where there are obvious risks for patient complications. The organisational patient safety management process still seems to allow deviation from established practice standards at times, and relies on individual-based corrective measures at the ‘bedside’ at times for good results. g ; 9 Kurtz SM, Lau E, Schmier J, et al. Infection burden for hip and knee arthroplasty in the United States. J Arthroplasty 2008;23:984–91. g ; 9 Kurtz SM, Lau E, Schmier J, et al. Infection burden for hip and knee arthroplasty in the United States. J Arthroplasty 2008;23:984–91. 10 WHO. Global guidelines for the prevention of surgical site infection, 2018. Available: https://apps.who.int/iris/bitstream/handle/10665/ 277399/9789241550475-eng.pdf?ua=1 [Accessed Jun 2021]. 10 WHO. Global guidelines for the prevention of surgical site infection, 2018. Available: https://apps.who.int/iris/bitstream/handle/10665/ 277399/9789241550475-eng.pdf?ua=1 [Accessed Jun 2021]. g 11 Parvizi J, Gehrke T, Chen AF. Proceedings of the International consensus on periprosthetic joint infection. Bone Joint J 2013;95- B:1450–2. 12 Swedish County Council’s Mutual Insurance Company. PRISS - Prosthesis related Infections Shall be Stopped 3.0, 2019. Available: https://lof.se/wp-content/uploads/Optimal-operationsmiljö.pdf [Accessed Jun 2021]. [ ] 13 Tong A, Sainsbury P, Craig J. Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int J Qual Health Care 2007;19:349–57. Å Acknowledgements The authors would like to acknowledge and thank the ORNs at the three hospitals for participating and the nursing management for making it possible to conduct the interviews. g p 14 von Vogelsang A-C, Swenne CL, Gustafsson Birgitta Åkesdotter, et al. Operating theatre nurse specialist competence to ensure patient safety in the operating theatre: a discursive paper. Nurs Open 2020;7:495–502. Contributors All authors conceived the study design. AN collected the data. All authors participated in the data analysis, interpretation and manuscript writing, and 6 Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604 copyright. on October 23, 2024 by guest. copyright. on October 23, 2024 b http://bmjopenquality.bmj.com/ BMJ Open Qual: first published as 10.1136/bmjoq-2021-001604 on 8 November 2021. Downloaded from Aorn J 2019;109:217–26. 23 Rothstein DH, Raval MV. Operating room efficiency. 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Ann Surg 2020;272:1164–70. 29 Sandelin A, Gustafsson Birgitta Å, . Operating theatre nurses’ experiences of teamwork for safe surgery. Nord J Nurs Res 2015;35:179–85. ; 47 McDermid F, Peters K, Jackson D, et al. Conducting qualitative research in the context of pre-existing peer and collegial relationships. Nurse Res 2014;21:28–33. 30 Cumin D, Skilton C, Weller J. Information transfer in multidisciplinary operating room teams: a simulation-based observational study. BMJ Qual Saf 2017;26:209–16. p 48 McEvoy P. Interviewing colleagues: addressing the issues of perspective, inquiry and representation. Nurse Res 2001;9:49–59. Nyberg A, et al. BMJ Open Quality 2021;10:e001604. doi:10.1136/bmjoq-2021-001604
https://openalex.org/W3037473845	https://hal.archives-ouvertes.fr/hal-02878613/file/CE_Discourses.pdf	English	null	A typology of circular economy discourses: Navigating the diverse visions of a contested paradigm	Resources, conservation and recycling	2,020	cc-by	21,272	A typology of circular economy discourses: Navigating the diverse visions of a contested paradigm Martin Calisto Friant, Walter J V Vermeulen, Roberta Salomone To cite this version: Martin Calisto Friant, Walter J V Vermeulen, Roberta Salomone. A typology of circular economy discourses: Navigating the diverse visions of a contested paradigm. Resources, Conservation and Recycling, 2020, 10.1016/j.resconrec.2020.104917. hal-02878613 To cite this version: Martin Calisto Friant, Walter J V Vermeulen, Roberta Salomone. A typology of circular economy discourses: Navigating the diverse visions of a contested paradigm. Resources, Conservation and Recycling, 2020, 10.1016/j.resconrec.2020.104917. hal-02878613 A R T I C L E I N F O The circular economy (CE) has recently become a popular discourse especially in government and corporate sectors. Given the socio-ecological challenges of the Anthropocene, the concept of CE could indeed help the transition to a sustainable, just and resilient future. However, the actual definition, objectives and forms of implementation of the CE are still unclear, inconsistent, and contested. Different actors and sectors are thus articulating circular discourses which align with their interests, and which often do not sufficiently examine the ecological, social and political implications of circularity. In this context, this research asks how to better na- vigate and analyse the history, complexity and plurality of circularity discourses by conceptually differentiating them in a comprehensive discourse typology. To answer this question a critical literature review has been carried out, which first, examines and reflects on the core challenges, gaps and limitations of the CE concept. Second, this research develops a comprehensive timeline of circularity thinking, which identifies and conceptually classifies 72 different CE-related concepts from the Global North and South (such as Gandhian and steady-state economics, buen vivir, doughnut economics and degrowth). This leads to the development of a typology of circularity discourses, which classifies circularity visions according to their position on fundamental social, technological, political and ecological issues. This research thus seeks to provide a basis for a more inclusive and comprehensive discussion on the topic, which opens the imaginary regarding the many circular futures that can exist and allows for a cross-pollination of ideas, policy options, strategies, practices and solutions. Keywords: Circular economy Circular society Sustainability Discourse analysis Critical literature review A typology of circular economy discourses: Navigating the diverse visions of a contested paradigm Martin Calisto Frianta,⁎, Walter J.V. Vermeulena, Roberta Salomoneb a Copernicus Institute of Sustainable Development, Faculty of Geosciences, Utrecht University b University of Messina, Department of Economics a Copernicus Institute of Sustainable Development, Faculty of Geosciences, Utrecht University b University of Messina, Department of Economics ⁎ Corresponding author. E-mail address: p.m.calisto@uu.nl (M. Calisto Friant). 1 Search conducted in www.google.com on the 11th of November 2019, using the advanced search option to search for all results before the 31st of December 2008. 2 Based on SCOPUS search for “circular economy” (abstract, keyword, title) search conducted on the 11th of November 2019. https://doi.org/10.1016/j.resconrec.2020.104917 Received 12 November 2019; Received in revised form 29 April 2020; Accepted 29 April 2020 ⁎ Corresponding author. E-mail address: p.m.calisto@uu.nl (M. Calisto Friant). 1 Search conducted in www.google.com on the 11th of November 2019, using the advanced search option to search for all results before the 31st of December 2008. 2 Based on SCOPUS search for “circular economy” (abstract, keyword, title) search conducted on the 11th of November 2019. 09213449/©2020Th Ath Pblihdb El i BVThi i til d th CCBYli https://doi.org/10.1016/j.resconrec.2020.104917 Received 12 November 2019; Received in revised form 29 April 2020; Accepted 29 April 2020 0921-3449/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). p @ 1 Search conducted in www.google.com on the 11th of November 2019, using the advanced search option to search for all results before the 31st of December 2008. 2 Based on SCOPUS search for “circular economy” (abstract, keyword, title) search conducted on the 11th of November 2019. HAL Id: hal-02878613 https://hal.science/hal-02878613v1 Submitted on 23 Jun 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Resources, Conservation & Recycling 161 (2020) 104917 Contents lists available at ScienceDirect E-mail address: p.m.calisto@uu.nl (M. Calisto Friant). 1 https://doi.org/10.1016/j.resconrec.2020.104917 Received 12 November 2019; Received in revised form 29 April 2020; Accepted 29 April 2020 ⁎ Corresponding author. E-mail address: p.m.calisto@uu.nl (M. Calisto Friant). 1 Search conducted in www.google.com on the 11th of November 2019, using the advanced search option to search for all results before the 2 Based on SCOPUS search for “circular economy” (abstract, keyword, title) search conducted on the 11th of November 2019. 0921-3449/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). 2. Methods Since this article aims to investigate the diversity of different circularity discourses, rather than developing an in-depth analysis of a specific aspect of the CE, a critical review is better suited to the objectives of this research as it can generally include a broader range of perspectives and theoretical positions (Greenhalgh et al., 2018; Snyder, 2019) .5 To establish a systematic and consistent typology we used several research questions as guidelines in our step-wise design process. First, what are the main challenges and shortcomings of the CE concept? Second, what are the historical origins and linkages of the CE with other concepts from the Global South and North alike? Third, what are the main conceptual differences and similarities of the core circularity discourses? By answering these three questions, this paper develops the first 2 × 2 typology of circularity discourses to date. While some papers do elaborate distinctions within CE thinking (see for example Blomsma, 2018; Blomsma and Brennan, 2017; D'Amato et al., 2019; Geissdoerfer et al., 2017; Homrich et al., 2018; Korhonen et al., 2018b; Kuzmina et al., 2019; Marin and De Meulder, 2018; Merli et al., 2018; Reike et al., 2018) no research proposes a systematic classification of circular discourses. This paper thus builds and expands on the work of those previous authors to fill this research gap and develops a discourse typology which contributes towards a better understanding and ana- lysis of the CE and helps contextualize and navigate the plurality of the concept and its manifold possibilities.i This paper was developed in four main steps, which build on each other and lead to the construction of the typology of circularity dis- courses presented in Section 4.2 (see Fig. 1).i The first step consists of a critical literature review of the CE and its challenges, gaps and limitations. This review does not focus on sys- tematically or bibliometrically exploring what has been written on the CE, as many recent systematic literature reviews have already done so (see for example Blomsma and Brennan, 2017; Geissdoerfer et al., 2017; Ghisellini et al., 2016; Homrich et al., 2018; Kalmykova et al., 2018; Korhonen et al., 2018b; Kühl et al., 2019; Merli et al., 2018; Murray et al., 2017; Prieto-Sandoval et al., 2018; Reike et al., 2018). Instead, it focuses on critically analysing the conceptual challenges of the CE and why they are important to address. 2. Methods There are no standard methods for developing a discourse typology as previous researchers have followed a variety of different approaches (e.g. Audet, 2016; Dryzek, 2013; Schwarz and Thompson, 1990; van Egmond and de Vries, 2011). This paper was built based on a cri- tical literature review,4 which is particularly valuable to identify con- ceptual gaps in the literature and to develop new theoretical perspec- tives from a broad range of different fields and perspectives (Grant and Booth, 2009; Greenhalgh et al., 2018; Saunders and Rojon, 2011; Snyder, 2019). Some authors argue that those conceptual limitations are not im- portant for practitioners, which need further empirical research rather than theoretical discussions (Kirchherr and van Santen, 2019). Never- theless, considering that the CE is still a relatively recent concept, there is still a strong necessity to build its theoretical foundations. Otherwise, the CE runs the risk of lacking systemic validity, critical social relevance and its claims and propositions might be unachievable on a relevant scale to effectively address the socio-ecological challenges of the 21st century. In this context, the CE concept could easily be discredited and disregarded as a new form of greenwashing or as an oxymoron, com- parable to green growth or ecological modernization (Gregson et al., 2015; Lazarevic and Valve, 2017; Monsaingeon, 2017; Valenzuela and Böhm, 2017). This research aims to address such conceptual risks and help actors better navigate and analyse the history, complexity and plurality of circularity visions by establishing a typology of circularity discourses. Such a typology can provide a basis for a more inclusive and comprehensive discussion on the topic, which opens the imaginary regarding the many circular futures that can exist and allows for a cross- pollination of ideas, policy options, strategies, practices and solutions. In general, the main weakness of critical literature reviews is the inherent subjectivity in the selection of literature (Snyder, 2019). A systematic literature review could reduce this bias by having strict criteria for the selection of literature, which enables a detailed analysis of a specific topic (Grant and Booth, 2009). However, a systematic re- view does not allow for the effective integration of grey and academic literature, as well as academic literature in languages other than Eng- lish, which are not effectively indexed in the main academic search engines such as Scopus and Web-of-Science (Albarillo, 2014; Morrison et al., 2012; Paez, 2017). 1. Introduction consumption, which closes the input and output cycles of the economy, the CE is expected to solve the problems of resource scarcity, bio- chemical flow disruption, and climate change, all while revitalizing local and regional economies (Batista et al., 2018; Delannoy, 2017; Stahel, 2010). The Circular Economy (CE) has become a “go-to concept” that has caught the attention of all sectors of society in the recent years, in- cluding academia, businesses, NGOs and governments (Lazarevic and Valve, 2017). Searching online for the “circular economy” concept in 2008 would only show 20,570 results, the same search now leads to over 5.74 million, thus surpassing the popularity of the many ideas that originated it, such as “industrial ecology” (1.01 million results), and “industrial symbiosis” (195,000), and the ideas that are directly related to it like “cradle to cradle” (3.14 million), “biomimicry” (2.47 million), and “performance economy” (224,000).1 While those ideals are very appealing, the CE concept is still under construction and debate and it still faces many challenges and research gaps to fulfil its promises. Indeed, it is a relatively new concept that is just recently catching academic attention. While there were only 116 academic articles published on the topic from 2001 to 2008, this number has grown exponentially to over 4900.2 Nevertheless, most of the CE discourse has actually been developed by actors in the govern- ment and private sectors, which have specific political and economic agendas, and have often used the CE as a narrative device for green- washing (Ampe et al., 2019; Korhonen et al., 2018b; Nylén and Salminen, 2019; Van den Berghe and Vos, 2019). Public policy predates Overall, the CE concept is viewed as a promising idea and ideal that has much to bring towards addressing challenges of the Anthropocene (Aurez et al., 2016; Geissdoerfer et al., 2017; Murray et al., 2017). By proposing a regenerative and restorative system of production and Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. most academic research, especially in China where the concept has been a national strategy as early as 2002 (McDowall et al., 2017; Qi et al., 2016). Overall, the CE discourse has been dominated by non- academic sectors, which are espousing many economic and environ- mental benefits from circular policies and business models (e.g. Ellen MacArthur Foundation, 2015; European Commission, 2015). 4 A critical literature review (also called integrative literature review) ”aims to assess, critique, and synthesize the literature on a research topic in a way that enables new theoretical frameworks and perspectives to emerge […] This type of review often requires a more creative collection of data, as the purpose is usually not to cover all articles ever published on the topic but rather to combine perspectives and insights from different fields or research traditions.” (Snyder, 2019, p.335-336) 1. Introduction However, these discourses have failed to build a systemic and holistic understanding of the social and sustainability implications of the CE (Millar et al., 2019; Moreau et al., 2017; Temesgen et al., 2019). Moreover, there is little discussion regarding the complex and con- troversial relationships between CE, energy, resources, biodiversity, entropy, and economic growth (Bruel et al., 2019; Cullen, 2017; Desing et al., 2020; Korhonen et al., 2018a). methods (Section 2). It then critically reviews the challenges of the CE concept (Section 3.1) and establishes a comprehensive historical time- line of circularity thinking (Section 3.2). The paper builds on these findings to differentiate circularity discourses based on the extent to which they address the identified challenges (Section 4.1). This is fol- lowed by the development of a new discourse typology, which classifies circularity discourses according to their position on fundamental socio- ecological issues (Section 4.2). Finally, a discussion Section 5 reflects on the conceptual and methodological implications of this research. 3 A Scopus search for “circular economy” AND “discourse analysis” (abstract, keyword, title), conducted on the 20th of December 2019, finds only 3 results. 2. Methods When conceptually defining each discourse type, the authors built on the results of previous research on the topic, in particular those, which have sketched other distinctions in circularity thinking (see for example Blomsma, 2018; Blomsma and Brennan, 2017; D'Amato et al., 2019; Geissdoerfer et al., 2017; Homrich et al., 2018; Korhonen et al., 2018b; Merli et al., 2018; Reike et al., 2018), and which have analysed circularity discourse in specific sectors (Colombo et al., 2019; Fratini et al., 2019; Kaźmierczyk, 2018; Kuzmina et al., 2019; Marin and De Meulder, 2018; Monsaingeon, 2017; Pardo and Schweitzer, 2018; Repo et al., 2018; Rijnhout et al., 2018; Valenzuela and Böhm, 2017; Vonk, 2018; Welch et al., 2017). The above steps ensured that the final typology would not be overly stereotypical or simplistic as it closely aligns both with previous research on discourse analysis and key debates on cir- cularity. Moreover, earlier versions of the discourse typology were presented in an academic conference and three academic workshops to test and improve it.6 These workshops involved around 15 to 25 aca- demic participants and allowed for the discussion of the discourse ty- pology and 4 circularity discourse types. They helped reduce inherent subjectivity in the construction of the discourse typology by collectively discussing the description of each discourse type and cross-checking their relation to the concepts in the timeline. Once the typology was finalized, it was used to classify the 72 concepts in the timeline (Fig. 3) as well as a list of 120 definitions of the CE7 to evaluate where the current and past circularity debates stand. ecological economics (Giampietro, 2019; Millar et al., 2019; Temesgen et al., 2019), environment and sustainability sciences (Korhonen et al., 2018a; Repo et al., 2018; Schröder et al., 2019a), resource efficiency (Lehmann et al., 2018), critical geography (Hobson and Lynch, 2016), engineering (Cullen, 2017), political ecology (Bihouix, 2014; Fressoz and Bonneuil, 2016), waste manage- ment (Velis, 2018), political sciences (Monsaingeon, 2017; Valenzuela and Böhm, 2017), business and management (Geisendorf and Pietrulla, 2018) etc. Search engines included Google Scholar, Scopus, and WorldCat: a total of 107 articles and books were thus reviewed (please see supplementary materials for full list). In the second step, a timeline of circularity thinking was elaborated based on a broad perspective of CE as an umbrella concept (Homrich et al., 2018). 6 The conference was the 25th International Sustainable Development Research Society (ISDRS) Conference in Nanjing, China on the 27th of June 2019, 2 of the workshops were held at the Copernicus Institute of Sustainable Development, Utrecht University, in May 2019 and December 2019, and a third workshop with the CRESTING project research community in Lisbon, Portugal in September 2019.i 2. Methods When conceptually defining each discourse type, the authors built on the results of previous research on the topic, in particular those, which have sketched other distinctions in circularity thinking (see for example Blomsma, 2018; Blomsma and Brennan, 2017; D'Amato et al., 2019; Geissdoerfer et al., 2017; Homrich et al., 2018; Korhonen et al., 2018b; Merli et al., 2018; Reike et al., 2018), and which have analysed circularity discourse in specific sectors (Colombo et al., 2019; Fratini et al., 2019; Kaźmierczyk, 2018; Kuzmina et al., 2019; Marin and De Meulder, 2018; Monsaingeon, 2017; Pardo and Schweitzer, 2018; Repo et al., 2018; Rijnhout et al., 2018; Valenzuela and Böhm, 2017; Vonk, 2018; Welch et al., 2017). The above steps ensured that the final typology would not be overly stereotypical or simplistic as it closely aligns both with previous research on discourse analysis and key debates on cir- cularity. Moreover, earlier versions of the discourse typology were presented in an academic conference and three academic workshops to test and improve it.6 These workshops involved around 15 to 25 aca- demic participants and allowed for the discussion of the discourse ty- pology and 4 circularity discourse types. They helped reduce inherent subjectivity in the construction of the discourse typology by collectively discussing the description of each discourse type and cross-checking their relation to the concepts in the timeline. Once the typology was finalized, it was used to classify the 72 concepts in the timeline (Fig. 3) as well as a list of 120 definitions of the CE7 to evaluate where the current and past circularity debates stand. circularity discourses (Table 1). Different 2 × 2 typologies were tested with each axis representing a core challenge identified in step 1 or a differentiating criterion developed in step 3. Different combinations were thus tried until a definitive version was established, which could effectively incorporate and differentiate all the circularity concepts presented in the timeline (Fig. 3). 2. Methods Literature was selected based on its relevance for answering the research question, publication date (with a specific focus on recent work) and importance (citation count, regardless of year). Moreover, to ensure breadth and diversity, literature from various fields was reviewed including industrial ecology (Bruel et al., 2019; Saavedra et al., 2018; Zink and Geyer, 2017), Moreover, this research finds that the many related concepts which the CE historically builds on can positively contribute to its limitations through the cross-pollination of solutions and ideas. This is particularly important now that the concept faces a period of “validity challenge” (Blomsma and Brennan, 2017, p.609), and needs to address some of its major critiques and limitations to propose a compelling, fair, resilient and sustainable future. This paper thus not only fills a literature gap on CE discourse analysis3 but also on the links between the CE and alter- native social discourses and ideas (Bruel et al., 2019; D'Amato et al., 2019; Ghisellini et al., 2016; Moreau et al., 2017; Prieto-Sandoval et al., 2018; Schröder et al., 2019b; Temesgen et al., 2019).i The paper is structured as follows: first, it describes the research 5 Reviewers have noted that a meta-synthesis method of literature review could overcome some of the limitations of a systematic literature, by adding expert consultations to search engine results to ensure a broad and diverse range of literature (see for example Kirchherr et al., 2016). 2 Fig. 1. Main steps in research methods. M. Calisto Friant, et al. Resources, Conservation & Recycling 161 (2020) 10 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. Fig. 1. Main steps in research methods. circularity discourses (Table 1). Different 2 × 2 typologies were tested with each axis representing a core challenge identified in step 1 or a differentiating criterion developed in step 3. Different combinations were thus tried until a definitive version was established, which could effectively incorporate and differentiate all the circularity concepts presented in the timeline (Fig. 3). 7 The set of definitions used are mainly those sampled by Kirchherr et al., 2017, which were supplemented with the addition of a few more recent defi- nitions from (Geisendorf and Pietrulla, 2018; Gregson et al., 2015; Korhonen et al., 2018a, 2018b; Prieto-Sandoval et al., 2018). Please see sup- plementary materials for further details. 6 The conference was the 25th International Sustainable Development Research Society (ISDRS) Conference in Nanjing, China on the 27th of June 2019, 2 of the workshops were held at the Copernicus Institute of Sustainable Development, Utrecht University, in May 2019 and December 2019, and a third workshop with the CRESTING project research community in Lisbon, Portugal in September 2019. 7 The set of definitions used are mainly those sampled by Kirchherr et al., 2017, which were supplemented with the addition of a few more recent defi- nitions from (Geisendorf and Pietrulla, 2018; Gregson et al., 2015; Korhonen et al., 2018a, 2018b; Prieto-Sandoval et al., 2018). Please see sup- plementary materials for further details. 3.1. Review of challenges and limitations of the circular economy This will inevitably increase demand for material re- sources, many of which could become inaccessible in less than 80 years, especially cobalt, lithium and nickel (Aurez et al., 2016; Fressoz and Bonneuil, 2016; Suh et al., 2017). Yet, these scarce critical raw mate- rials used for renewable energy currently have very low recycling rates so various CE strategies are needed to prevent material shortages, such as refurbishing, recycling, lifetime extension and consumption reduc- tion (Bengtsson et al., 2018; Bihouix, 2014; Lapko et al., 2019; Monsaingeon, 2017). For instance, there is little clarity regarding entropy and the laws of thermodynamics as applied to a CE (Mayumi and Giampietro, 2019; Rammelt and Crisp, 2014). Since materials degrade in quantity and quality each time they are cycled or used, they cannot be circulated indefinitely (Korhonen et al., 2018a; Reuter et al., 2019). This means that to establish a perfect CE, where all resource inputs come from recovered or renewable materials, a general reduction in material de- mand, and economic throughput is necessary (Giampietro, 2019; Korhonen et al., 2018a). On the other hand, it is also worth noting that the CE could be an avenue for energy saving for some material resource flows as many secondary materials (mainly metals) can be obtained at much lower energy costs compared to virgin ones (Aurez et al., 2016). Moreover, improving waste management and eliminating landfilling can lead to lower methane emissions, thus contributing to climate change mitiga- tion (Hawken, 2017; Jurgilevich et al., 2016). The interactions between CE, energy, climate change and material resources are henceforth complex and need further research to build sustainable pathways to- wards zero-carbon circular economies (Bleischwitz and Miedzinski, 2018). Due to the immense challenges and limitations of recycling and recovery activities, a fully CE might be just as illusory as a “perpetual motion machine” (Cullen, 2017). Even if a perfect circularity of mate- rials flows were possible, this would require a capping global resource use at a certain sustainable level, so the economy can run only on re- covered and renewable resources. 3.1. Review of challenges and limitations of the circular economy 3.1.2. Challenge 2: the materials, energy and biodiversity nexus The relationship between materials, energy, biodiversity, and cir- cularity is a critical area that needs further research. Tackling climate change, biodiversity loss and resource scarcity indeed involves many complex trade-offs and synergies (Bleischwitz and Miedzinski, 2018).l The major research gaps and critiques of the CE have been grouped into the 5 following topics, which will be examined in this section: 1 Systemic thinking on entropy, growth, capitalism and decoupling f Energy plays a key role in the cycling of material flows as it is needed to recycle, repair, refurbish or remanufacture any product or material (Cullen, 2017). Wastes (such as end-of-life tyres, biofuel pel- lets, food waste, and wastewater) can also play a key role in energy provision (through energy-from-waste operations) and by doing so, they reduce dependence from fossil fuels (Lehmann, 2018). However, energy recovery competes with higher value recovery options (such as recycling, composting or refurbishing) and generates significant greenhouse gas emissions (Bihouix, 2014). A mismanaged CE transition could thus lead to an increase in energy demand and greenhouse gas emissions (Monsaingeon, 2017). y g py, g , p p g 2 The materials, energy and biodiversity nexus 2 The materials, energy and biodiversity nexus 3 Evaluating and assessing the full impacts of a circular economy 4 Governance, social justice, and cultural change 4 Governance, social justice, and cultural change 5 Alternative visions of circularity 3.1.1. Challenge 1: systemic thinking on entropy, growth, capitalism and decoupling 3.1.1. Challenge 1: systemic thinking on entropy, growth, capitalism and decoupling There is no agreed general economic or social theory underlying the CE. It is a useful concept for organizing regenerative and restorative production and consumption systems, but it is not based on any eco- nomic model or philosophical theory (Velis, 2018). While this makes the concept simpler and easier to promote and adopt, it also means that it faces key challenges, inconsistencies, and limitations in its under- standing, application and its systemic validity (Geissdoerfer et al., 2017; Korhonen et al., 2018b; Lazarevic and Valve, 2017; Reike et al., 2018). Furthermore, transitioning to fully renewable energy grid will re- quire a large amount of material resources to build the new infra- structure such as wind turbines, solar panels, smart grids, electric cars, trains and buses etc. (Moreau et al., 2019; Nieto et al., 2019; Reuter et al., 2019). 8 Figure for 2015 considering a World Bank global aggregation measure that uses 2011 PPP and $5,50/day poverty line (World Bank, 2019). 9 Productivism is as system based on ever-expanding productivity and eco- nomic growth as the main purpose of human organization, it includes (footnote continued) capitalism but also state communism as implemented in the USSR (Audier, 2019). 10 It is worth noting that many other resource nexus perspectives exist. The academic literature typically speaks of a water, food, energy nexus (Del Borghi et al., 2020), a water, food, energy, land and materials nexus (Bleischwitz and Miedzinski, 2018) and more recently an urban nexus of “food, water, energy and waste treatment systems” (S. Lehmann, 2018, p47) . Here a new nexus approach is formulated based on the interactions, synergies and interrelations which are most relevant for a circular society. 10 It is worth noting that many other resource nexus perspectives exist. The academic literature typically speaks of a water, food, energy nexus (Del Borghi et al., 2020), a water, food, energy, land and materials nexus (Bleischwitz and Miedzinski, 2018) and more recently an urban nexus of “food, water, energy and waste treatment systems” (S. Lehmann, 2018, p47) . Here a new nexus approach is formulated based on the interactions, synergies and interrelations which are most relevant for a circular society. 3. Results Kallis et al., 2018; Latouche, 2009). 3.1. Review of challenges and limitations of the circular economy Resources, Conservation & Recycling 161 (2020) 104917 Resources, Conservation & Recycling 161 (2020) 104917 3. Results 3. Results M. Calisto Friant, et al. (footnote continued) capitalism but also state communism as implemented in the USSR (Audier, 2019). 3.1. Review of challenges and limitations of the circular economy Yet considering the large unmet needs of over 45% of the global population which remains in poverty worldwide8 (World Bank, 2019), capping material resource use has critical geopolitical dimensions and necessitates an essential re- consideration of normative questions regarding global justice, well- being and world-wide wealth redistribution (Arnsperger and Bourg, 2017; Bengtsson et al., 2018; Schröder et al., 2019a).fi The third dimension to the abovementioned resource nexus between materials and energy is biodiversity.10 A zero-carbon CE can lead to increased demand for natural resources such as wood, bio-fuels, bio- polymers, natural fibres, and land for wind, solar and tidal energy (Heck et al., 2018; Suh et al., 2017). This is especially the case if bio- technology, biomaterials and bio-based energy play a central role in a decarbonized “circular bioeconomy” (OECD, 2018). It is thus essential to balance an increased demand for natural resources and renewable energy with efforts in biodiversity conservation and restoration to maintain the biophysical health of the planet and the ecosystem ser- vices on which life depends (von Weizsäcker and Wijkman, 2017). There is a generally recognized planetary boundary that identifies the need to conserve at least 75% of the earth's natural ecosystems Furthermore, there is insufficient investigation on whether and how the CE could lead to an absolute, global decoupling of economic growth from environmental degradation (Antikainen et al., 2018). The question of growth is perhaps the largest elephant in the room for the CE. While proponents in the public and private sector argue that a CE would lead to over 600 billion euros in yearly economic gains for Europe alone (Ellen MacArthur Foundation, 2015), this relationship is very unclear (Korhonen et al., 2018a). Due to the inevitability of entropy and the inexistent evidence of absolute decoupling, there is no reason to think that a CE can operate in a context of continued economic growth (Hickel and Kallis, 2019; Jackson, 2016; Mayumi and Giampietro, 2019; Parrique et al., 2019; Ward et al., 2016). As capit- alism cannot operate in a context of degrowth, this would mean that a fully CE is also inherently incompatible with the current productivist economic system9 (Arnsperger and Bourg, 2017; Audier, 2019; 8 Figure for 2015 considering a World Bank global aggregation measure that uses 2011 PPP and $5,50/day poverty line (World Bank, 2019). capitalism but also state communism as implemented in the USSR (Audier, 2019). 2. Methods The timeline builds on those previously devel- oped by Blomsma and Brennan (2017) and Reike et al. (2018) and further adds to them to expand the debate on the CE. To elaborate the timeline, the results of the previous critical review were first carefully analysed, especially by examining what other ideas and theories were commonly connected to the CE. Snowball sampling (Handcock and Gile, 2011) was used to widen the focus to other similar sustainability discourses, especially focusing on the most influential work in the area. Books and articles from on closely related concepts were thus examined such as “permacircular economy” (Arnsperger and Bourg, 2017), “performance economy” (Stahel, 2010), “cradle to cradle” (McDonough and Braungart, 2002), “degrowth” (D'Alisa et al., 2014) etc. Conceptual diversity, plurality and breadth was sought by re- viewing literature from the Global South and North alike as well as concepts from both practitioners and academics. A complete list of 72 different CE-related concepts was thus established. A key originating book or influential article was then reviewed for each concept to ana- lyse its relation to circularity and to organise each idea in different historical and conceptual groups (Fig. 3).i The third step is a classification of circularity discourses according to their level of complexity (Table 1), meaning the extent which each discourse addresses the complex challenges identified in step 1. Five levels of complexity and a set of differentiating criteria for each level were thus established based on those challenges, which include issues of temporal and geographic scales, sustainability dimensions, and on- tology. This allows for a clearer and more consistent distinction of the various concepts presented in the timeline (Table 2). Finally, in the fourth step, a typology of circularity discourses was developed based on previous classifications of environmental dis- courses (Audet, 2016; Dryzek, 2013; Mann, 2018; Schwarz and Thompson, 1990; van Egmond and de Vries, 2011) and their adaptation to the particularities of the CE. The typology draws upon the findings of the previous steps by integrating the most important challenges iden- tified in step 1 as well as the core criteria for the classification of 3 M. Calisto Friant, et al. 3.1. Review of challenges and limitations of the circular economy 9 Productivism is as system based on ever-expanding productivity and eco- nomic growth as the main purpose of human organization, it includes 4 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. (Steffen et al., 2015). Currently, only 62% of natural ecosystems remain and the transition to a circular and zero-carbon economy could further worsen this situation, especially if the complex interactions between energy, biodiversity and material resources are not adequately dealt with (Bihouix, 2014; Heck et al., 2018; Raworth, 2017). higher levels of overall resource consumption in the economy (Junnila et al., 2018). This is known as the Jevon's paradox and it has key implications in the realization of a CE that does not end up causing more negative than positive impacts (Kjaer et al., 2019). Even some product-service systems (PSS), which promote the access to products and services (as opposed to ownership), have had limited environ- mental benefits due to the abovementioned rebound effect (Hobson and Lynch, 2016; Junnila et al., 2018; Kjaer et al., 2019). Moreover, biodiversity provides with key solutions to global pro- blems by reducing soil erosion, improving human health, contributing to climate change adaptation (through ecosystem-based disaster risk reduction strategies), climate change mitigation (through carbon se- questration), improving water quality and quantity (through watershed conservation and restoration), improving soil health (through re- generative agriculture), reducing air pollution (through urban greening), improving waste-water treatment (through constructed wetlands), and inspiring human creativity and innovation (Benyus, 1998; Delannoy, 2017). These nature-based solutions must thus be better integrated with regenerative and restorative CE practices (Del Borghi et al., 2020; Jurgilevich et al., 2016; Reynaud et al., 2019). Moreover, a CE can also lead to reduced demand for goods through longer use-rates, reuse, repair, recycling, and refurbishing strategies as well as simple-living behaviours, all of which can significantly reduce environmental pressures (Bengtsson et al., 2018; Jackson, 2016; ; ). The interactions between energy, biodiversity and material resources are graphically represented in Fig. 2. The development of clear indicators and assessment mechanisms to measure circularity, while accounting for this rebound effect, is thus a complex issue that needs to be resolved to ensure that circularity claims actually lead to ecological benefits (Antikainen et al., 2018; Corona et al., 2019; Manninen et al., 2018). 3.1.3. Challenge 3: evaluating and assessing the full impacts of a circular economy 3.1.3. Challenge 3: evaluating and assessing the full impacts of a circular economy 3.1.3. Challenge 3: evaluating and assessing the full impacts of a circular economy Holistically assessing and evaluating the sustainability impacts of circular systems is another large challenge. Research has found that many production systems that define themselves as circular can lead to greater environmental impacts than their linear counterparts (such as biofuels and biopolymers) (Hobson and Lynch, 2016; Monsaingeon, 2017; Velis, 2018; Zink and Geyer, 2017). Moreover, a CE approach that focuses on eco-efficiency creates a rebound effect, where reduced costs for one product or service leads to increased de- mand for it, while also creating saving that incentivize consumption in other areas (Zink and Geyer, 2017). Thus, efficiency gains lead to 3.1. Review of challenges and limitations of the circular economy While some CE impact studies exist in China and Northern Europe, more research is needed to fully understand the outcomes of circular projects and solutions (Kalmykova et al., 2018; Saavedra et al., 2018; Winans et al., 2017) and especially for result-orientated PSS, which could have a high sustain- ability potential if they are well designed and implemented (Kühl et al., 2019). The ecological footprint indicator might be a useful tool in this re- gard, as it allows the measurement of the overall impacts of human activities, beyond punctual eco-efficiency improvements (Junnila et al., 2018; Kaźmierczyk, 2018; Rijnhout et al., 2018). The better integration of circularity and footprint indicators is thus key to ensure circularity interventions actually reduce the pressure on the Earth's biophysical limits (Arnsperger and Bourg, 2017; Bruel et al., 2019; Temesgen et al., 2019). 3.1.4. Challenge 4: governance, social justice, and cultural change They thus open up entirely new forms of con- ceiving democracy, waste, well-being, society and nature (Calisto Friant and Langmore, 2015; D'Alisa et al., 2014; Kothari et al., 2019). Moreover, there is a general lack of discussion regarding social and environmental justice aspects related to the CE. In a review of 114 definitions, Kirchherr et al. (2017) found that only 18–20% include social equity considerations. Critical questions regarding, who controls CE technologies and patents, and how the economic costs and benefits should be distributed both within and between countries, have thus received very little attention. Those are nonetheless vitally important questions that will determine whether the CE will lead to more mean- ingful jobs, closer communities, greater social equity and global soli- darity or rather to increased precarity, inequality, and neo-colonialism (Arnsperger and Bourg, 2017; Bihouix, 2014; Schröder et al., 2019a). All in all, the CE could become a profitable industry owned by a few corporations in a handful of countries rather than a transformative movement that benefits all of humankind (Monsaingeon, 2017). Buddhism, Taoism and Confucianism also have strong ecological components, which have not been sufficiently related to the CE (Subramanian et al., 2018). Confucianism and Taoism played a key role in the early adoption of the CE in China as part of the creation of a “har- monious society” and an “ecological civilization” (Jin, 2008; Naustdalslid, 2014). Japan has also been implementing circular policies since the early 2000s through its innovative “Fundamental Plan for Es- tablishing a Sound Material-Cycle Society” (Hara and Yabar, 2012; Hotta, 2011; Takahashi, 2020) and more recently with the “regional circular and ecological sphere” policy, which addresses key territorial and socio-ecological synergies (Japanese Ministry of the Environment, 2018). The Buddhist inspired Gross National Happiness Index (GNI) of Bhutan also deserves greater attention as it shows how new metrics that go be- yond the Gross Domestic Product (GDP) can be developed and adapted to include key circularity criteria (Rinzin et al., 2007; Verma, 2017). Yet, more remains to be written on the philosophical components of a CE and how they can relate to different worldviews. i In his latest book, Thomas Piketty proposes a CE that circulates property and capital to redistribute resources and counter capitalism's inherent accumulative tendencies (Piketty, 2019). Further discussion on this form of CE is necessary to foster a circularity transition that is socially, economically and ecologically sustainable. 3.1.4. Challenge 4: governance, social justice, and cultural change 2316 are in environmental sciences, 1753 are in engineering, 1191 are in energy sciences, and only 804 are in social sciences or humanities).11 By overlooking social considerations, CE research is proposing a tech- nological path to sustainability that many have criticized for being overly optimistic regarding the speed of technological transitions and the capacity of society to integrate disruptive innovations, which challenge vested interests (Bihouix, 2014; Feola, 2019a; Fressoz and Bonneuil, 2016; Jackson, 2016; Latouche, 2009). This approach also fails to recognize the massive socio-cultural change that a CE entails by transforming consumption and production structures based on materialism, convenience, and ownership to ones based on col- laborative consumption, sharing economies and use-value (Frenken, 2017; Hobson, 2019; Lazarevic and Valve, 2017; Pomponi and Moncaster, 2017). When some of those social and cultural topics are ad- dressed in the literature, it is dominantly done through commercial ap- proaches, such as new business models for the private sector rather than from the perspective of a transformative social and solidarity economy (with some notable exceptions such as Baruque-Ramos et al., 2017; Chaves Ávila and Monzón Campos, 2018; Gutberlet et al., 2017; Hobson and Lynch, 2016; Moreau et al., 2017; Schröder et al., 2019a). Yet this is a key topic as evidenced by the work of Kirchherr et al. (2018) which found that practitioners see cultural barriers as the main barriers to a CE transition. There is also little work on indigenous discourses on circularity and alternative concepts from the Global South such as “ubuntu” (Shumba, 2011), “ecological swaraj” (Kothari et al., 2014), “buen vivir”/ “suma qamaña” (Calisto Friant and Langmore, 2015) and the “Buddhist middle path” (Rinzin, 2006), which also share the goal of building re- generative systems that respect, sustain and restore the natural cycles of the earth. After all, circularity, in traditional hunter-gatherer, agri- cultural and pastoral societies, has existed for much of humankind's presence on planet earth and still exists in many parts of the world today (Giampietro, 2019). This has yet to be recognized by the literature, which is hence missing the opportunity to build key synergies and learn from radically different epistemological and ontological frameworks. Moreover, indigenous discourses often have the added value of being radically pluralistic and ecocentric as opposed to the anthropocentrism and ethnocentrism of most western environmental discourses (Kothari et al., 2019). 11 Based on a Scopus search for “circular economy” (abstract, keyword, title) conducted on the 11th of November 2019. 3.1.4. Challenge 4: governance, social justice, and cultural change 3.1.4. Challenge 4: governance, social justice, and cultural change Another important challenge, which is often under-addressed in the CE literature to date, is the social dimension, especially with regards to issues of governance, justice, and cultural change (Geissdoerfer et al., 2017; Hobson, 2019; Korhonen et al., 2018a; Millar et al., 2019; Moreau et al., 2017; Schröder et al., 2019a; Temesgen et al., 2019). A Scopus search reveals that less than 17% of articles on the CE are from social science and humanities (of a total of 4901 articles on the CE: Fig. 2. Interactions of the Energy, Materials, Biodiversity Nexus (synergies are marked in green arrows, interactions with possible trade-offs are marked with red arrows). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 2. Interactions of the Energy, Materials, Biodiversity Nexus (synergies are marked in green arrows, interactions with possible trade-offs are marked with red arrows). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 5 5 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. compatible within the earth's system boundaries. Academics have indeed found core synergies between these concepts; especially literature on degrowth, which could complement the CE's lack of social dimension and system-wide thinking on entropy and biophysical limits (D'Amato et al., 2019; Ghisellini et al., 2016; Hobson and Lynch, 2016; Schröder et al., 2019b). Degrowth and simple living scholars also help conceive of cir- cularity through a lens of sufficiency, conviviality, and social justice ra- ther than being overly focused on technological innovation and eco-effi- ciency (Alexander, 2015; Caillé, 2015; Kothari et al., 2019). This sufficiency approach has recently gained some support in the CE litera- ture as it can lead to a slowing of resource loops, with significant sus- tainability benefits (Bengtsson et al., 2018; Bruel et al., 2019; Hayward and Roy, 2019; Hobson and Lynch, 2016; Schröder et al., 2019b). Yet a recent review of 327 academic articles on the CE found that less than 10% of articles include this approach (Homrich et al., 2018). 3.1.4. Challenge 4: governance, social justice, and cultural change The governance and political considerations of a CE also deserve greater attention and study. Power plays a key role in the future of a zero-carbon CE transition as it determines who controls the discourse, who takes decisions and who will benefit from them (Hobson and Lynch, 2016; Lazarevic and Valve, 2017; Schröder et al., 2018). This is why it is key to establish a democratic and deliberative governance system for a CE to ensure that everyone is involved in its construction and that its benefits reach the most vulnerable. Yet, those political considerations are rarely taken into account by the literature on CE, which has mostly dealt with design, technological, managerial or business-led solutions (as evidenced by Fratini et al., 2019; Millar et al., 2019; Moreau et al., 2017). The Gilet Jaune movement illustrates the risks with a sustainability transition that does not sufficiently include political and social justice elements (Deléage, 2019; Laurent, 2019). f Overall, the research on degrowth and non-western visions of sus- tainability could bring key insights in the first and most important “loops” in the CE value-retention hierarchy: refuse, reduce, reuse and repair (Reike et al., 2018), all of which can lead to the sustainable slowing of resource cycles (Homrich et al., 2018). 3.2. Timeline of circularity thinking 3.1.5. Challenge 5: alternative visions of circularity 3.1.5. Challenge 5: alternative visions of circularity There is limited research on alternative approaches to circularity such as degrowth (Kallis et al., 2018), steady-state (Daly, 1977), and simple living/voluntary simplicity (Alexander, 2015) concepts, which have a rich academic literature and share the same objective regarding the necessity to transform towards regenerative socio-economic structures which are A timeline of the CE thinking and its related concepts, based on a broad understanding of the EC as an umbrella concept (Homrich et al., 2018), is presented in Fig. 3. The timeline builds on the categorizations proposed by Reike et al. (2018) and by Blomsma and Brennan (2017), which were expanded to include a plurality of concepts from western and non-western perspectives alike. Fig. 3 thus allows to better situate the concept, both in its rich historical origins and in its complex theo- retical diversity. This helps illustrate the manifold conceptualizations of 6 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. M. Calisto Friant, et al. Fig. 3. Timeline of Circularity Concepts and Ideas (please see supplementary material for details).17 7 M. Calisto Friant, et al. Resources, Conservation & Recycling 161 (2020) 104917 persist as a contention, as different positions end up “agreeing to dis- agree” (Blomsma and Brennan, 2017, p.609). circularity as well as the reformist vs transformational circularity schools of thought, which shape the current debate on the topic (Reike et al., 2018). The 72 concepts in this timeline are further ana- lysed in Sections 4.1 and 4.2 to classify them based on their key socio- ecological considerations and to examine how they address the complex challenges that the CE concept is presently facing.i In this critical moment, two broad movements of the CE concept can be seen: first, Circularity 3.1, which represents reformist discourses that operate within the boundaries of the capitalist system (e.g., Allwood et al., 2011; Fullerton, 2015; Pauli, 2010; Rifkin, 2013), and second, Circularity 3.2, which represents transformational discourses seeking wholesale transformation of the socio-economic order (e.g., Arnsperger and Bourg, 2017; Kothari et al., 2014; Latouche, 2018; Trainer and Alexander, 2019). 4.1. Classification of circularity discourses according to their level of complexity Table 1 shows the different levels of complexity of circularity dis- courses depending on the extent to which they address the challenges reviewed in Section 3.1: Circularity 2.0, represents the beginning of an “excitement period” (1980–2010) (Blomsma and Brennan, 2017, p608), where a diversity of innovative ideas start to see waste as a valuable input for other processes (Reike et al., 2018). It is the time when the concept of a CE was first coined by Pearce and Turner (1989) and when many related ideas, po- licies, and business models emerged, including “industrial ecology” (Frosch and Gallopoulos, 1989), “industrial symbiosis” (Chertow, 2000), “product-service system” (Goedkoop et al., 1999), “reverse logistics” (Rogers and Tibben-Lembke, 1998), and “extended producer responsi- bility” (Lindhqvist, 2000). These ideas often take inspiration from nature to build new technologies and innovations that connect the output and input sides of the economy and make industries work like natural eco- systems. Since this period coincides with the growth of neoliberalism, most of these ideas were established and implemented through market- driven approaches and public-private partnerships (Monsaingeon, 2017) which didn't give much attention to the main challenges discussed in Section 3.1, except for challenge 2 on the resource nexus. • Columns (a) and (b), represent spatial and temporal scales, the importance of which was evidenced in challenges 1 and 3 discussing global resource limits and the rebound effect. f • Column (c) shows which pillars of sustainability (people, planet, prosperity)12 are included. The significance of this was seen in challenge 4, where the importance of social justice and political considerations was highlighted. • Column (d) distinguishes ontological (anthropocentric vs eco- centric) and epistemological perspectives (ethnocentric vs plural), which were discussed in challenge 5. • Column (e) relates to the complex interlinkages of the resource nexus, which was discussed in challenge 2. • Column (f) refers to the questions of economic growth, capitalism and decoupling, which was analysed in challenge 1.l • Columns (g) and (h) reflect the core objectives and narratives and column (i) shows where each circularity concept group from the timeline (Fig. 3) fits. With Circularity 3.0 (1990-present) the beginning of a compre- hensive socio-economic approach to waste, resources, production and consumption emerged; which often builds on the objectives of the Rio Declaration on Environment and Development (UN, 1992). 4. Synthesis and reflection Now that the core challenges, the conceptual origins and the di- versity of the CE have been reviewed, the next section synthesizes and reflects on these findings to analyse and differentiate the plurality of circularity discourses. The conceptual challenges highlighted in Section 3.1 are an effective starting point to unpack this diversity. They are thus the basis used for distinguishing circularity discourses ac- cording to their level of complexity (Section 4.1). This leads to the circularity discourse typology which allows for a clearer differentiation, navigation and comprehension of this contested paradigm (Section 4.2). During this preamble stage, a body of technical literature on waste management was also developed, which represents Circularity 1.0 ap- proaches that deal with waste as a problem to be managed through end- of-pipe technologies (Reike et al., 2018). This is thus when the first waste management and recycling systems were developed for various waste streams (Reike et al., 2018; Takahashi, 2020). These concepts were mostly focused on specific technological innovations and thus didn't address the main challenges evidenced in Section 3.1. 12 The people, planet, prosperity (PPP) framework represent a broad con- sensus on the core pillars of sustainability as evidenced by the review of Vermeulen (2018). 3.1.5. Challenge 5: alternative visions of circularity Both discourse types include issues of planetary boundaries, the rebound effect, social justice, and good governance (as discussed in challenges 2, 3 and 4), however, they vary in their views regarding the capacity of capitalism to overcome resource limits and decouple ecological degradation from economic growth (as evidenced in challenge 1) as well as topics of epistemological and on- tological pluralism (challenge 5) (please see the supplementary mate- rials for more details on each concept in the timeline). The first period is a preamble stage (1945 to 1980), where discus- sions regarding resource limits and the ecological impacts of human activities became widespread thanks to key publications such as “The Tragedy of the Commons” (Hardin, 1968), “the Limits to Growth” (Meadows et al., 1972), and “Overshoot” (Catton, 1980). This phase is also ripe with a diversity of transformative proposals from various perspectives including Gandhian economics (Kumarappa, 1945), Bud- dhist economics (Schumacher, 1973), socialism (Commoner, 1971), anarchism (Illich, 1973), ecological economics (Daly, 1977; Georgescu- Roegen, 1971), political ecology (Gorz, 1980), and eco-design (Papanek, 1972). During this time, Boulding (1966) wrote “The Eco- nomics of the Coming Spaceship Earth”, which is often considered to be the first reference to a CE-like system (Antikainen et al., 2018; Geissdoerfer et al., 2017; Murray et al., 2017; Prieto-Sandoval et al., 2018; Winans et al., 2017). He calls for a “spaceman economy”, where “all outputs from consumption would constantly be recycled to become inputs for production” (Boulding, 1966, p5). These concepts had a strong understanding of planetary limits and gave a great attention to all the issues discussed in Section 3.1, including decoupling (challenge 1), resource trade-offs (challenge 2), rebound effects (challenge 3), social justice (challenge 4) and alternative visions of sufficiency (challenge 5). 4.1. Classification of circularity discourses according to their level of complexity During this period the original CE concept was further developed by new ideas including “the natural step” (Robèrt, 2002), “the performance economy” (Stahel, 2010), “cradle to cradle” (McDonough and Braungart, 2002), and “natural capitalism” (Hawken et al., 1999). However, from about 2010 onwards, it is also a “validity challenge period” (Blomsma and Brennan, 2017, p.609), where many incon- sistencies and conceptual challenges of the CE must be resolved. From this point, the concept can either cohere, by resolving its theoretical challenges, collapse, as inconsistencies become insurmountable, or To distinguish discourses that go beyond market-based solutions and economic considerations and see circularity as a holistic social transformation, the term circular society is proposed; this applies to complexity levels 4 and 5. A circular society defines discourses with a vision of circularity where not only resources are circulated in 8 8 M. Calisto Friant, et al. Resources, Conservation & Recycling 161 (2020) 104917 Circularity discourse complexity. Circularity vision Complexity level a) Temporal scale b) Spatial scale c) Sustainability factors included d) ontology and epistemology e) Perspective on the resource nexus f) Views on capitalism and decoupling g) Main goal/objective h) Narrative i) Circularity concept group Circular Society 5. Very long term: multiple generations (beyond 50 years) Macro-scale: planet Earth People, Planet, Prosperity Ecocentric and plural Changing consumption and production patterns to keep energy, biodiversity and material resources within safe planetary limits Sceptical regarding the possibility of decoupling and the sustainability of capitalism Maintaining socio- ecological health and wellbeing for present and future generations of human and non- human life The earth is borrowed from future generations of living beings, humans must preserve, respect, restore and share it in a fair manner, even if that entails changing lifestyles and consumption patterns Mainly Circularity 3.2 and most Precursors 4. Long term: 1 to 2 generations (20–50 years) Macro-scale: planet Earth People, Planet, Prosperity Anthropocentric and ethnocentric Balancing trade-offs and synergies to keep energy, biodiversity and material resources within safe planetary limits Believe in the possibility of decoupling and the sustainability of capitalism Preserving social well- being and the biophysical health of the earth system in line with the SDGs Humans must ensure justice, fairness and participation in the sustainable stewardship of the earth, even if that entails redistributing and changing consumption patterns Mainly Circularity 3.1 Circular Economy 3. 4.1. Classification of circularity discourses according to their level of complexity Long term: one generation (10–25 years) Macro-scale: planet Earth Planet, Prosperity Anthropocentric and ethnocentric Balancing trade-offs and synergies to keep energy, biodiversity and material resources within safe planetary limits Believe in the possibility of decoupling and the sustainability of capitalism Maintaining the biophysical health of the earth system Reducing humanity's overall ecological footprint and balancing resource limits and constraints is key to ensure the stability of the biosphere and long-term economic prosperity Mainly Circularity 2.0 2. Mid-term: 1 to 2 government planning cycles (5 to 10 years) Meso-scale (country, region, industrial park, city) Planet, Prosperity Anthropocentric and ethnocentric Optimizing and securing material, natural and energy resources, especially for critical raw materials Believe in the possibility of decoupling and the sustainability of capitalism Securing and preserving critical resources and materials Strategically maximising eco-efficiency and balancing resource use is necessary to maintain resource security and ensure geopolitical stability Mainly Circularity 1.0 and 2.0 1. Short term: single product life cycle (1 to 2 years) Micro-scale (single product, service, or firm) Planet, Prosperity Anthropocentric and ethnocentric Optimising material and energy resource flows in product design. Believe in the possibility of decoupling and the sustainability of capitalism Capturing opportunities to lower both environmental impacts and economic costs. Ensuring optimum resource efficiency through eco- innovation leads to win-win solutions that reduce ecological harm and increase economic value Mainly Circularity 1.0 and 2.0 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. Fig. 4. Conceptual Differentiation between Circular Economy and Circular Society. M. Calisto Friant, et al. Resources, Conservation & Recycling 161 (2020) 104917 Fig. 4. Conceptual Differentiation between Circular Economy and Circular Society. limited), and hierarchists, who see nature as tolerant (resilient but only up to some extent) (Schwarz and Thompson, 1990).f sustainable loops, but also wealth, knowledge, technology and power is circulated and redistributed throughout society (see Fig. 4). These discourses thus comprehensively include the three pillars of sustain- ability and see circularity as a holistic transition, where issues of poli- tical empowerment and social justice also have to be addressed. The term circular economy, in contrast to this, focuses on circulating re- sources alone and applies for complexity levels 1 to 3, which largely deal with circularity through a technical lens of ecological and material efficiency alone. 4.2. Development of a circularity discourse typology While many are proposing a “deliberately vague but un- controversial” (Lazarevic and Valve, 2017, p60) discourse on the CE as a strategy to gain widespread support in the short term, this could lead to a depoliticised CE, which does little towards tackling the systemic socio-ecological challenges of the Anthropocene (Korhonen et al., 2018b; Millar et al., 2019; Valenzuela and Böhm, 2017). Thus, it is key to propose alternative visions, which tackle the key conceptual chal- lenges evidenced in Section 3. As Latouche (2018) rightly pointed out, the core challenge for global sustainability is to “decolonize the ima- ginary” and allow other futures to emerge. In the same manner, Korten (2015) speaks of “changing the story to change the future”, Escobar (2018) argues for a “pluriversal imagination” needed for en- tirely different forms of “world making”, and Feola (2019b) calls for the “unmaking of capitalism” to “make space” for a diversity of alter- natives. A typology of circularity discourses must thus not only help better distinguish different circular discourses but also allow for an expansion of the imaginary regarding other possible circular futures.i The second typological axis was developed from challenge 1 on capit- alism, economic growth and decoupling (column (f) in Table 1). Recent research has found that this could be the most crucial element to transition discourses, as it deals with the ability, or inability, of the current socio- economic system to prevent ecological collapse by decoupling economic growth from environmental degradation (eco-economic decoupling)13 (Feola, 2019a; Fergnani, 2019; Giampietro, 2019; Hickel and Kallis, 2019; Parrique et al., 2019). The second typological differentiation thus distin- guishes whether discourses are optimist or sceptical about the capacity of technology and innovation to overcome the major ecological challenges of the Anthropocene before an irreversible socio-ecological collapse occurs. This differentiation is similar to the distinction between Mann's (2018) prophets and wizards, but it adds a stronger systemic and political dimension to it. While other classifications of environmental discourses have been developed previously, none has been specifically designed to compre- hensively distinguish circularity discourses. Other environmental dis- course typologies include Dryzek's (2013), which divides reformist versus radical positions with prosaic versus imaginative positions leading to four core discourses: environmental problem solving (prosaic and reformist), green radicalism (imaginative and radical), survivalism (prosaic and radical), and sustainability (imaginative and reformist). 13 Eco-economic decoupling is defined here as the absolute decoupling of environmental degradation from economic growth, meaning growing GDP while reducing absolute environmental impacts from production and con- sumption activities (Kjaer et al., 2019). 4.1. Classification of circularity discourses according to their level of complexity Moreover, when discussing CE as a general umbrella concept, this article uses term circularity to comprehensively include all its historically related concepts and ideas (as seen in Fig. 3). Con- sidering the importance of “policy labels, keywords and framing” for sustainability transitions (Silva et al., 2016, p. 224), these differentia- tions can help acknowledge and address the complex ecological, so- ciological and political implications of a circular future. Mann (2018) differentiates between prophets who call for urgent cut-backs in consumption to stay within planetary limits and wizards who propose optimistic technological solutions. Moreover, Audet (2016) distinguishes between technocentrist and localist transition discourses, the first being focused on scientific innovation, and the later on bottom-up social transformation. Nevertheless, none of the abovementioned discourse typologies applies perfectly to circularity, as they are either too narrow, leaving some circularity discourses out; or too general and do not allow for a clear differentiation of the circularity concepts presented in Fig. 3. The typology of CE discourses presented in Fig. 5 draws inspiration from the division of environmental discourses developed by the above authors and adapts them to the CE by integrating the results of this research.i The first typological axis was developed from challenge 4, on social justice and governance, which many authors have identified as one of the most important issues for a circular future (Geissdoerfer et al., 2017; Hobson, 2019; Korhonen et al., 2018a; Millar et al., 2019; Moreau et al., 2017; Schröder et al., 2019a; Temesgen et al., 2019). This axis thus builds on the distinction between circular society and circular economy presented in Section 4.1 by dividing holistic from segmented discourses. Holistic discourses comprehensively integrate the social, ecological and political considerations of circularity (like circular society visions). Segmented discourses, on the other hand, have a homogeneous perspective and a uniform focus on only eco- nomic and technical components of circularity (like circular economy vi- sions). This differentiation is similar to Dryzek's (2013) distinction between prosaic vs imaginative discourses but it is specifically focused on the cir- cularity challenges reviewed in this article, and especially the distinction between circular economy and circular society concepts. 14 This article follows the value-retention hierarchy (also called R-hierarchy, R-imperatives or simply R's) established by Reike, Vermeulen and Witjes (2018): R0 refuse, R1 reduce, R2 reuse/resell, R3 repair, R4 refurbish, R5 remanufacture, R6 re-purpose, R7 recycle materials, R8 recover energy, R9 re- mine. 4.2.1. Reformist circular society the Club of Rome (von Weizsäcker and Wijkman, 2017) as well as cities like Amsterdam (as evidenced by Fratini et al., 2019). Reformist Circular Society has a lot in common to Dryzek's (2013) sustainability category of discourses. The name of this discourse type derives from Reike et al. (2018) reformist CE distinction combined with the circular society concept as described in Section 4.1. Holistic and optimistic discourses propose a mix of behavioural and technological change, leading to an abundant, fair, and sustainable future where scarcity and environmental overshoot has been dealt with by impressive social, economic, industrial and environmental innova- tions. While they believe important socio-cultural change is necessary, and new forms of public participation and inclusion are needed, they do not see a fundamental contradiction between capitalism and sustain- ability. Reformist Circular Society discourses thus argue that the current system can be deeply reformed toward circularity and believe that so- cial and economic innovation can lead to a sufficient level of eco-eco- nomic decoupling to prevent a widespread ecological collapse. Reformist Circular Society discourses promote a variety of circular so- lutions such as industrial symbiosis, PSS, ecodesign and biomimicry, and they integrate the 3 main components of sustainability in their discourse, which is often framed around achieving the sustainable de- velopment goals while remaining within safe planetary boundaries. This discourse type considers solutions throughout all the value reten- tion options of the CE, yet it gives a stronger focus to intermediate loops such as R2 reuse/resell, R3 repair, R4 refurbish, R5 remanufacture, R6 re-purpose, and R7 recycle.14 All Circularity 3.1 concepts fall within this discourse type, as well as the positions of various NGOs and non- profits like Circle Economy (Verstraeten-Jochemsen et al., 2018) and Fig. 5. Circularity discourse typology. 4.2.1. Reformist circular society Fig. 5. Circularity discourse typology. 4.2. Development of a circularity discourse typology Schwarz and Thompson (1990) build on the cultural theory of risk by adding a vision on the fragility of nature to the divide between cultural rationalities This leads to four core environmental discourses: fatalist, who see nature as capricious (unpredictable and un- controllable), individualist, who see nature as benign (resilient and abundant), egalitarians, who see nature as ephemeral (fragile and 10 M. Calisto Friant, et al. Resources, Conservation & Recycling 161 (2020) 104917 4.2.2. Transformational circular society Holistic and sceptical discourses propose an entirely transformed social system where individuals gain a renewed and harmonious con- nection with the Earth and their communities. A general economic downscaling and a philosophy of sufficiency leads to simpler, slower and more meaningful lives. Local production is emphasized, especially through cooperative and collaborative economic structures and by using agroecological techniques and open-source innovations and technologies that do not harm the biosphere nor deplete its limited resources, such as 3D printing, solar panels, wind turbines, cooperative P2P platforms, etc. This discourse type thus gives a stronger focus to the shorter loops in the CE value retention hierarchy, especially R0 refuse, R1 reduce, R2 reuse/resell, R3 repair, R4 refurbish, R5 remanufacture, and R6 re-purpose. Transformational Circular Society discourses also place a core emphasis on changing materialistic, anthropocentric, pa- triarchal, individualistic and ethnocentric worldviews to more holistic, plural, and inclusive ones. They also propose to redistribute global re- sources from nations and social sectors that grossly overshoot their ecological footprint to those that do not. Transformational Circular Society discourses emphasize direct participation and citizen inclusion in the democratic construction of the future, often through novel me- chanisms of bottom-up governance. All Circularity 3.2 concepts and most Precursors fall in the Transformational Circular Society discourse 14 This article follows the value-retention hierarchy (also called R-hierarchy, R-imperatives or simply R's) established by Reike, Vermeulen and Witjes (2018): R0 refuse, R1 reduce, R2 reuse/resell, R3 repair, R4 refurbish, R5 remanufacture, R6 re-purpose, R7 recycle materials, R8 recover energy, R9 re- mine. 11 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. capitalism”,15 in which investors, entrepreneurs and venture capitalists see green solutions and business models as new opportunities for capital expansion (Fletcher, 2019, 2012). Many states and corporations are thus already using a framing of scarcity to grab land and resources in the Global South and develop infrastructures and technologies to ensure resource security (Cavanagh and Benjaminsen, 2018; Mehta et al., 2019). There is also a growing number of NGOs, think tanks and gov- ernments using a discourse of climate change, scarcity and over- population to protect geopolitical power, resources and prosperity from migrant people from the Global South (Hendrixson and Hartmann, 2019). 15 The term “disaster capitalism” originates from Naomi Klein, which defines it as “orchestrated raids on the public sphere in the wake of catastrophic events, combined with the treatment of disasters as exciting marketing opportunities” (cited in Fletcher, 2012, p.99) this crisis-driven narrative have been used to control natural resources and commodify nature (Fletcher, 2019, 2012). 4.2.5. Circularity discourses and concepts through history bl h i i f h f Table 2 presents the position of each concept from the timeline in relation to the discourse typology. The most widespread discourse in the literature is Transformational Circular Society (42% of reviewed concepts), followed by Reformist Circular Society (28% of reviewed concepts), Technocentric Circular Economy (26% of reviewed concepts) and Fortress Circular Economy (4% of reviewed concepts). On the other hand, revising 120 academic, government and practitioner definitions of the CE reveals that 101 of them (84%) fall in the Technocentric Cir- cular Economy discourse type, 14 in Reformist Circular Society (12%), 3 in Fortress Circular Economy (2.5%), and 2 in the Transformational Cir- cular Society (1.5%) (please see supplementary materials for details). There is thus a discrepancy between the diversity of holistic CE-related concepts in the literature, and the most common definitions of the CE term, which are generally situated within segmented and optimist dis- course types. The present status quo of the CE discourse is thus in the Technocentric Circular Economy discourse type, despite of the significant literature on other circular discourses. This is in line with results of D'Amato et al. (2019), which found that academics had a more de- growth or post-growth oriented perspective on circularity than main- stream CE propositions (D'Amato et al., 2019). 4.2.2. Transformational circular society This narrative is clear in a Pentagon-commissioned report arguing that wealthy nations may “build defensive fortresses around their countries […] to hold back unwanted starving im- migrants” and preserve their resources (Schwartz and Randall, 2003 p. 18). Fortress Circular Economy discourses are similar to Schwarz and Thompson's (1990) fatalists, and Dryzek's (2013) survivalists. Moreover, like fatalists and survivalists, the proponents of this discourse type do not always engage in mainstream debate since they have rather cynically realist visions. Yet, they might often be the underlying focus of many business and government discussions on circularity, especially when they are based on a narrative of geopolitical resource security, over- population and economic competitiveness. type, as well as various social movements, such as the Great Transition Initiative and the Transition Towns Network (as evidenced by Feola and Jaworska, 2019), local bottom-up circular initiatives such as De Ceuvel in Amsterdam (as evidenced by Hobson, 2019) and R-Urbain in Paris (as evidenced by Marin and De Meulder, 2018), and many indigenous movements form the Global South (Kothari et al., 2019). These dis- courses are similar to Dryzek's (2013) green radicalism as well as Schwarz and Thompson's (1990) egalitarians. The name of this discourse type derives from Reike et al. (2018) transformational CE distinction combined with the circular society concept as described in Section 4.1. 4.2.3. Technocentric circular economy Segmented and optimistic discourses propose an era of green- growth and technological advancements, which allow for increasing levels of prosperity while reducing humanity's ecological footprint. These discourses thus expect that circular innovations can lead to an absolute eco-economic decoupling to prevent ecological collapse. To do so many win-win solutions are promoted such as PSS, EPRs, biomimicry reverse logistics, industrial symbiosis, remanufacturing, refurbishing, big data, and eco-design, as well as controversial in- novations such as carbon capture and storage, artificial intelligence, geoengineering, and synthetic biology. This discourse type thus gives a stronger focus to the larger loops in the CE value retention hier- archy, especially R4 refurbish, R5 remanufacture, R6 re-purpose, R7 recycle, R8 recover energy and R9 re-mine. These discourses are common in European government policies (as evidenced by Colombo et al., 2019; Kaźmierczyk, 2018; Pardo and Schweitzer, 2018; Repo et al., 2018; Rijnhout et al., 2018), CE de- velopment plans in cities such as London (as evidenced by Fratini et al., 2019), corporate strategies such as Apple's (as evi- denced by Valenzuela and Böhm, 2017; Vonk, 2018), business con- sultancies such as McKinsey and some international organizations including the World Economic Forum, the International Resource Panel, and the OECD (IRP, 2019; OECD, 2018; WEF et al., 2016). These institutions focus mostly on new technologies, innovations and business models as avenues for green growth, without mentioning social justice and participatory governance. Circularity 1.0 and 2.0 concepts fall within the Technocentric Circular Economy discourse type, which has a lot in common with Audet's (2016) technocentrist discourses. The name, of this discourse type, in fact, derives from Audet's (2016) technocentrists, combined with the circular economy concept as described in Section 4.1. 4.2.4. Fortress circular economy Segmented and sceptical discourses have a vision of a future where scarce resources, overpopulation and biophysical limits require strong cohesive measures. These discourses thus seek to impose sufficiency, population controls and resource efficiency from the top down to ra- tionally confront global scarcity and limits, yet they do not deal with questions of wealth distribution and social justice. This is evident in the texts of precursors such as Catton (1980), Ehrlich (1968), and Hardin (1968), all of which build on Malthusian theories of over- population and resource scarcity to advocate for strong population control and materials efficiency strategies. This discourse type thus considers solutions throughout the entire CE value retention hierarchy (R0-R9). These positions have often been criticised as sexist, elitist, and ethnocentric as they involve white, male, scientists from the Global North imposing sufficiency and limits to populations, which for the most part, had very little to do with the crisis at hand (Dryzek, 2013; Fressoz and Bonneuil, 2016). These type of discourses have also his- torically existed in authoritarian regimes such as in Nazi Germany and the German Democratic Republic, which developed "Kreislauföko- nomie" (circulatory economy) policies to conserve and recycle resources in conditions of geopolitical conflict, economic strife and resource scarcity (Corvellec et al., 2020). More recently, the ecological concerns and resource limits of the 21st century have led to a growing “disaster It is also worth noting that there has been a shift in circularity discourses through time. A plurality of Transformational Circular Society discourses prevailed in the 1960s and 1970s, as most precursors had a strong understanding of planetary limits and comprehensively ad- dressed the main social and systemic challenges of a circular future. Later, in the 1990s and early 2000s, Technocentric Circular Economy discourses of circularity 1.0 and 2.0 became dominant and focused on sophisticated technical innovations instead of wholescale socio-political transformations. This is closely synchronous with the rise of neoliber- alism and its market-based approach to environmental issues. Since the 2008 economic crisis, Reformist and Transformational Circular Society discourses have become more widespread, showing a slowing faith in the market and a re-examination of the socio-political dimensions of circularity. 12 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. Table 2 Concepts within each circularity discourse type. Discourse Concepts from the Timeline Reformist Circular Society (20 concepts) N. 20 Circularity 3.1 concepts: 1. 4.2.4. Fortress circular economy Rio Declaration on Environment and Development (UN, 1992) 2. Regenerative design (Lyle, 1994) 3. Natural Capitalism (Hawken et al., 1999) 4. Sound Material-Cycle Society (Government of Japan, 2000) 5. Cyclical Economy (Young et al., 2001) 6. Materials Matter (Geiser, 2001) 7. Cradle to Cradle (McDonough and Braungart, 2002) 8. The Natural Step (Robèrt, 2002) 9. Performance Economy (Stahel, 2010) 10. Blue economy (Pauli, 2010) 11. Material Efficiency (Allwood et al., 2011) 12. Third industrial revolution (Rifkin, 2013) 13. Eco-system economy (Scharmer and Kaufer, 2013) 14. Regenerative capitalism (Fullerton, 2015) 15. Sharing Economy (Frenken, 2017) 16. Doughnut Economics (Raworth, 2017) 17. Symbiotic Economy (Delannoy, 2017) 18. Social Circular Economy (Social Circular Economy, 2017) 19. Spiral Economy (Ashby et al., 2019) 20. Coviability (Barrière et al., 2019) Transformational Circular Society (30 concepts) N. 13 precursor concepts: 1. Gandhian economics (Kumarappa, 1945) 2. The Economics of the Coming Spaceship Earth (Boulding, 1966) 3. The entropy law and the economic process (Georgescu- Roegen, 1971) 4. The Closing Circle (Commoner, 1971) 5. Social Ecology (Bookchin, 1971) 6. Ecological Design (Papanek, 1972) 7. Limits to Growth (Meadows et al., 1972) 8. Small is Beautiful (Schumacher, 1973) 9. Conviviality (Illich, 1973) 10. Steady-state economics (Daly, 1977) 11. Permaculture (Mollison and Holmgren, 1978) 12. Décroissance (Gorz, 1980) 13. Deep Ecology (Næss and Rothernberg 1989) N. 17 Circularity 3.2 concepts: 1. Transition Movement (Hopkins, 2008) 2. Degrowth (Latouche, 2009) 3. Eco-socialism (Löwy, 2011) 4. Laudato Si’ (Pope Francis, 2015) 5. Transition design (Irwin, 2015) 6. Economy for the Common Good (Felber, 2015) 7. Post-growth (Jackson, 2016) 8. Permacircular Economy (Bourg, 2018), 9. Voluntary Simplicity (Trainer and Alexander, 2019) 10. Convivalism (Caillé, 2019) 11. Buen Vivir/ Sumark Kawsay (Government of Ecuador, 2008) 12. Ubuntu (Shumba, 2011) 13. Ecological Civilization (Zhang et al., 2011) 14. Ecological Swaraj (Kothari et al., 2014) 15. Suma Qamaña / Vivir Bien (Artaraz and Calestani, 2015) 16. Buddhist, Confucian and Taoist ecology (Arler, 2018) 17. Radical Pluralism/ Pluriverse (Kothari et al., 2019) Technocentric Circular Economy (19 concepts) N. 15 concepts from Circularity 2.0: 1. Industrial Ecology (Frosch and Gallopoulos, 1989) 2. Circular Economy (Pearce and Turner, 1989) 3. Eco-design /Design for environment (Ryan et al., 1992) 4. Cyclic Economy (Tibbs, 1993) 5. Industrial Metabolism (Ayres and Simonis, 1994) 6. Cleaner Production (Baas, 1995) 7. Reverse Logistics (Rogers and Tibben-Lembke, 1998) 8. Eco-industrial parks and networks (Côté and Cohen- Rosenthal, 1998) 9. Biomimicry (Benyus, 1998) 10. 5. Discussion Technocentric Circular Economy discourses focus on implementable technical innovations, which can transform the industrial production system without having to change social-economic power relations. Technocentric Circular Economy visions are thus practical and applicable, which makes them appealing for a broad range of actors seeking win- win solutions to reconcile environmental and economic objectives. However, Technocentric Circular Economy discourses fail to deal with all challenges identified in Section 3, as they do not address issues of en- tropy, planetary limits, rebound effects, and the social implications of circularity, and only sometimes deal with challenge 2 on the resource nexus. They thus fall within complexity level 1 to 3 of Table 1, de- pending on their spatial and temporal scale and their perspective on the resource nexus. Technocentric Circular Economy discourses might hence be unappealing to social and environmental groups seeking a more holistic, inclusive and systemic response to the socio-ecological chal- lenges of the 21st century. This paper's approach involved first investigating the current limits of the discussion on the CE (Section 3.1), and then recognising that the term is actually much older and much more diverse than what is usually conceived (Section 3.2). Through this broader view of circularity as an umbrella concept, the authors developed a new circularity discourse typology, which attempts to unpack and navigate the full complexity of its ideas (Section 4). This section reflects on the conceptual and methodological implications of this research. Table 2 N. 3 precursor concepts: 1. The tragedy of the Commons (Hardin, 1968) 2. The Population Bomb (Ehrlich, 1968) 3. Overshoot (Catton, 1980) N. 3 precursor concepts: 1. The tragedy of the Commons (Hardin, 1968) 2. The Population Bomb (Ehrlich, 1968) 3. Overshoot (Catton, 1980) presented in Section 4.1 (please see Table 1). presented in Section 4.1 (please see Table 1). 4.2.4. Fortress circular economy Product Service System (Goedkoop et al., 1999) 11. Extended Producer Responsibility (Lindhqvist, 2000) 12. Industrial Symbiosis (Chertow, 2000) 13. Closed-loop Supply Chain (Guide et al., 2003) 14. Biobased Economy / Bioeconomy (OECD, 2004) 15. The Biosphere Rules Unruh, 2008 N. 4 Circularity 1.0 concepts: 1. Waste-Water Treatment (Holcomb, 1970) 2. Integrated Solid Waste Management and Recycling (Levick and Davies, 1975) 3. Bio-Digestion (Hughes, 1975) 4. Energy Recovery (Boyle, 1977) Fortress Circular Economy (3 concepts) N. 3 precursor concepts: 1. The tragedy of the Commons (Hardin, 1968) 2. The Population Bomb (Ehrlich, 1968) 3. Overshoot (Catton, 1980) 5.2. Methodological implications and challenges of building a discourse typology They address some systemic chal- lenges (1, 2 and 3), but instead of proposing socially desirable solutions, they seek to manage and/or take advantage of the crisis in a top-down manner. Fortress Circular Economy discourses are thus clearly not as universally desirable as they do not address social, cultural and gov- ernance considerations (challenges 4 and 5). However, they nonetheless play a key role in shaping circularity debates, especially in geostrategic policy and business circles as they are the most realistic of all discourses due to their rational and unidealistic understanding of systemic con- ditions. As the socio-ecological crisis of the Anthropocene worsens and climate change intensifies, Fortress Circular Economy positions might become much more widely accepted, especially in a context where “it is easier to imagine an end to the world than an end to capitalism”16 (Fisher, 2009). Yet, these discourses can easily lead to a divided and unequal world of haves and have-nots; a type of eco-apartheid (Malleson, 2016) (or fortress Europe/North America) where only a few nations can invest in new circular solutions and gain access to the technologies and means to a materially affluent life (Monsaingeon, 2017; Valenzuela and Böhm, 2017). In fact, in a 2019 report, Philip Alston, the UN Special Rapporteur on extreme poverty and human rights, has already spoken of a “climate apartheid scenario in which the wealthy pay to escape overheating, hunger, and conflict, while the rest of the world is left to suffer” (Alston, 2019, p. 14). While 26 of the 72 concepts in the timeline (36%) were from non- academic origins18, and various circularity positions from government, civil society or business practitioners were reviewed (E.g., European Commission, 2015; IRP, 2019; OECD, 2018; Schwartz and Randall, 2003; Verstraeten-Jochemsen et al., 2018; von Weizsäcker and Wijkman, 2017; WEF et al., 2016), the majority of literature in this research originates from academia. This represents another limitation of this paper. Future research can use the typology as a theoretical framework to analyse discourses from the public and private sectors. This could help uncover different circularity discourses and better un- derstand their main strengths and weaknesses as well as test, improve and update the discourse typology here proposed. 5.2. Methodological implications and challenges of building a discourse typology 5.2. Methodological implications and challenges of building a discourse typology There are inevitable simplifications involved in the development of a discourse typology. Each concept within the typology is thus much more diverse than can be evidenced in this research. For instance, there is a large diversity of visions of Degrowth (Kallis et al., 2018) and the Bioeconomy (Hausknost et al., 2017). This discourse typology cannot distinguish all the intricate sub-types within each discourse, rather it shows their commonalities and simplifies their complexity so they can be understood in relation to other discourses. Further research can build on this to analyse how different academic, public and private discourses fit within this discourse typology. In addition to this, Table 1 can help distinguish the diversity of Technocentric Circular Economy proposals, by classifying them in complexity levels 1 to 3. Transformational Circular Society discourses address all the 5 chal- lenges identified in Section 3 by seeking a wholescale transformation of the entire socio-economic system and not just its industrial model. They are thus in complexity level 5 of Table 1. Transformational Circular Society discourses have a rational analysis of current planetary limits and the structural contradictions of the capitalist system and propose a utopic vision that is appealing for many social and environmental movements. However, they might be disregarded by mainstream de- bates for being overly idealist regarding the likelihood of fundamental socio-cultural change and the probability of a post-capitalist future. Some discourses might not always be easily distinguishable black or white propositions and could present multiple shades and nuances. Indeed, some discourses could be “hybrids”, which include elements of a number of the 4 discourse types presented in this article. Further case study research on specific circular sectors or stakeholders, using this typology should acknowledge this complexity in their analysis of cir- cular discourses, policies and practices. A particular stakeholder might thus have a prominently Technocentric Circular Economy discourse with moderate notes of Reformist Circular Economy. Moreover, some actors might have discrepancies between their practices and their discourses, such as having a Reformist Circular Society discourse and Fortress Circular Economy practices. Future research can address these complexities by combining this typology with methodological tools such as Q-methods and corpus-based discourse analysis. In contrast to this, Fortress Circular Economy discourses are neither optimist about the possibility of eco-economic decoupling nor about fundamental socio-cultural change. 17 It is worth noting that the timeline does not strictly follow the dates pro- posed by Reike et al. (2018) or Blomsma and Brennan (2017) as it is a new proposition. Moreover, each time period overlaps, showing that ideological phases do not necessarily have strict start and end date but rather gradients with moments of prominence and decline. A detailed and interactive version of this timeline is available in the following web-page: http://cresting.hull.ac.uk/ impact/circularity-timeline. 18 Waste-Water Treatment, Solid Waste Management and Recycling, Bio- Digestion, Energy Recovery, Cyclic Economy, Reverse Logistics, Biomimicry, Product Service System, Extended Producer Responsibility, Bioeconomy, Rio Declaration on Environment and Development, Natural Capitalism, Sound Material-Cycle Society, Cyclical Economy, The Biosphere Rules, Performance Economy, Blue economy, Buen vivir/Sumak Kawsay, Regenerative capitalism, Social Circular Economy, Transition Movement, Laudato Si', Economy for the Common Good, Ubuntu, Ecological Civilization, Suma Qamaña/Vivir Bien. 16 Fisher attributes this quote to both Fredric Jameson and Slavoj Žižek (Fisher, 2009) 17 It is worth noting that the timeline does not strictly follow the dates pro- posed by Reike et al. (2018) or Blomsma and Brennan (2017) as it is a new proposition. Moreover, each time period overlaps, showing that ideological phases do not necessarily have strict start and end date but rather gradients with moments of prominence and decline. A detailed and interactive version of this timeline is available in the following web-page: http://cresting.hull.ac.uk/ impact/circularity-timeline. 18 Waste-Water Treatment, Solid Waste Management and Recycling, Bio- Digestion, Energy Recovery, Cyclic Economy, Reverse Logistics, Biomimicry, Product Service System, Extended Producer Responsibility, Bioeconomy, Rio Declaration on Environment and Development, Natural Capitalism, Sound Material-Cycle Society, Cyclical Economy, The Biosphere Rules, Performance Economy, Blue economy, Buen vivir/Sumak Kawsay, Regenerative capitalism, Social Circular Economy, Transition Movement, Laudato Si', Economy for the Common Good, Ubuntu, Ecological Civilization, Suma Qamaña/Vivir Bien. 5.1. Conceptual implications of each circularity discourse type Each one of the above circularity discourse types has different conceptual strengths and weaknesses, especially in relation to the 5 main challenges identified in Section 3.1 (1. growth, entropy and de- coupling, 2. materials-energy-biodiversity nexus, 3. CE impact-assess- ment and the rebound-effect, 4. socio-political implications of the CE, 5. alternative visions of circularity) as well as the 5 levels of complexity Reformist Circular Society discourses answer many of these concerns, particularly in terms of social justice, participatory governance and the 13 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. enclosure, commodification, and marketization of nature and the commons, which replicates global environmental injustices and ends up making circularity “a luxury” (Schröder et al., 2019b, p13). resource nexus (challenges 2 and 4) and sometimes acknowledge the issues revolving around the rebound effect (challenge 3). Reformist Circular Society discourses thus perhaps have the most largely appealing vision of circularity, as they add a human dimension and seek to re- concile capitalism with a just and sustainable future for all. However, these discourses fail to confront questions of entropy, economic growth, decoupling and epistemological and ontological pluralism (challenges 1 and 5). Reformist Circular Society discourses are thus within complexity level 4 of Table 1 and might be unappealing to social movements with plural and ecocentric perspectives. References Albarillo, F., 2014. Language in Social Science Databases: english Versus Non-English Articles in JSTOR and Scopus. Behav. Soc. Sci. Libr. 33, 77–90. https://doi.org/10. 1080/01639269.2014.904693.fi Alexander, S., 2015. Sufficiency economy: enough, for Everyone. Forever. Simplicity Institute Publishing, Melbourne, Australia.fi Allwood, J.M., Ashby, M.F., Gutowski, T.G., Worrell, E., 2011. Material efficiency: a white paper. Resour. Conserv. Recycl. 55, 362–381. https://doi.org/10.1016/j. resconrec.2010.11.002. Alston, P., 2019. Climate change and poverty : report of the special rapporteur on extreme poverty and human rights 21 p. Ampe, K., Paredis, E., Asveld, L., Osseweijer, P., Block, T., 2019. A transition in the Dutch wastewater system? The struggle between discourses and with lock-ins. J. Environ. Policy Plan. 1–15. https://doi.org/10.1080/1523908X.2019.1680275. One core challenge and implication of these results is that eviden- cing discursive differences could bring opposing discourses apart, ra- ther than together. Research on deliberative democracy and colla- borative decision making has shown that a better understanding of conflicting ideas can actually promote respect, trust, innovation, and consensual cooperation (Calisto Friant, 2019; Dryzek et al., 2019; Friend and Hickling, 2005; Schwarz and Thompson, 1990). By un- packing and navigating different discourses of circularity, this article thus hopes to promote greater inclusiveness, collaboration and plur- alism in the debate and implementation of this contested paradigm. Antikainen, R., Lazarevic, D., Seppälä, J., 2018. Circular Economy: origins and Future Orientations. In: Lehmann, H. (Ed.), Factor X Challenges, Implementation Strategies and Examples for a Sustainable Use of Natural Resources, pp. 115–129. https://doi. org/10.1007/978-3-319-50079-9_7. Arler, F., 2018. Revitalizing traditional chinese concepts in the modern ecological civi- lization debate. Open J. Philos. 8, 102–115. https://doi.org/10.4236/ojpp.2018. 82009. É Arnsperger, C., Bourg, D., 2017. Écologie intégrale : Pour Une société Permacirculaire. PUF, Paris. Artaraz, K., Calestani, M., 2015. Suma qamaña in Bolivia: indigenous understandings of well-being and their contribution to a post-neoliberal paradigm. Lat. Am. Perspect. 42, 216–233. https://doi.org/10.1177/0094582X14547501. Ashby, A., Callegaro, A.M., Adeyeye, K., Granados, M., 2019. The Spiral Economy: A Socially Progressive Circular Economy Model? Springer, Cham, pp. 67–94. https:// doi.org/10.1007/978-3-030-15066-2_5. The authors encourage the use, adaptation and improvement of this discourse typology to further build on this work, which can be seen as a continuous participatory thought process with other scholars and practitioners. 5.2. Methodological implications and challenges of building a discourse typology Considering that the CE is still a contested concept with many public and private actors competing to influence its meaning and interpretation, this is a parti- cularly important avenue for future research.i f Considering the strengths and weaknesses of each circularity dis- course, there is great value in opening up the debate on circularity to allow for a more complex discussion of the core challenges of a circular future. Indeed, some discourses might lack realism, while others might lack feasibility. A cross-pollination of ideas and perspectives is thus beneficial to develop better policies, practices and research projects. Future transdisciplinary research and participatory policymaking with deliberative mechanism can help bring all discourses to the table and establish more inclusive, legitimate, achievable and sustainable circular futures. On the other hand, if a more plural debate on circularity is not held, there is a high risk that a depoliticised discourse of circularity dilutes the complexity of the present socio-ecological crisis. This simple and uncontroversial discourse of circularity could create many new business opportunities for some, by expanding capital accumulation into the realm of waste materials and bio-resources. Yet, it will likely create a rebound effect and, thus, do very little towards actually re- ducing humanity's ecological footprint. Moreover, it can lead to the A final limitation of this research is that it is mostly based on desk research. Another method to build a discourse typology could be 14 Resources, Conservation & Recycling 161 (2020) 104917 M. Calisto Friant, et al. interests or personal relationships that could have appeared to influ- ence the work reported in this paper. through a set of workshops and focus group discussions with various practitioners and academics. The limitations of a participatory ap- proach are the complexities in obtaining a diverse enough sample of stakeholders and adequately covering discourses from many different countries and continents. They can thus easily result in typologies that only apply to certain cultural or geographical contexts or only represent a limited range of discourses. Nonetheless, for future research on the topic, there is significant potential to hold participatory workshops where the discourse typology here developed is used as the theoretical basis to analyse the discourse and practices of specific CE sectors and actors. This would allow for innovative research that unpacks different practitioner discourses of the CE and can help validate, adapt and im- prove the discourse typology here developed. Acknowledgements This research was funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 765198. A previous version of this paper was presented at the 25th International Sustainable Development Research Society (ISDRS) Conference in Nanjing, China on the 27th of June 2019 and was published in its proceedings with the following title: “advan- cing a critical research agenda on the circular economy”. We would like to thank the anonymous reviewers for their detailed and helpfull comments and suggestions and well as Ton Markus for the design of the figures. We would also like to thank Pauline Deutz, Kieran Campbell- Johnston, Kaustubh Thapa and Natasha Klein for their constructive reviews of earlier versions of this paper. Finally, we would like to thank our peers from the CRESTING project, the International Sustainable Development Research Society and the Copernicus Institute of Sustainable Development, which provided invaluable comments and suggestions during workshops where earlier versions of this research were presented. 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https://openalex.org/W2911843571	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0210949&type=printable	English	null	Enhanced activity of pyramidal neurons in the infralimbic cortex drives anxiety behavior	PloS one	2,019	cc-by	12,292	RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Berg L, Eckardt J, Masseck OA (2019) Enhanced activity of pyramidal neurons in the infralimbic cortex drives anxiety behavior. PLoS ONE 14(1): e0210949. https://doi.org/10.1371/ journal.pone.0210949 Citation: Berg L, Eckardt J, Masseck OA (2019) Enhanced activity of pyramidal neurons in the infralimbic cortex drives anxiety behavior. PLoS ONE 14(1): e0210949. https://doi.org/10.1371/ journal.pone.0210949 Editor: Alexandra Kavushansky, Technion Israel Institute of Technology, ISRAEL Received: August 17, 2018 Accepted: January 6, 2019 Published: January 24, 2019 Received: August 17, 2018 Accepted: January 6, 2019 Published: January 24, 2019 Laura Berg1, Josephine Eckardt2, Olivia Andrea MasseckID1,3* 1 Advanced Fluorescence Microscopy, Ruhr University Bochum, Bochum, Germany, 2 Department of Systems Neuroscience Ruhr University Bochum, Bochum, Germany, 3 University of Bremen, Synthetic Biology, Bremen, Germany * masseck@uni-bremen.de * masseck@uni-bremen.de a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract We show that in an animal model of anxiety the overall excitation, particularly in the infralim- bic region of the medial prefrontal cortex (IL), is increased and that the activity ratio between excitatory pyramidal neurons and inhibitory interneurons (AR PN/IN) is shifted towards exci- tation. The same change in AR PN/IN is evident for wildtype mice, which have been exposed to an anxiety stimulus. We hypothesize, that an elevated activity and the imbalance of excitation (PN) and inhibition (IN) within the neuronal microcircuitry of the prefrontal cortex is responsible for anxiety behaviour and employed optogenetic methods in freely moving mice to verify our findings. Consistent with our hypothesis elevation of pyramidal neuron activity in the infralimbic region of the prefrontal cortex significantly enhanced anxiety levels in several behavioural tasks by shifting the AR PN/IN to excitation, without affecting motor behaviour, thus revealing a novel mechanism by which anxiety is facilitated. Enhanced activity of pyramidal neurons in the infralimbic cortex drives anxiety behavior Laura Berg1, Josephine Eckardt2, Olivia Andrea MasseckID1,3* Introduction Anxiety disorders belong to the most common mental illnesses within the western world affecting a total amount of about 20% of the human population, a lifetime prevalence of nearly 30% and with a steadily increasing occurrence [1–3]. One of the key areas involved in the expression of anxiety and fear, beside the hippocampus and the amygdala, is the infalimbic region (IL) of the medial prefrontal cortex (mPFC) [4–9], which is the rodent homolog to the anterior cingulate cortex of humans. In contrast to fear, which is a specific in time limited response to a threatening stimulus, often investigated with fear conditioning, anxiety is a sus- tained negative emotional state, in which unknown or uncertain situations are encountered with defensive behaviour [10]. Copyright: © 2019 Berg 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. Enhanced activity of pyramidal neurons drives anxiety effects, i.e. anxiogenic or anxiolysis, on innate fear and learned fear, pointing to a highly com- plex role of the mPFC in anxiety [15,16]. One striking hypothesis for neuronal mechanism underlying various psychiatric diseases, is that changes in cortical excitation inhibition (E/I) balance are responsible for observed cogni- tive deficits [3]. Neuromodulatory systems, such as the noradrenergic or in particular the serotonergic sys- tem seem to contribute to the development and manifestation of anxiety [17,18]. For example, anxiety disorders such as panic, posttraumatic stress, social phobia or obsessive-compulsive disorder are commonly treated by selective serotonin reuptake inhibitors (SSRIs) suggesting an important role of 5-HT in these diseases [19]. As the serotonergic system projects to all parts of the well known tripartite anxiety circuitry, namely the ventral Hippocampus, the Amygdala and the prefrontal cortex [4,20–22]it is likely that the serotonergic system might be potent modulator of anxiety behaviour. The mPFC receives massive input from the seroto- nergic system and 5-HT affects postsynaptic excitatory, as well as inhibitory neurons mainly by 5-HT1A receptors [8,17]. Low levels of 5-HT1A receptor have been associated with the manifestation and development of anxiety disorders [18] and 5-HT1A knock mice are a well established genetic model of anxiety [23–25]. A previous study indicates that 5-HT1A heterore- ceptor expression in cortex and hippocampus is primarily responsible for the anxiety pheno- type [26]. In conjunction, 5-HT1A knock out mice show abnormalities within the GABA- glutamate system of the prefrontal cortex [27] pointing again to an imbalance of excitatory and inhibitory neurotransmission within the prefrontal cortex, probably mediated by distur- bances in the 5-HT system. However, so far the underlying neuronal mechanisms are still largely unknown and despite numerous studies on 5-HT signalling and anxiety, their involve- ment in modulating the ratio between excitatory pyramidal and inhibitory interneurons activ- ity (AR PN/IN) has not been scrutinized. Material and methods All experiments in this study were approved by the Institutional Animal Research Facility (Lande- samt fu¨r Natur, Umwelt und Verbraucherschutz NRW). Mice were housed in standardized cages and maintained on a 12:12 (h) light-dark cycle (lights on from10.00 pm-10.00am) in a tempera- ture and humidity controlled scantainer. Food and water were available ad libitum. All behavioral experiments were performed in the main activity phase of the animals. Experiments were per- formed in C56Bl/6J mice (Jackson Laboratory) and in two different transgenic mousselines with a C56Bl/6J background obtained from collaborators. For characterizing the activity pattern of dif- ferent neuron types during anxiety 5-HT1A(-/-) mice (kindly provided by L. Tecott, University of California San Fransisco)[23] were used. To specifically express ChR2 or a fluorescent control plasmid (td-tomato) in pyramidal neurons Nex-Cre(-/-) mice were used (kindly provided by K. Nave and S. Goebbels, Max Planck Institute fu¨r Experimentelle Medizin) [28]. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work received support from a Grant awarded to Olivia Masseck (MA4692/3-2) from the DFG, http://www.dfg.de/. The funders had no role in study design, data collection and analysis, decision t publish, or preparation of the manuscript. Studies have repeatedly shown, that neuronal excitability is increased in the prefrontal cor- tex of patients with anxiety disorders and in animal models of anxiety [9,11,12]. Consequently, mainly disturbances in GABAergic neurotransmission have been associated with the manifes- tation and appearance of anxiety disorders [4,13]. Treatments of choice for generalized anxiety disorders are in many cases benzodiazepine, which act on GABAergic receptors and enhance inhibitory neurotransmission in general [14]. But also contradictory result have been obtained, lesions of the mPFC, or inhibition did not affect anxiety behaviour at all or elicit even opposing Competing interests: The authors have declared that no competing interests exist. 1 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Enhanced activity of pyramidal neurons drives anxiety All surgical procedures were performed under anesthesia with Isofluran (Initial 5%, 2% for surgery). Pressure injections of virus or tracer, by customized glass pipettes (tip diameter 10μm), were performed at various depths to ensure proper virus spreading within the target area. For optogenetic experiments a customized ceramic ferrule (Thorlabs, 1,25mm diameter with 230μm bore hole, CFLC230-10) with an opitical fiber (Thorlabs, FT200EMT, length 1.8mm, diameter 200μm) was implanted directly above the IL region for optical stimulation. Therefor the skull was prepared with the primer and adhesive of Optibond FL (Kerr) and the implant was fixed with Gradia direkt—Flo dental cement (Henry Schein, 103322) and cured with UV light. After surgery animals received subcortical injection of Carprofen (2 mg/kg) for analgesia and 0.1ml glucose. Animals were from now on housed individually and were allowed to recover at least for 14 days before behavioral experiments were performed. To identify pro- jections between the prefrontal cortex and the serotonergic system and vice versa, as also between the prefrontal cortex and the amygdala, we injected Fluorogold dissolved in NaCl into the dorsal raphe, the IL region or the amygdala respectively. Mice were perfused after 5–7 days of retrograde transport and injection sites were verified by fluorescence microscopy and immunhistochemistry. Viral vectors and surgery 3–6 month old C56Bl/6J mice were stereotaxically injected with Fluorogold (Thermo Fisher, H22845) for retrograde tracing into the dorsal raphe nucleus (AP -4.5 mm, ML 0 mm, DV -2.5mm, -2mm, -1.5mm), the IL region (AP + 1.66 mm, ML 0.3 mm left, DV -2mm, -1.8mm, -1.6mm) or the amygdala (AP -1.58 mm, ML -3 mm left, DV -3.8mm). Nex-Cre(-/-) mice were injected with AAVs, encoding for Cre depended expression to target ChR2 (AAV9.EF1a. DIO.hChR2(H134R)-eYFP.WPRE.hGH (Adgene 20298, Penn Vector Core)) or td-tomato (pAAV1.CAG.FLEX.tdTomato.WPRE.bGH (Penn Vector Core, Adgene 51503)) to pyramidal neurons in the IL region. 2 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Behavioral experiments We used male mice for all behavioral experiments. Before implantation mice were housed in groups. Each mouse was tested only once in each behavioral test and at least one week was in between different anxiety tests. Trials between groups were always interleaved. Elevated-Plus maze. For the Elevated-Plus Maze 5-HT1A(-/-) mice were transported indi- vidually from the mouse facility to the experimental room. Prior to the experiment mice were marked with a green label to be later recognized by the tracking software (TDT). The Elevated- Plus maze consists of two open (total length 33,5cm, 5cm wide) and two closed arms (total length 33,5cm with a 17cm high wall, 5cm wide) all connected to a center platform in the mid- dle. After 10 minutes of habituation to the experimental room, mice were placed at the center of the maze facing the open arm directed to the experimenter. Its behavior was recorded for 5 minutes and analyzed by a customized Matlab routine. For optogenetic stimulation of pyrami- dal neurons in the cortex of Nex-Cre(-/-) mice, optogenetic implants were connected via a sleeve and optical cable to the light source (473 nm LED, Pizmatix, Optogenetics-LED-STSI). After habituation in an extra waiting cage (10 min) mice were placed in the center of the EPM, directly facing the experimenter. The behavior was recorded and analyzed by Ethovision XT (Noldus) for 18 minutes with alternating 3-minutes off and on trials [31]. In three trials (1,3,5) no light stimulation occurred, whereas in all remaining trials (2,4,6) light stimulation was on. Videos were analyzed by the tracking software EthoVision XT. Time mice spend on the open arms, open arm entries and distance moved in total was quantified. Open-Field. For the openfield test mice were transported individually from the mouse facility to the experimental room. Optogenetic implants were connected via a sleeve and opti- cal cable to the stimulating light source (473 nm LED, Prizmatix, Optogenetics-LED-STSI). After connecting animals to the light stimulation fiber, mice were allowed to habituate in an extra waiting cage, which was positioned outside of the testing arena. The Open-Field arena is a squared plexiglasbox with sandblasted walls (50x50x50cm). After 10 minutes of habituation mice were placed into the left lower corner, facing towards the cen- ter. In total, mice behavior was recorded for 20min. Obtained data was analyzed offline by EthoVision XT (Noldus). Immunohistochemistry For immunohistochemical analysis mice were perfused transcardial after exposure to different conditions: 5-HT1A(-/-) mice and Wt littermates were either sacrificed naïve, i.e. directly from their homecages, or 90 minutes after a 5 minute exposure to the Elevated-Plus maze (anxiety stimulus). Nex-Cre(-/-) mice were either sacrificed naïve, 90 after 5 minutes exposure to the Open-Field, or 90 minutes after 5 minutes within the Open-Field plus light stimulation (473 nm, 20Hz) [29,30]. Mice were euthanized with an overdose of Ketamin (0,33mg/g) i.p. and brains were post fixed in 4% PFA in PBS overnight and processed in 30% sucrose for cryosectioning. Obtained brain slices (40μm) were washed 3x times in PBS. After blocking in 3% donkey serum in 0.3% PBST slices were stained overnight in 1,5% donkey serum in 0.3% PBST at room temperature with rabbit-anti-c-Fos (226003, Synaptic Systems), mouse-anti-Gad67 (MAB5406, Merck Millipore) and sheep-anti-TPH (MBS502127, MyBioSoure) for the DRN and rabbit-anti-c-Fos (226003, Synaptic Systems, 1:1000), mouse-anti-Gad67 (MAB5406, Merck Millipore, 1:500) and goat-anti-CamKIIa (sc-5391, Santa Cruz Biotechnology, 1:300) for the IL region and amygdala. The next day, after 3 additional washing steps in PBS, slices were incubated, for 1 h at room temperature, with the secondary antibody. For the 5-HT1A(-/-) mice Cye3-donkey-anti-rabbit (711-165-152, Jackson ImmunoResearch, 1:500), Alexa488-donkey-anti-sheep (A-11015, Thermo Fisher, 1:500) and DyLight647-donkey-anti-mouse (715-605-150, Jackson ImmunoR- esearch, 1:500) for the DRN and Cye3-donkey-anti-rabbit (711-165-152, Jackson ImmunoRe- search, 1:500), Alexa488-donkey-anti-goat (A-11055, Thermo Fisher, 1:500) and DyLight647- donkey-anti-mouse (715-605-150, Jackson ImmunoResearch, 1:500) for the IL region. For the Nex-Cre(-/-)-ChR2-eYFP mice it was DyLight405-donkey-anti-sheep (DkxSh-003- F405NHSX, ImmunoReagents, 1:500), DyLight550-donkey-anti-rabbit (SA5-10039, Thermo Fisher, 1:500) and Alexa647-donkey-anti-mouse (715-605-150, Jackson ImmunoResearch, 1:500) for the DRN and DyLight405-donkey-anti-goat (705-475-003, Jackson ImmunoRe- search, 1:500), DyLight550-donkey-anti-rabbit (SA5-10039, Thermo Fisher, 1:500) and Alex- a647-donkey-anti-mouse (715-605-150, Jackson ImmunoResearch, 1:500) for the IL region and the amygdala. For the Nex-Cre(-/-)-dtomato mice it was DyLight405-Donkey-anti-sheep (DkxSh-003-F405NHSX, ImmunoReagents, 1:500), DyLight488-donkey-anti-mouse 3 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Enhanced activity of pyramidal neurons drives anxiety (DkxMu-003-D488NHSX, ImmunoReagents, 1:500) and Alexa647-donkey-anti-rabbit (711- 605-152, Jackson ImmunoResearch, 1:500) for the DRN and DyLight405-donkey-anti-goat (705-475-003, Jackson ImmunoResearch, 1:500), DyLight488-donkey-anti-mouse (DkxMu- 003-D488NHSX, ImmunoReagents, 1:500) and Alexa647-donkey-anti-rabbit (711-605-152, Jackson ImmunoResearch, 1:500) for the IL region and the amygdala. Slices were mounted on Superfrost Plus microscope slides (J1800AMNZ, Thermo Scien- tific) with Roti-Mount FluorCare (HP19.1, Carl Roth) and Cover slips (24x60 mm #1, Menzel- Gla¨ser) and analyzed with a laser scanning confocal microscope (SP5, or SP8, Leica). Immunohistochemistry Brian slices from fluorogold injected brains were either directly mounted on Superfrost Plus microscope slides (J1800AMNZ, Thermo Scientific) with Roti-Mount FluorCare contain- ing DAPI (HP19.1, Carl Roth) and Cover slips (24x60 mm #1, Menzel-Gla¨ser), or stained against TPH in the DRN or CamKII in amygdala and IL with the same antibodies as for the Nex-Cre mice. Enhanced activity of pyramidal neurons drives anxiety Novelty-Suppressed feeding. For the Novelty-Suppressed Feeding test mice were deprived from food for 24h but had access to water ad libitum. On the testing day mice were transported individually from the facility to the experimental room. Optogenetic implants were connected via a sleeve and optical cable to the stimulating light source (473 nm LED, Prizmatix, Optogenetics-LED-STSI). After connecting to the light stimulation fiber, mice were allowed to habituate in an extra waiting cage, which was positioned outside of the testing arena. After 10 minutes of habituation mice were placed in the left lower corner of the Open field arena (see openfield test). The floor of the openfield was covered with litter and a standard food pellet, situated on a 5x5 cm filter paper, was placed in the middle of the center. Record- ings were stopped as soon as mice started feeding, defined as mice holding the food pellet in their fore pads and biting into it. During the experiment mice were either stimulated by light (20Hz, 1,mW) (Liu et al. 2014, Zhao et al., 2011), or only connected to the optical fiber without performing light stimulation. Subsequently, mice were allowed to eat their food pellet for addi- tional 5 minutes in their home cage. Afterwards the food pellet was weighted to calculate the amount of food intake. For analysis time to feed, and time in the center of the openfield during the first minute were taken into consideration. Data analysis. Data were analyzed by EthoVision XT (Noldus), ImageJ, SigmaPlot, Sig- maStat. All obtained images were processed in ImageJ for contrast and brightness. Overlays of c-Fos with either Gad67 or CamKIIa/TPH were created and all cells of the specific types were counted in ImageJ, which expressed c-Fos, as also all c-Fos positive cells in total. Heatmaps were produced in EthoVision XT, where color codes represent time spent in each location. Data extraction and collection was done automatically from recorded videos by EthoVision XT. Data were then further analyzed in SigmaPlot and tested for normality. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Behavioral experiments Each experiment consisted of four 5-minute trials with alternating light on and light off [31]. In two trials (2,4) light stimulation was switched on (20Hz, 1mW) [29,30], in the remaining trials (1,3) no stimulation was done. The arena was divided into 16 equal squares, whereby the 4 inner squares build the center (25x25cm). Videos were analyzed automatically with a tracking software (Noldus) for time spend in the center of the open field, entries to the center and distance traveled. 4 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 C-fos expression in the dorsal raphe nucleus during anxiety (C) colocalization of c-fos expression in GABAergic and serotonergic cells (analysis of 42 brain slices from 3 brains per group). N naive, A anxiety. N(Gad67) Wt 3.34±0.37, 1A-ko 5.88±0.53, Mann Whitney Rank Sum test, p = 0.003, A(Gad67) Wt 11.4±1.25, 1A-ko 15.98±1.52, Mann Whitney Rank Sum, p = 0.01, N(TPH) Wt 8.19±0.81, 1A-ko 20.29±1, two tailed t-test t = -8.595, p0.001, A(TPH) Wt 26.69±2.38 1A-ko 60.48±3.58, Mann Whitney Rank Sum test, p0.001. (D) total amount of c-fos expressing cells. Wt naïve 22.14±2.06, 1A-ko naïve 43.43±2.47, two-tailed t-test t = -5.978, p0.001, Wt anxiety 74.47±5.48, 1A-ko anxiety 141.14±7.8 Mann Whitney Rank Sum test p0.001 (E) activity ratio between serotonergic neurons and interneurons (AR 5-HT/ GABA) calculated as ratio between c-fos positive serotonergic and c-fos positive GABAergic neurons. Wt naïve 2.77±0.33, Wt anxiety 2.79 ±0.19, Mann Whitney Rank Sum test, p = 0.372, 1A-ko naïve 4.11±0.27, 1A-ko anxiety 4.76±0.36, Mann Whitney Rank Sum test, p = 0.238. Values are mean ± S.E.M. indicate significant differences (p 0.01), indicate significant differences (p 0.001). https://doi.org/10.1371/journal.pone.0210949.g001 Fig 1. AR 5-HT/GABA in the serotonergic system is not ch HT/GABA in the serotonergic system is not changed due to anxiety. (A) experimental workflow of c-fos stainings. DRN l f k d d G d fl l b l f GA A ( ) T fl l b l f Fig 1. AR 5-HT/GABA in the serotonergic system is not changed due to anxiety. (A) experimental workflow of c-f dorsal raphe nucleus, c fos marked in red, Gad67 fluorescence labeling of GABAergic neurons (green), TPH fluorescence labeling of serotonergic neurons (red). (B) confocal images showing c-fos expression in wildtype (Wt) and 5-HT1A(-/-) knockout mice (1A-ko). C-fos in red. Insets show high magnification. Scale bar 150μm, insets 100 μm. (C) colocalization of c-fos expression in GABAergic and serotonergic cells (analysis of 42 brain slices from 3 brains per group). N naive, A anxiety. N(Gad67) Wt 3.34±0.37, 1A-ko 5.88±0.53, Mann Whitney Rank Sum test, p = 0.003, A(Gad67) Wt 11.4±1.25, 1A-ko 15.98±1.52, Mann Whitney Rank Sum, p = 0.01, N(TPH) Wt 8.19±0.81, 1A-ko 20.29±1, two tailed t-test t = -8.595, p0.001, A(TPH) Wt 26.69±2.38 1A-ko 60.48±3.58, Mann Whitney Rank Sum test, p0.001. (D) total amount of c-fos expressing cells. C-fos expression in the dorsal raphe nucleus during anxiety To test now whether disturbed serotonergic neurotransmission might contribute to changes in cortical activity patterns we performed immunohistochemical analyses of neuronal activity in 5-HT1A(-/-) knockout mice (Fig 1), which exhibit enhanced anxiety levels in comparison to wildtype mice (S1 Fig). We examined anxiety behaviour in two behavioural tests, the Elevated Plus Maze (EPM) and the Novelty Suppressed Feeding test (NSF), both tests revealed enhanced anxiety in 5-HT1A(-/-) knockout mice. In the EPM time spent in open arms (Wt 47.91±6.12s; 1A-ko 31.59±4.34s, p = 0.036) and open arm entries (Wt 10.6±1.25; 1A-ko 7.28 ±0.66, p = 0.015) were significantly decreased (S1B and S1C Fig), while the overall distance moved (Wt 3859.34±979.26cm, 1A-ko 4373±669.08cm, p = 0.487) was not affected at all (S1D Fig). Just like in the EPM also the NSF confirmed enhanced anxiety in 5-HT1A(-/-) knockout mice, time spent in the center of the arena (Wt = 27.32±3.81s, 1A-ko = 17.57±2.6s, p = 0.042) is significantly decreased and time till feeding showed a trend towards longer waiting times (Wt 118.56±19.16s, 1A-ko 146.69±36.67s, p = 0.772) (S1F and S1G Fig). Having confirmed increased anxiety in 5-HT1A(-/-) we started to compare neuronal activ- ity, i.e. c-fos expression, in wildtype and 5-HT1A(-/-) knockout mice in naïve (homecage) and fearful situations (Elevated-Plus Maze) (Fig 1A). For this, animals were subjected to either a neutral situation, i.e. remaining in their homecage (naïve) or underwent an anxiety provoking Elevated-Plus Maze test for 5 min followed by additional 90 min in their homecage, in which c-fos expression reaches its maximum. Brains were removed and coronal sections of the DRN and IL were immunolabelled for the neuronal activity marker c-fos (Fig 1A). In the literature extensive interconnectivity between the serotonergic system, mainly the dorsal raphe nucleus (DRN) and the prefrontal cortex is described [32,33]. To verify this interconnectivity we PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 5 / 19 Enhanced activity of pyramidal neurons drives anxiety Fig 1. AR 5-HT/GABA in the serotonergic system is not changed due to anxiety. (A) experimental workflow of c-fos stainings. DRN dorsal raphe nucleus, c-fos marked in red, Gad67 fluorescence labeling of GABAergic neurons (green), TPH fluorescence labeling of serotonergic neurons (red). (B) confocal images showing c-fos expression in wildtype (Wt) and 5-HT1A(-/-) knockout mice (1A-ko). C-fos in red. Insets show high magnification. Scale bar 150μm, insets 100 μm. https://doi.org/10.1371/journal.pone.0210949.g001 C-Fos expression in the infralimbic cortex in anxiety To test now whether anxiety may originate in the prefrontal cortex we examined c-fos expres- sion in the IL (Fig 2). Again we found a highly significant increase of total c-fos in fearful situa- tion in Wt, as well as in 5-HT1A(-/-) mice (Wt naïve 11.64±0.45, 1A-ko naïve 32.74±1.45, p0.001, Wt anxiety 179.9±4.6, 1A-ko anxiety 211.33±5.36, p0.001.) (Fig 2A–2D). The num- ber of c-fos positive cells is increased in pyramidal (CamkII) and GABAergic interneurons (Fig 2C and 2D). But now a significant change in the ratio between active glutamatergic pyra- midal neurons and active interneurons (AR PN/IN) between naïve conditions and anxiety is evident in Wt mice (Fig 2E). In Wt mice the AR PN/IN is nearly doubled (Wt naïve 2.24±0.17, Wt anxiety 3.8± 0.18, p0.001) during anxiety (Fig 2E), suggesting that a certain balance between excitation and inhibition, in the IL is important in the regulation of anxiety behav- iour. Consequently, in 5-HT1A(-/-) knockout mice this AR PN/IN values are already elevated in naïve conditions and do not further increase in anxiety (1A-ko naïve 5.53±0.42 1A-ko anxi- ety 4.2±0.18, p = 0.291)(Fig 2E).This would suggest that probably enhanced anxiety levels in 1A-ko and Wt mice is triggered by a change in cortical E/I balance. Enhanced activity of pyramidal neurons drives anxiety decided to perform tracer injections in the IL and DRN (S2 Fig). Injections of fluorogold in the IL revealed retrogradly stained neurons in the DRN (S2A Fig) and specific expression of td-tomato in pyramidal neurons of NEX-Cre revealed anterogradly labelled fiber terminals in the DRN (S2B Fig). Vice versa, fluorogold injections in the DRN labelled pyramidal neurons in the IL region (S2C Fig). Once we have revisited extensive connections between the DRN and the IL, we focused our analysis of the activity marker c-fos in naïve and fearful situations first within the DRN. Our analysis revealed an increase of total c-fos expression in TPH and GABAergic neurons in anxiety provoking situations in Wt as well as in 5-HT1A(-/-) mice (Fig 1B, 1C and 1D). Whereby in all conditions and cell types the number of c-fos is increased in 5-HT1A(-/-) mice (Wt naïve 22.14±2.06, 1A-ko naïve 43.43±2.47, p0.001, Wt anxiety 74.47±5.48, 1A-ko anxi- ety 141.14±7.8 p0.001) (Fig 1C and 1D), probably due to missing feedback inhibition of the 5-HT1A receptor [34]. However, ratios between c-fos expressing serotonergic (TPH) and c-fos expressing GABAergic neurons is not changed due to anxiety in Wt (Wt naïve 2.77±0.33, Wt anxiety 2.79±0.19, p = 0.372) as well as in 5-HT1A(-/-) mice (1A-ko naïve 4.11±0.27, 1A-ko 24.76±0.36, p = 0.238) (Fig 1E). Hence no significant changes in AR 5-HT/GABA between naïve and anxiety condition is present in the DRN (Fig 1E). C-fos expression in the amygdala during anxiety As the serotonergic system and the infralimbic cortex are both interconnected with the amyg- dala (S3 Fig) we decided to analyse also c-fos expression with in the amygdala in naive and anxiety conditions. As expected we found a highly significant increase of total c-fos in fearful situation in mice (naïve 26.07±1.69, Wt anxiety 82.9±4,87, p0.001.) (Fig 3B–3E). The num- ber of c-fos positive cells is increased in GABAergic interneurons as well as in CamKII positive neurons (Fig 3D and 3E). Overall ratios between c-fos expressing glutamatergic and c-fos expressing GABAergic neurons (AR Glu/GABA) are not changed in the amygdala due to anxi- ety (Wt naïve 1.59±0.083, Wt anxiety 1.81±0.07, p = 0.078) (Fig 3F). C-fos expression in the dorsal raphe nucleus during anxiety Wt naïve 22.14±2.06, 1A-ko naïve 43.43±2.47, two-tailed t-test t = -5.978, p0.001, Wt anxiety 74.47±5.48, 1A-ko anxiety 141.14±7.8 Mann Whitney Rank Sum test p0.001 (E) activity ratio between serotonergic neurons and interneurons (AR 5-HT/ GABA) calculated as ratio between c-fos positive serotonergic and c-fos positive GABAergic neurons. Wt naïve 2.77±0.33, Wt anxiety 2.79 ±0.19, Mann Whitney Rank Sum test, p = 0.372, 1A-ko naïve 4.11±0.27, 1A-ko anxiety 4.76±0.36, Mann Whitney Rank Sum test, p = 0.238. Values are mean ± S.E.M. indicate significant differences (p 0.01), indicate significant differences (p 0.001). 6 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Optogenetic excitation of pyramidal neurons in the IL promotes anxiety To directly probe the role of increased cortical excitation, i.e. elevation of AR PN/IN in the IL, we optogenetically excited specifically pyramidal neurons during the OFT and the NSF and analysed c-fos immunolabeling subsequently (Fig 4A). We expressed ChR2 in pyramidal 7 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Fig 2. Elevation of cortical excitation during anxiety behavior. (A) experimental workflow of c-fos stainings. IL infralimbic cortex, Gad67 fluorescence labeling of GABAergic neurons (green), CamKII fluorescence labeling of pyramidal neurons (blue). (B) confocal images showing c- fos expression in Wt and 1A-ko. C-fos in red. Insets show high magnification. Scale bar 150μm, insets 100μm. (C) colocalization of c-fos expression in GABAergic and pyramidal cells (analysis of 42 brain slices in 3 brains per group). N(GAD67) Wt 1.82±0.13, 1A-ko 3.52±0.2, Mann Whitney Rank Sum test, p0.001, A(GAD67) Wt 16.1±0.84, 1A-ko 23.92±1.07, Mann Whitney Rank Sum test, p0.001, N(CamKII) Wt 3.68±0.2, 1A-ko 13.4±0.61, Mann Whitney Rank Sum test, p0.001, A(CamKII) Wt 55.64±1.29, 1A-ko 90.19±2.55, Mann Whitney Rank Sum test, p0.001. (D) total amount of c-fos expressing cells. Wt naïve 11.64±0.45, 1A-ko naïve 32.74±1.45, Mann Whitney Rank Sum test, p0.001, Wt anxiety 179.9±4.6, 1A-ko anxiety 211.33±5.36, Mann Whitney Rank Sum test, p0.001. (E) activity ratio between pyramidal neurons and interneurons (AR PN/IN), calculated as ratio between c-fos positive pyramidal and c-fos positive GABAergic neurons. Wt naïve 2.24±0.17, Wt anxiety 3.8±0.18, Mann Whitney Rank Sum p0.001, 1A-ko naïve 4.53±0.42,1A-ko anxiety 4.2±0.18 Mann Whitney Rank Sum p = 0.291, Wt naïve:1A-ko naïve, Mann Whitney Rank Sum test p0.001. Values are mean ± S.E.M. indicate significant differences (p 0.001). Red boxes show significant difference between Wt and 1A-ko. https://doi org/10 1371/journal pone 0210949 g002 Enhanced activity of pyramidal neurons drives anxiety Enhanced activity of pyramidal neurons drives anxiety Fig 2. Elevation of cortical excitation during anxiety behavior. (A) experimental workflow of c-fos stainings. IL infr Fig 2. Elevation of cortical excitation during anxiety behavior. (A) experimental workflow of c-fos stainings. IL infralimbic cortex, Gad67 fluorescence labeling of GABAergic neurons (green), CamKII fluorescence labeling of pyramidal neurons (blue). (B) confocal images showing c- fos expression in Wt and 1A-ko. C-fos in red. Insets show high magnification. Scale bar 150μm, insets 100μm. (C) colocalization of c-fos expression in GABAergic and pyramidal cells (analysis of 42 brain slices in 3 brains per group). Optogenetic excitation of pyramidal neurons in the IL promotes anxiety N(GAD67) Wt 1.82±0.13, 1A-ko 3.52±0.2, Mann Whitney Rank Sum test, p0.001, A(GAD67) Wt 16.1±0.84, 1A-ko 23.92±1.07, Mann Whitney Rank Sum test, p0.001, N(CamKII) Wt 3.68±0.2, 1A-ko 13.4±0.61, Mann Whitney Rank Sum test, p0.001, A(CamKII) Wt 55.64±1.29, 1A-ko 90.19±2.55, Mann Whitney Rank Sum test, p0.001. (D) total amount of c-fos expressing cells. Wt naïve 11.64±0.45, 1A-ko naïve 32.74±1.45, Mann Whitney Rank Sum test, p0.001, Wt anxiety 179.9±4.6, 1A-ko anxiety 211.33±5.36, Mann Whitney Rank Sum test, p0.001. (E) activity ratio between pyramidal neurons and interneurons (AR PN/IN), calculated as ratio between c-fos positive pyramidal and c-fos positive GABAergic neurons. Wt naïve 2.24±0.17, Wt anxiety 3.8±0.18, Mann Whitney Rank Sum p0.001, 1A-ko naïve 4.53±0.42,1A-ko anxiety 4.2±0.18 Mann Whitney Rank Sum p = 0.291, Wt naïve:1A-ko naïve, Mann Whitney Rank Sum test p0.001. Values are mean ± S.E.M. indicate significant differences (p 0.001). Red boxes show significant difference between Wt and 1A-ko. Fig 2. Elevation of cortical excitation during anxiety behavior. (A) experimental workflow of c-fos stainings. IL infralimbic cortex, Gad67 fluorescence labeling of GABAergic neurons (green), CamKII fluorescence labeling of pyramidal neurons (blue). (B) confocal images showing c- fos expression in Wt and 1A-ko. C-fos in red. Insets show high magnification. Scale bar 150μm, insets 100μm. (C) colocalization of c-fos expression in GABAergic and pyramidal cells (analysis of 42 brain slices in 3 brains per group). N(GAD67) Wt 1.82±0.13, 1A-ko 3.52±0.2, Mann Whitney Rank Sum test, p0.001, A(GAD67) Wt 16.1±0.84, 1A-ko 23.92±1.07, Mann Whitney Rank Sum test, p0.001, N(CamKII) Wt 3.68±0.2, 1A-ko 13.4±0.61, Mann Whitney Rank Sum test, p0.001, A(CamKII) Wt 55.64±1.29, 1A-ko 90.19±2.55, Mann Whitney Rank Sum test, p0.001. (D) total amount of c-fos expressing cells. Wt naïve 11.64±0.45, 1A-ko naïve 32.74±1.45, Mann Whitney Rank Sum test, p0.001, Wt anxiety 179.9±4.6, 1A-ko anxiety 211.33±5.36, Mann Whitney Rank Sum test, p0.001. (E) activity ratio between pyramidal neurons and interneurons (AR PN/IN), calculated as ratio between c-fos positive pyramidal and c-fos positive GABAergic neurons. Wt naïve 2.24±0.17, Wt anxiety 3.8±0.18, Mann Whitney Rank Sum p0.001, 1A-ko naïve 4.53±0.42,1A-ko anxiety 4.2±0.18 Mann Whitney Rank Sum p = 0.291, Wt naïve:1A-ko naïve, Mann Whitney Rank Sum test p0.001. Values are mean ± S.E.M. indicate significant differences (p 0.001). Red boxes show significant difference between Wt and 1A-ko. 8 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Enhanced activity of pyramidal neurons drives anxiety Fig 3. Optogenetic excitation of pyramidal neurons in the IL promotes anxiety AR Glu/GABA in the basolateral amygdala is not changed due to anxiety. (A) experimental workflow of c-fos stainings in the amygdala. AMY amygdala, BLA basolaterale amygdala, Ce central amygdala, DR dorsal raphe, IL infralimbic cortex, La lateral amygdala. Gad67 fluorescence labeling of GABAergic neurons (green), CamKII fluorescence labeling of pyramidal neurons (blue), c-fos fluorescence labeling of c-fos expressing cells (red). (B) confocal images showing c-fos expression in the amygdala of naïve and anxiety treated mice. Gad67 fluorescence labeling of GABAergic neurons (green), CamKII fluorescence labeling of pyramidal neurons (blue), c-fos fluorescence labeling of c-fos expressing cells (red). Scale bar 250μm, Insets high magnification, scale bar 100μm. (C) total amount of c-fos expressing cells (analysis of 56 brain slices in 4 brains for naïve conditions and 42 brain slices in 3 brains for the anxiety condition): naïve 26.07±1.69, Anx 82.9±4.87, Mann Whitney Rank Sum test p0.001. (D) colocalization of c-fos expression in GABAergic cells: Naïve 5.95±0.44, Anx 13.45±0.98, Mann Whitney Rank Sum test p0.001. (E) colocalization of c-fos expression in pyramidal cells: naïve 8.72±0.55, Anx 22.57±1.34, Mann Whitney Rank Sum test p0.001. (F) activity ratio between glutamatergic neurons and interneurons (AR Glu/GABA), calculated as ratio between c-fos positive pyramidal and c-fos positive GABAergic neurons: naïve 1.59±0.08, Anx 1.81±0.07, Mann Whitney Rank Sum test, p = 0.075 n.s. Values are mean ± SEM. indicate significant differences (p0.001). https://doi org/10 1371/journal pone 0210949 g003 Fig 3. AR Glu/GABA in the basolateral amygdala is not changed due to anxiety. (A) experimental workflow of c-fos stainings in the amygdala. AMY amygdala, BLA basolaterale amygdala, Ce central amygdala, DR dorsal raphe, IL infralimbic cortex, La lateral amygdala. Gad67 fluorescence labeling of GABAergic neurons (green), CamKII fluorescence labeling of pyramidal neurons (blue), c-fos fluorescence labeling of c-fos expressing cells (red). (B) confocal images showing c-fos expression in the amygdala of naïve and anxiety treated mice. Gad67 fluorescence labeling of GABAergic neurons (green), CamKII fluorescence labeling of pyramidal neurons (blue), c-fos fluorescence labeling of c-fos expressing cells (red). Scale bar 250μm, Insets high magnification, scale bar 100μm. (C) total amount of c-fos expressing cells (analysis of 56 brain slices in 4 brains for naïve conditions and 42 brain slices in 3 brains for the anxiety condition): naïve 26.07±1.69, Anx 82.9±4.87, Mann Whitney Rank Sum test p0.001. (D) colocalization of c-fos expression in GABAergic cells: Naïve 5.95±0.44, Anx 13.45±0.98, Mann Whitney Rank Sum test p0.001. Optogenetic excitation of pyramidal neurons in the IL promotes anxiety (E) colocalization of c-fos expression in pyramidal cells: naïve 8.72±0.55, Anx 22.57±1.34, Mann Whitney Rank Sum test p0.001. (F) activity ratio between glutamatergic neurons and interneurons (AR Glu/GABA), calculated as ratio between c-fos positive pyramidal and c-fos positive GABAergic neurons: naïve 1.59±0.08, Anx 1.81±0.07, Mann Whitney Rank Sum test, p = 0.075 n.s. Values are mean ± SEM. indicate significant differences (p0.001). https://doi.org/10.1371/journal.pone.0210949.g003 https://doi.org/10.1371/journal.pone.0210949.g003 neurons of the left IL (S4 Fig) and implanted an optical fiber directly above the IL to specifi- cally increase pyramidal neuron activity and therefore increase the AR PN/IN of the IL region (S4 Fig). In the OFT we alternated light stimulation with control phases in which no light stim- ulation occurred (Fig 4B and 4C). Stimulation of pyramidal neurons resulted in decreased center times (Off1 39.49±6.9s, On1 19.87±4.47s, Off2 28.13±8.55s, On2 23.42±9.32s) in com- parison to the first control phase (Fig 4B). During the second control phase anxiety behaviour did not recover completely, but showed only a non-significant increase in center duration times. Control mice, in which dt-tomato was only expressed in pyramidal neurons, showed no alteration in anxiety behaviour due to light stimulation (S5A Fig). Light stimulation had no effects on motor behaviour (S4B and S5A Figs) and did not induce long lasting changes in anx- iety behaviour (S5B Fig). Next we scrutinized the influence of excitation of IL pyramidal neu- rons in the NSF. Now we stimulated pyramidal neurons permanently during the execution of neurons of the left IL (S4 Fig) and implanted an optical fiber directly above the IL to specifi- cally increase pyramidal neuron activity and therefore increase the AR PN/IN of the IL region (S4 Fig). In the OFT we alternated light stimulation with control phases in which no light stim- ulation occurred (Fig 4B and 4C). Stimulation of pyramidal neurons resulted in decreased center times (Off1 39.49±6.9s, On1 19.87±4.47s, Off2 28.13±8.55s, On2 23.42±9.32s) in com- parison to the first control phase (Fig 4B). During the second control phase anxiety behaviour did not recover completely, but showed only a non-significant increase in center duration times. Control mice, in which dt-tomato was only expressed in pyramidal neurons, showed no alteration in anxiety behaviour due to light stimulation (S5A Fig). Light stimulation had no effects on motor behaviour (S4B and S5A Figs) and did not induce long lasting changes in anx- iety behaviour (S5B Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Optogenetic excitation of pyramidal neurons in the IL promotes anxiety Anx EXP:Anx+L EXP M Whit R k S t t 0 001 A L EXP A L CT t t il d t t t t 2 714 0 009 (H) l li ti f f i i Enhanced activity of pyramidal neurons drives anxiety Fi 4 El d i l AR PN/IN b l d i i b h i (A) i ChR2 id l i l d i l AR PN/IN balance drives anxiety behavior. (A) targeting ChR2 to pyramidal cortical neurons and experimental cortex (B) group data for openfield experiments in ChR2 injected animals (EXP), center duration Off1 39.49±6.9s, On1 . Elevated cortical AR PN/IN balance drives anxiety behavio Fig 4. Elevated cortical AR PN/IN balance drives anxiety behavior. (A) targeting ChR2 to pyramidal cortical neurons and experimental workflow. IL infralimbic cortex (B) group data for openfield experiments in ChR2 injected animals (EXP), center duration Off1 39.49±6.9s, On1 19.87±4.47s, Off2 28.13±8.55s, On2 23.42±9.32s, Off1:On1 two tailed t-test, t = 2.286, p = 0.033, Off1:Off2 Mann Whitney Rank Sum test p = 0.168, Off1:On2 Mann Whitney Rank Sum test p = 0.049, distance moved Off1 2703.09±292.65cm, On1 3113.4±491.15cm, Off2 3331.86 ±482.62cm, On2 3082.17±658,61cm, Off1:On1 Mann Whitney Rank Sum test p = 0.743, Off1:Off2 two tailed t-test t = 0.52, p = 0.609, Off1:On2 Mann Whitney Rank Sum p = 0.646, n = 11 mice. (C) example of raw data from an individual mouse during an openfield test. (D) group data for novelty suppressed feeding, time till feeding CT no light 112.42±14.9s, CT light 129.38±22.96s, EXP no light 93.38±17.41s, EXP light 225.86 ±32.76s, CT light:EXP light, two tailed t-test, t = -2.461, p = 0.029, EXP no light:EXP light Mann Whitney Rank Sum test p = 0.004, food intake CT no light 0.12±0.02g, CT light 0.15±0.02g, EXP no light 0.16±0.03, EXP light 0.15±0.02 CT light:EXP light Mann Whitney Rank Sum test, p = 1, EXP no light:EXP light Mann Whitney Rank Sum test p = 0.955. (E) example of raw data from an individual mouse during novelty suppressed feeding test. (F) total amount of c-fos expressing cells, naïve EXP 47.11±3.25, CT 83.86±5, Anx EXP 160.14±10.14, CT 231.21±9.93, Anx+L EXP 323.38±13.61, CT 192.11±9.51. Anx EXP:Anx+L EXP two tailed-t-test t = -9.709, p0.001, Anx+L EXP:Anx+L CT two tailed-t-test t = -8, p0.001. Optogenetic excitation of pyramidal neurons in the IL promotes anxiety Next we scrutinized the influence of excitation of IL pyramidal neu- rons in the NSF. Now we stimulated pyramidal neurons permanently during the execution of PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 9 / 19 Enhanced activity of pyramidal neurons drives anxiety Fig 4. Elevated cortical AR PN/IN balance drives anxiety behavior. (A) targeting ChR2 to pyramidal cortical neurons and experimental workflow. IL infralimbic cortex (B) group data for openfield experiments in ChR2 injected animals (EXP), center duration Off1 39.49±6.9s, On1 19.87±4.47s, Off2 28.13±8.55s, On2 23.42±9.32s, Off1:On1 two tailed t-test, t = 2.286, p = 0.033, Off1:Off2 Mann Whitney Rank Sum test p = 0.168, Off1:On2 Mann Whitney Rank Sum test p = 0.049, distance moved Off1 2703.09±292.65cm, On1 3113.4±491.15cm, Off2 3331.86 ±482.62cm, On2 3082.17±658,61cm, Off1:On1 Mann Whitney Rank Sum test p = 0.743, Off1:Off2 two tailed t-test t = 0.52, p = 0.609, Off1:On2 Mann Whitney Rank Sum p = 0.646, n = 11 mice. (C) example of raw data from an individual mouse during an openfield test. (D) group data for novelty suppressed feeding, time till feeding CT no light 112.42±14.9s, CT light 129.38±22.96s, EXP no light 93.38±17.41s, EXP light 225.86 ±32.76s, CT light:EXP light, two tailed t-test, t = -2.461, p = 0.029, EXP no light:EXP light Mann Whitney Rank Sum test p = 0.004, food intake CT no light 0.12±0.02g, CT light 0.15±0.02g, EXP no light 0.16±0.03, EXP light 0.15±0.02 CT light:EXP light Mann Whitney Rank Sum test, p = 1, EXP no light:EXP light Mann Whitney Rank Sum test p = 0.955. (E) example of raw data from an individual mouse during novelty suppressed feeding test. (F) total amount of c-fos expressing cells, naïve EXP 47.11±3.25, CT 83.86±5, Anx EXP 160.14±10.14, CT 231.21±9.93, Anx+L EXP 323.38±13.61, CT 192.11±9.51. Anx EXP:Anx+L EXP two tailed-t-test t = -9.709, p0.001, Anx+L EXP:Anx+L CT two tailed-t-test t = -8, p0.001. Anx anxiety paradigm, Anx+L anxiety paradigm with light stimulation (G) colocalization of c-fos expression in GABAergic neurons (Gad67) naïve EXP 9.93±0.76, CT 16.57±1.17, Anx EXP 21.5±1.2, CT 29.14±1.23, Anx+L EXP 34.12±1.84, CT 27.71±1.49. Optogenetic excitation of pyramidal neurons in the IL promotes anxiety Anx anxiety paradigm, Anx+L anxiety paradigm with light stimulation (G) colocalization of c-fos expression in GABAergic neurons (Gad67) naïve EXP 9.93±0.76, CT 16.57±1.17, Anx EXP 21.5±1.2, CT 29.14±1.23, Anx+L EXP 34.12±1.84, CT 27.71±1.49. Anx EXP:Anx+L EXP Mann Whitney Rank Sum test, p0.001, Anx+L EXP:Anx+L CT two tailed-t-test t = -2.714, p = 0.009. (H) colocalization of c-fos expression in pyramidal neurons (CamKII) naïve EXP 16.04±1.14, CT 28.21±2.1, Anx EXP 56.68±3.52, CT 71.57±2.98, Anx+L EXP 120.08±542, CT 65.46 ±3.26. Anx EXP:Anx+L EXP Mann Whitney Rank Sum test, p0.001, Anx+L EXP:Anx+L CT Mann Whitney Rank Sum test p0.001. (I) activity ratio between pyramidal neurons and interneurons (AR PN/IN), calculated as ratio between c-fos positive pyramidal and c-fos positive PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 10 / 19 Enhanced activity of pyramidal neurons drives anxiety GABAergic neurons. Naïve EXP 1.63±0.05, CT 1.7±0.04, Anx EXP 2.65±0.08, CT 2.47±0.05, Anx+L EXP 3.59±0.1 CT 2.38±0.04. Anx EXP: Anx +L EXP two tailed t-test t = -7.377, p0.001, Anx+L EXP: Anx+L CT Mann Whitney Rank Sum test p0.001.Values are mean ± S.E.M. indicate significant differences (p 0.05), indicate significant differences (p 0.01), indicate significant differences (p 0.001). GABAergic neurons. Naïve EXP 1.63±0.05, CT 1.7±0.04, Anx EXP 2.65±0.08, CT 2.47±0.05, Anx+L EXP 3.59±0.1 CT 2.38±0.04. Anx EXP: Anx +L EXP two tailed t-test t = -7.377, p0.001, Anx+L EXP: Anx+L CT Mann Whitney Rank Sum test p0.001.Values are mean ± S.E.M. indicate significant differences (p 0.05), indicate significant differences (p 0.01), indicate significant differences (p 0.001). https://doi.org/10.1371/journal.pone.0210949.g004 the NSF. After 24 h of food deprivation animals were challenged with a food pellet situated in the center of an OFT. Control animals, expression of dt-tomato with and without light stimu- lation, as well as ChR2 injected mice without light stimulation showed significant shorter latencies to start feeding than ChR2 injected mice with light stimulation (CT no light 112.42 ±14.9s, CT light 129.38±22.96s, EXP no light 93.38±17.41s, EXP light 225.86±32.76s) (Fig 4D and 4E) as also higher center duration times (S5C Fig). While excitation of pyramidal neurons increased latencies to feed more than twofold (EXP no light 93.38±17.41s, EXP light 225.86 ±32.76s) (Fig 4D) it divided center time duration by four (S5C Fig). Overall food intake was not changed among groups (CT no light 0.12±0.02g, CT light 0.15±0.02g, EXP no light 0.16 ±0.03, EXP light 0.15±0.02 CT) (Fig 4D). C-Fos expression indicates that an elevated AR PN/IN in the IL induces anxiety Subsequent immunohistochemical analyses in the IL revealed again increased c-fos expression in glutamatergic and GABAergic neurons in fearful situation (Fig 4F–4I), with even higher neuronal activity in glutamatergic neurons during additional light stimulation (naïve EXP 47.11±3.25, Anx EXP 160.14±10.14, Anx+L EXP 323.38±13.6). In control animals light stimu- lation had no further effects (Anx CT 231.21±9.93, ANX+L CT 192.11±9.51) (Fig 4F–4I). Con- sequently AR PN/IN is increased during light stimulation in experimental mice (Anx EXP 2.65±0.08, Anx+L EXP 3.59±0.1) and did not further change in control mice (AnxCT 2.47 ±0.05, Anx+L CT 2.38±0.04) (Fig 4I). Hence shifting the activity ratio between excitatory pyra- midal neurons and inhibitory interneurons in the IL towards excitation promotes anxiety behaviour. To ensure, that optogenetic stimulation within the IL does not induce any stimula- tion artefacts within the DRN, which probably promote anxiety behaviour we additionally ana- lysed AR 5-HT/GABA in the DRN during optogenetic stimulation in the IL. Light stimulation in the IL does not induce any significant changes in the activity ratio of the DRN (S7 Fig), emphasizing the pivotal role of IL cortex in the modulation of anxiety levels. Optogenetic excitation of pyramidal neurons in the IL promotes anxiety In addition we also conducted the EPM in ChR2 injected and control mice (S6 Fig). But unfortunately we noticed that during the execution of the EPM 42% of experimental and control animals fall off the maze. These animals had to be excluded from our analyses. All other animals, which did not fall off immediately had difficul- ties to walk on the open arms: they regularly slipped off from the open arms and then started consequently to avoid them categorically. Due to this behaviour control animals and experi- mental animals started to avoid open arms during all consecutive trials (S6A–S6C Fig). As this observed behaviour was completely independent from stimulation protocols (light or no light) or from the injected virus (ChR2 or control fluorophore) (S6D–S6F Fig), we think decreased open arm times in experimental, as well as in control mice do not permit to make any state- ment on the anxiety behaviour in the EPM. Taken together our results demonstrate that exci- tation of IL pyramidal neurons during the OFT and NSF enhance anxiety (Fig 4B–4E). Circuitry of anxiety Previous studies have identified the prefrontal cortex as part of a tripartite anxiety network, consisting of the amygdala, the ventral hippocampus (vHPC) and the prefrontal cortex. Manipulations within this network are able to chance anxiety behaviour [4,20–22]. During the expression of anxiety, activity between the vHPC and the prefrontal cortex are highly synchro- nized and power in theta frequencies (4–12 Hz) is markedly increased in an anxiogenic envi- ronment [22,35]. Adhikari et al. (2011) hypothesize that place fields within the vHPC are well suited to encode the emotional valence of the environment and can provide contextual infor- mation to the mPFC [35], which in turn can act on the amygdala to modulate exploratory behaviour. In general the PFC is associated with a role in active inhibition of behaviour (Shah and Treit 2003) and it is an intriguing hypothesize that the PFC is by this connectivity able to modulate the expression of anxiety behaviour. Single unit recordings in mPFC have revealed that neurons in the mPFC encode safe and aversive places in the EPM [36], pointing again to the involvement of PFC neurons in guiding exploratory behaviour. Our results are in conjunction with studies that report large increases in theta power and prefrontal cortex firing rates in 5-HT1A(-/-) knockout and high avoidance Wt mice during anxiety [35,37]. Presumably, in our experiments an acute optogenetic increase in pyramidal neuronal activity amplified theta synchrony, which in turn leads then to an enhanced expres- sion of anxiety by the generalization of aversiveness throughout the anxiety probing test. This might also explain, that in contrast to a study by Fuchikami et al. 2015, in which a continuous light stimulation of layer 5 pyramidal neurons over 60 minutes one day prior to testing induced anxiolytic and antidepressant behavioural responses [38], acute light stimulation of pyramidal neurons in all cortical layers during a stressful situation is anxiogenic. Acute changes in pyramidal neuron activity might directly act on anxiety behaviour by modulating the encoding of safe vs. aversive places, whereas spine sprouting in the prefrontal cortex by ketamine or optogenetic stimulation could probably prime the prefrontal cortex to less inhibi- tion of exploratory behaviour. Enhanced activity of pyramidal neurons drives anxiety sufficient to resemble this phenotype and drives anxiety behaviour. In conjunction with cur- rent models of anxiety our study provides further evidence that a functional decrease of seroto- nergic neurotransmission at postsynaptic sites and subsequent disinhibition of IL pyramidal neurons may be a pathogenic mechanism of anxiety [9,26]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Discussion By combining behavioural and immunohistochemistry studies we could show, that in a model of anxiety an imbalance of excitatory pyramidal neurons and inhibitiory interneuron activity in the IL is evident and that optogenetic elevation of the pyramidal neuron activity in the IL is PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 11 / 19 Conclusion Our findings suggest that an exact balance between excitatory pyramidal neurons and inhibi- tory interneurons in the IL of the prefrontal cortex is a prerequisite to maintain healthy anxiety responses and that an increased activity of cortical pyramidal neurons can drive anxiety. Fol- low-up studies have to proof if shifting this ratio towards interneuron activity will already be anxiolytic or if specifically, 5-HT1A signalling in the IL is needed to maintain normal anxiety levels. Nevertheless an oversimplification should be avoided, i.e. excitation and inhibition might be regulated differently at different circuits levels, for example neurons in different microcircuits targeting other brain areas could be modulated independently and excitatory and inhibitory inputs might even vary at different cell compartments [53]. Our study further supports that a dysregulation of AR PN/IN in cortical areas, presumably caused by disturbed serotonergic signalling, is one possible cause of generalized anxiety giving new insights into underlying mechanism of anxiety. Serotonin and anxiety Numerous studies have investigated the influence of serotonergic neurotransmission on anxi- ety. One of the most famous genetic mouse models of anxiety is a 5-HT1A(-/-) receptor knock- out mouse line. This mouse line provides further evidence for a close link of the serotonergic system with anxiety. Pharmacogenetic studies revealed that an increase in serotonergic neuro- nal activity results in elevated anxiety (Teissier et al. 2015) whereas depletion of 5-HT reduces anxiety [18,45]. As serotonin preferentially acts on 5-HT1A receptors of GABAergic interneu- rons, the net effect of serotonin release into the cortex results in inhibition of GABAergic inter- neurons and therefore in a disinhibition of pyramidal neurons, which is associated with an increase in pyramidal neuron activity [46]. Hence serotonin would be a potential candidate to initiate a transition from balanced excitation/inhibition ratios to enhanced excitation. Our observations of an enhanced AR PN/IN in 5-HT1A(-/-) mice is in accordance with former stud- ies, which reported an important role of 5-HT1A receptors in tuning E/I balance [47]. However, pre- and postsynaptic effects of serotonin in the prefrontal cortex might act dif- ferently on anxiety. A study by Kjaerby et al. 2016 convincingly showed that presynaptic 5-HT1B receptors are able to selectively supress hippocampal and callosal input leading to an inhibition of prefrontal theta oscillations and in turn to reduced anxiety mediated avoidance [48]. It is likely that disturbed serotonergic neurotransmission could cause changes in the bal- ance between excitatory and inhibitory neuronal activity ratios by altering synaptic input strength or by incorrect gating of inputs. Postsynaptically mainly 5-HT1A receptors, located on pyramidal, as well as on GABAergic interneurons, are expressed in the prefrontal cortex. Several studies, in humans and animal models have shown that, especially dysfunction of postsynaptic 5-HT1A receptors is associated with anxiety [49–52]. Albert et al. 2014 propose an anxiety model, in which either too low or too high levels of serotonin will induce anxiety by an increased pyramidal neuron output, mediated by the highly sensitive 5-HT1A receptor [18]. Our studies support this anxiety model and underlines further the necessity of balanced E/I in the prefrontal cortex for healthy anxiety behaviour. The role of the prefrontal cortex in anxiety Contradictory results have been obtained regarding the role of the PFC in anxiety. Studies report a decrease in anxiety after lesioning of the PFC [6,39], but also anxiogenic behaviour was observed after inhibition of the PFC [15,16]. In the course of this conflicting results differ- ential roles of subdivisions of the PFC, i.e. the IL and prelimbic cortex (PL) were proposed [40]. Pharmacological manipulations of inhibitory and excitatory neurotransmission within the PFC indicated that enhanced excitatory inputs to the PFC, i.e. increased glutamate or decreased GABA neurotransmission and therewith an imbalance of excitation inhibition, sup- port anxiety behaviour [9]. These results are in tight conjunction with our results and underlie the notion that disturbances in cortical excitation inhibition balance promote pathological conditions and are one possible cause of anxiety disorders [41–44]. Two recent studies have revealed the importance of projections and connections to the IL in extinction learning [7] pointing to the complex role of the PFC in anxiety and fear regulation and underlining proba- bly different mechanism in innate anxiety and acquired anxiety. 12 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Enhanced activity of pyramidal neurons drives anxiety Supporting information DRD dorsal raphe drsal part, DRV dorsal raphe ventral part, IL infralimbic cortex. (PDF) S2 Fig. Connectivity of the IL and DRN. (A) experimental workflow for retrograde tracing of DRN-IL projection neurons and confocal images showing fluorogold stained cell bodies in the DRN. Only the left IL was injected. Fluorogold stained cell bodies in the DRN (yellow) and immunohistochemical labeling of TPH positive neurons (blue). Scale bar 1mm. Insets show high magnification. Scale bar 50μm. Arrow heads indicate exemplarily double-positive cells. (B) experimental workflow for anterograde tracing of IL-DRN projections and confocal image showing expression of td-tomato in fibers terminating in the DRN. TPH stained cell bodies in the DRN (blue) and fiber terminals (red). Scale bar 500μm. Insets show high magnification. Gad67 stained cell bodies in the DRN (green) and fiber terminals (red). Scale bar 50μm. (C) experimental workflow for retrograde tracing of IL-DRN projection neurons and confocal image showing fluorogold stained cells bodies (yellow) in the IL. Only the left IL was in injected. Fluorogold stained cell bodies in the IL (yellow) and immunohistochemical labeling of CamKII positive neurons (blue). Scale bar 1mm. Insets show high magnification. Scale bar 50μm. Arrows indicate double-positive cells. DRD dorsal raphe drsal part, DRV dorsal raphe ventral part, IL infralimbic cortex. (PDF) S3 Fig. Connectivity of the amygdala with the IL and the DRN. (A) experimental workflow for anterograde tracing of the left IL-amygdala projections and confocal image showing expression of td-tomato fibers from the IL terminating in the left amygdala (top) or the right amygdala (bottom). CamKII stained cell bodies in the amygdala (blue). Gad67 stained cell bodies in the amygdala (green) and fiber terminals from the IL (red). Scale bar 250μm. Insets of high magnification show either the colocalization of CamKII or Gad67 with fiber terminals arising from the IL. Scale bar 100 μm. (B) experimental workflow for retrograde tracing of IL- amygdala projection neurons and confocal images showing fluorogold stained cell bodies (yel- low), flurogold was injected in the left amygdala. Fluorogold stained cell bodies in the IL (yel- low) and immunohistochemical labeling of CamKII positive neurons (blue). Scale bar 500μm. Insets show colocalization of fluorogold and CamKII in high magnification, scale bar 100μm. (C) experimental workflow for retrograde tracing of DRN-amygdala projection neurons and confocal images showing fluorogold stained cell bodies (yellow), fluorogold was injected in the left amygdala. Supporting information S1 Fig. Enhanced anxiety in 5-HT1A(-/-) knockout mice. (A) setup Elevated-Plus Maze (EPM) (B) group data for the EPM, time in open arms: Wt 47.91±6.12s, 1A-ko 31.59±4.34s, two-tailed t-test, t = 2.207, p = 0.026, Wt n = 10, 1A-ko n = 18 (C) group data for the EPM open arm entries: 10.6±1.25, 1A-ko 7.28±0.66, two-tailed t-test, t = 2.604, p = 0.015, n = 10, 1A-ko 13 / 19 13 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Enhanced activity of pyramidal neurons drives anxiety n = 18. (D) group data for distance moved Wt 3859.34±979.26cm, 1A-ko 4373±669.08cm, Mann Whitney Rank Sum test, p = 0.487, Wt n = 8, 1A-ko n = 18. (E) setup novelty suppressed feeding (NSF). (F) group data for NSF time in center Wt = 27.32±3.81s, 1A-ko = 17.57±2.6s, two-tailed t-test, t = 2.193, p = 0.042, Wt n = 8, 1A-ko = 12. (G) group data NSF time till feed- ing Wt 118.56±19.16s, 1A-ko = 146.69±36.67s, Mann Whitney Rank Sum test, p = 0.772, Wt n = 10, 1A-ko = 16. (H) NSF food intake, Wt 0.1±0.015g, 1A-ko 0.13±0.027g, Mann Whitney Rank Sum test, p = 0.613. Values are mean ± S.E.M. indicate significant differences (p 0.05). (PDF) S2 Fig. Connectivity of the IL and DRN. (A) experimental workflow for retrograde tracing of DRN-IL projection neurons and confocal images showing fluorogold stained cell bodies in the DRN. Only the left IL was injected. Fluorogold stained cell bodies in the DRN (yellow) and immunohistochemical labeling of TPH positive neurons (blue). Scale bar 1mm. Insets show high magnification. Scale bar 50μm. Arrow heads indicate exemplarily double-positive cells. (B) experimental workflow for anterograde tracing of IL-DRN projections and confocal image showing expression of td-tomato in fibers terminating in the DRN. TPH stained cell bodies in the DRN (blue) and fiber terminals (red). Scale bar 500μm. Insets show high magnification. Gad67 stained cell bodies in the DRN (green) and fiber terminals (red). Scale bar 50μm. (C) experimental workflow for retrograde tracing of IL-DRN projection neurons and confocal image showing fluorogold stained cells bodies (yellow) in the IL. Only the left IL was in injected. Fluorogold stained cell bodies in the IL (yellow) and immunohistochemical labeling of CamKII positive neurons (blue). Scale bar 1mm. Insets show high magnification. Scale bar 50μm. Arrows indicate double-positive cells. Supporting information Fluorogold stained cell bodies in the IL (yellow) and immunohistochemical labeling of TPH positive neurons (blue). Scale bar 500μm. Insets show colocalization of fluoro- gold and TPH in high magnification, scale bar 100μm. Arrows indicate examples for double positive cells. AMY amygdala, DR dorsal raphe, DRD dorsal raphe dorsal part, DRV dorsal raphe central part, IL infralimbic cortex, (PDF) S4 Fig. Injections sites and expression in pyramidal neurons. (A) ChR2 or td-tomato was unilateral delivered into the left IL, optical fibers were placed in the IL and optical stimulation with blue (473 nm) light was restricted to the IL. (B) Confocal images showing expression of 14 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Enhanced activity of pyramidal neurons drives anxiety ChR2 (left, yellow) and td-tomato (right, red) in the IL. Cell nuclei are marked with DAPI (blue). Scale bar 1mm. (C) Insets from b showing high magnification of the IL region. Scale bar 150μm. Cell nuclei are marked with DAPI (blue) (D) Top row, Colocalization of ChR2 (yellow) with pyramidal neurons (CamKII in blue). Scale bar 150μm. Bottom row, high magni- fication of above confocal images showing in detail the injected and non-injected side. Scale bar 100μm. Arrow heads indicate exemplarily double-positive cells. (E) Top row, ChR2 (yel- low) is not expressed in GABAergic neurons (Gad67 in green). Scale bar 150μm. Bottom row, high magnification of above confocal images showing in detail the injected and non-injected side. Scale bar 100μm. IL infralimbic cortex, PL prelimbic cortex. (PDF) S5 Fig. Control experiments. (A) group data for the OFT in control mice. Center duration Off1 16.73±2.65s, On1 16.02±1.89s, Off2 12.02±1.76s, On2 13.04±2.58s, Mann Whitney Rank Sum Off1:On1 p = 0.868, Off1:Off2 p = 0.263, Off1:On2 p = 0.33. Distance moved Off1 3399.69 ±296.77cm, On1 3210.6±446.9cm, Off2 3030.28±513.83cm, On2 2955.82±617.7, Mann Whitney Rank Sum test Off1:On1 p = 0.171, Off1:Off2 p = 0.081, Off1:On2 p = 0.028, n = 15. (B) group data for the NSF. Center duration CT no light 5.8±2.01s, CT light 8.85±3.11s, EXP no light 7.93 ±2.79s, EXP light 1.55±0.81s. CT no light:CT light two tailed t-test, t = -0.844, p = 0.417, EXP no light:EXP light Mann Whitney Rank Sum test p = 0.035, CT light:EXP light Whitney Rank Sum p = 0.053. CT no light n = 6, CT light n = 7, EXP no light n = 6, EXP light n = 7. Supporting information (C) group data for persistent effects of anxiety in CT and EXP mice. Two days after the OFT mice were again challenged in the openfield. Neither freezing, nor center times were significantly different in CT or EXP mice, indicating that optogenetic stimulation did not have long-term effects on anxiety behavior. Freezing: CT 3.36±1.16s, EXP 5.4±2, two tailed t-test t = -0.957, p = 0.364, Center duration CT 6.89±0.89s, EXP 6.17±1.38s, two tailed t-test t = 0,461, p = 0.656, CT n = 7, EXP n = 4. Values are mean ± S.E.M. indicate significant differences (p 0.05). (PDF) S6 Fig. Nex-Cre mice failed to perform Elevated-Plus maze test. (A) setup Elevated-Plus Maze and light stimulation protocol. (B) group data for “slip off’s” (slipping off with back paws from the floor of the open arm) for experimental and control animals. Only mice, which stayed all trials in the EPM are considered. Slip offs in first light off phase are reason for later avoidance of open arms in all groups: Off1 EXP 1.625±0.6, CT 2.2±0.79, Off2+3 EXP 0.125 ±0.125, CT 0±0, On1+2+3 EXP 0.625±0.26, CT 0.1±0.1. Pie chart of mice falling from the EPM 42,42% fall down and only 57,57% were able to stay in the EPM during all trials. (C) group data for the experimental group in the EPM, time in open arms: Off1 73.91±12.22s, On1 36.15±14.65s, Off2 15.61±6.23s, On2 19.49±7.51s, Off3 9.36±4.44s, On3 7.96±3.47s, n = 12, two-tailed t-test Off1:On1 p = 0,041, t = 2.168. (E) group data for the control group in the EPM, time in open arms: Off1 86.92±12.74s, On1 33.78±14.38s, Off2 18.01±11.61s, On2 16.41 ±9.61, Off3 11.36±4.01, On3 5.43±2.07, n = 11, Mann Whitney Rank Sum test Off1:On1 p = 0.009. (F) group data for distance moved by experimental animals, no significant differ- ences were evident during trials: Off1 679.96±71.63cm, On1 712.24±112.82cm, Off2 717.49 ±97.39cm, On2 782.51±81.11cm, Off3 722.11±68.60cm, On3 663.90±106.57cm, n = 12. (G) group data for distance moved by control animals, no significant differences were evident dur- ing trials: Off1 705.11±88.36cm, On1 789.45±77.53cm, Off2 724.74±80.49cm, On2 676.57 ±111.99cm, Off3 716.99±132.47cm, On3 663.03±132.46cm, n = 11. Values are mean ± SEM. indicate significant differences (p = 0.05), indicate significant differences (p0.01). CT control animals, EXP experimental animals. (PDF) 15 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210949 January 24, 2019 Enhanced activity of pyramidal neurons drives anxiety S7 Fig. Supporting information E/I balance in the serotonergic system is not changed due to optogenetic activation of cortical pyramidal cells. (A) total amount of c-fos expressing cells in the DRN: naïve EXP 49.43±2.91, CT 48.29±3.28, Anx EXP 81.93±3.68, CT 77.21±3.26, Anx+L EXP 73.88±4.33, CT 79.54±5.12, two-tailed t-test EXP naïve: EXP Anx p0.001, t = -6.928, n = 28, two-tailed t-test CT naïve:CT Anx p0.001, t = -6.253, n = 28. (B) colocalization of c-fos expression in GABAergic neurons (Gad67): naïve EXP 5.64±0.4, CT 5.79±0.49, Anx EXP 8.71±0.54, CT 8.11 ±0.5, Anx+L EXP 7.78±0.51, CT 8.0±0.66, two-tailed t-test EXP naïve:EXP Anx p0.001, t = 4.539, n = 28, two-tailed t-test CT naïve:CT Anx p = 0.002, t = -3.286, n = 28. (C) colocaliza- tion of c-fos expression in pyramidal neurons (CamKII): naïve EXP 11.32±18.21, CT 11.21 ±0.82, Anx EXP 20.18±1.04, CT 17.61±0,78, Anx+L EXP 18.21±0.92, CT 17.39±1.26, Mann Whitney Rank Sum test EXP naïve:EXP Anx p0.001, n = 28, two-tailed t-test CT naïve:CT Anx p0.001, t = -5.663, n = 28. (D) Excitation/Inhibition balance, calculated as ratio between c-fos positive serotonergic neurons as main projection neurons and c-fos positive GABAergic neurons, no significant difference is evident between conditions: naïve EXP 2.16±0.11, CT 2.04±0.09, Anx EXP2.44±0.12, CT 2.32±0.13, Anx+L EXP 2.52±0.16, CT 2.28±0.09. Values are mean ± SEM. indicate significant differences (p = 0.01), indicate significant differences (p0.001). Anx anxiety condition, Anx+L anxiety condition with light stimulation, CT control animals, EXP experimental mice injected with ChR2. (PDF) Acknowledgments We thank Prof. K. Nave and Dr. S. Goebbels for kindly providing NEX-Cre mice and Prof. Tecott for 5-HT1A (-/-) mice. We thank Anja Meyer-Berhorn and Rebecca Overbeck for their help to document immunohistochemical stainings of the amygdala. We are grateful to the Herlitze laboratory, which provided laboratory space and mice. Author Contributions Conceptualization: Laura Berg, Olivia Andrea Masseck. Conceptualization: Laura Berg, Olivia Andrea Masseck. Data curation: Laura Berg. Formal analysis: Laura Berg. Funding acquisition: Olivia Andrea Masseck. Investigation: Laura Berg, Josephine Eckardt. Methodology: Laura Berg, Olivia Andrea Masseck. Project administration: Olivia Andrea Masseck. Resources: Olivia Andrea Masseck. 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https://openalex.org/W3091456879	https://www.e3s-conferences.org/10.1051/e3sconf/202019204015/pdf	English	null	Prognosys of dangerous geodynamic phenomena at the Kun-Manye field based on the analysis of digital elevation models	E3S web of conferences	2,020	cc-by	2,948	* Corresponding authors: v-i-usikov@yandex.ru E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” https://doi.org/10.1051/e3sconf/202019204015 E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” https://doi.org/10.1051/e3sconf/202019204015 E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Prob © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Prognosys of dangerous geodynamic phenomena at the Kun-Manye field based on the analysis of digital elevation models Vitaly Usikov1* 1 Mining Institute FEB RAS, 51 Turgenev st., Khabarovsk, 680000, Russia y 1 Mining Institute FEB RAS, 51 Turgenev st., Khabarovsk, 680000, Russia Abstract. The Kuhn-Magnyo copper-nickel deposit is located in the southeast of the Aldan-Stanovoy shield. Based on the results of geological exploration, the object is assessed as promising. The ore field, within which the deposit is located, is confined to a complex junction of several regional tectonic structures. The area has an intensely dissected relief, increased seismicity. Therefore, an assessment of the possibility of dangerous geodynamic phenomena during its operation is required. This work describes its preliminary assessment based on the results of morphometry on digital elevation models (DEM). Several morphostructural elements have been identified that directly determine the geodynamics in the area of the earth's crust containing the deposit. The result of the study revealed a potential hazard. Key words: geodynamics, geotectonics, digital elevation model, DEM, morphostructure, tectonic flow, relief, lineament 2 Methodology The technique is based on the construction and analysis of digital elevation models based on the SRTM30, GTOPO30, SRTM03 DEMs [5]. To identify global and regional structures, DEMs are used based on SRTM30, GTOPO30, containing the elevations of the relief, distributed on a regular grid with a step of 30 arc seconds (on the ground, this corresponds to a distance of about 1 km along the meridian and in detail to a scale of 1: 1,000,000). SRTM03 matrices have a step of 3 arc seconds, which on the ground corresponds to 90 m along the meridian and a scale of 1: 100,000. Most of the work is done in QGIS in conjunction with GRASS GIS. Some operations are more convenient to perform in other computer applications. For example, shaded relief maps or in the form of an anaglyph image are more successfully generated in Microdem [6], DEM in axonometric projection in 3DEM [7] or Landserf [8], relief profiles in Microdem or Landserf. p j p The most productive way to use a DEM is to analyze a volumetric image using an anaglyphic map (an example of an anaglyph map is shown in Fig. 1). Therefore, the following sequence of actions is performed: the matrix is loaded into Microdem, a map is generated there in three versions: anaglyphic, as well as shaded color and gray. These images are saved in GeoTiff format with georeferencing information in the header. Then they are loaded into QGIS, where thematic vector maps and schemes are created from the anaglyph image. For data output, a different basis is used: shaded ("flat") bump maps, colored or, more often, in a gray palette. The interpretation of the DEM is based on the concept of tectonic flows [9, 10, 11], adapted for the East Asia region [12]. The main part of the work was carried out with a large-scale DEM 1: 100,000 based on the SRTM03 DEM. It was used to calculate and build: a map of selective contours of the relief; a volumetric relief map in the form of an anaglyphic image; with their help, a map of the relief lineaments was compiled (Fig. 2). The distribution areas of metagabbro and the zones of mineralization and ore bodies located in them were plotted on the maps (based on materials by V.A. 1 Introduction The study area is located practically at the junction of three tectonic structural units: the Ilyun structural-facies zone of the Aldan region, the ledge of the early Archean base of the Stanovoy fold system and the Udsko-May structural-facies zone of the Stanovoy system. The area of the Kurumkansky ore field is confined to the junction of the Janinsky and Tuxanian blocks of the crystalline basement of the Aldan-Stanovoy shield. The Kun-Manyo field is located within several isolated, linearly elongated in the north-western direction areals of mafic-ultramafic intrusions. Ore bodies are composed of disseminated sulfide mineralization [1, 2]. The intrusions belong to the Kunmanyon complex of gabbro-norite- pyroxenite-peridotite formation [3, 4]. The morphological features of ore bodies force at least part of the reserves to be mined underground. Considering the structural position and the significant vertical range of mineralization, the possibility of hazardous geodynamic phenomena should be assumed. The degree of study of the territory from the point of view of neotectonics and geodynamics is low. Therefore, a preliminary assessment was made based on the morphostructural features of the areas. E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” https://doi.org/10.1051/e3sconf/202019204015 E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” 2 Methodology Guryanov [3]) Based on the entire set of data, a morphotectonic map of the region and adjacent areas was compiled (Fig 3) ( g ) Fig.1. DEM of a part of the Far east - region based on the SRTM30 DEM, visualized as an anaglyph elevation map. 1 - the various sites Fig.1. DEM of a part of the Far east - region based on the SRTM30 DEM, visualized as an anaglyph elevation map. 1 - the various sites 2 2 E3S Web of Conferences 192, 04015 (2020) E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” https://doi.org/10.1051/e3sconf/202019204015 VIII International Scientific Conference “Problems of Complex Development of Georesources” Fig. 2. Plan of the location of the Kun-Manye ore bodies. Basis: map of sparse contours based on DEM SRTM03. 1 - contour of the ore region, 2 - mafic-ultramafic rocks of the Kun-Manyon complex with sulfide mineralization (ore bodies and deposits), 3 - metagabbro (after V.A.Guryanov, 2012); contoure: 1 - 800 m, 2 - 1200 m, 3 - 1500 m, 4 - 1800 m; 4 - relief lineaments. Oblique hatching covers the system-controlling tectonic structure Fig. 3. Morphotectonic scheme of the Kun-Manye ore region and adjacent areas based on the DEM visualized as a shaded relief map (taking into account the data of V.A. 1 - epicenters of earthquakes; 2 - contour of the ore field; 3 - hydraulic network; 4 - discharges; 5 - line of separation of the hanging side from the recumbent reverce thrust; 6 - rear edge of the reverce thrust hanging side; 7 - fronts of thrust faults; 8 - currently active tectonic disturbances of unclear kinematics; 9 - contours of rigid tectonic massivs Fig. 2. Plan of the location of the Kun-Manye ore bodies. Basis: map of sparse contours based on DEM SRTM03. 1 - contour of the ore region, 2 - mafic-ultramafic rocks of the Kun-Manyon complex with sulfide mineralization (ore bodies and deposits), 3 - metagabbro (after V.A.Guryanov, 2012); contoure: 1 - 800 m, 2 - 1200 m, 3 - 1500 m, 4 - 1800 m; 4 - relief lineaments. Oblique hatching covers the system-controlling tectonic structure Fig. 2. Plan of the location of the Kun-Manye ore bodies. Basis: map of sparse contours based on DEM SRTM03. 3 Results and discussion The relief lineaments have a significant multidirectionality, although among the most extended one can distinguish a diagonal network: elements of the SE strike, having an average azimuth of approximately 119 ° and structures directed almost normal to them, northeast. In fig. 2 and 3, one can also see that over most of the area there is a breaks of the relief. All this indicates a change in the direction of lateral movement of the upper layers of the earth's crust, complicated by chaotic movements of tectonic scales relative to each other. In accordance with the accepted hypothesis, this area is located in the northern part of the Amur tectonic flow [12]. The current drift vector of the tectonic flow is southeast, as evidenced by the predominant direction of the strike of the ridges - depressions and morphostructures intersecting them. In more detail, the method for assessing the direction of movement is described in [13, 14, 15.]. Based on the interpretation, two main cycles of horizontal movements of the tectonic flow can be distinguished in the described area. The first is a submeridional drift in the southern, apparently, direction. Sublatitudinal and submeridional lineaments are indicators of this movement. In some places, they are broken by diagonal structures, which reflect the movement of the second cycle - in the southeast direction, which continues to this day. The results of GPS observations [16] are independent confirmation of the modern drift. Apparently, chaotic fluctuations of individual tectonic scales are superimposed on the general drift. Based on the foregoing, it is natural to assume that the main direction of the horizontal compression vector is southeast. The results of earlier studies in other areas of the Amur Stream suggest that the time of the beginning of the second cycle - the end of the Cretaceous - the beginning of the Paleogene [12]. In the course of work on the study of the relief structure in the region, the presence of rigid tectonic massifs within the limits of tectonic flows was revealed. Visually, on the DEM, they are confidently distinguished as flattened, plateau-like morphostructures within the orogenic belts (areas with a less dissected and smoothed relief in comparison with neighboring, usually surrounding areas). The most clearly such objects are displayed on the anaglyph elevation map, as well as on the contour map. E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” https://doi.org/10.1051/e3sconf/202019204015 E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” 2 Methodology 1 - contour of the ore region, 2 - mafic-ultramafic rocks of the Kun-Manyon complex with sulfide mineralization (ore bodies and deposits), 3 - metagabbro (after V.A.Guryanov, 2012); contoure: 1 - 800 m, 2 - 1200 m, 3 - 1500 m, 4 - 1800 m; 4 - relief lineaments. Oblique hatching covers the system-controlling tectonic structure covers the system-controlling tectonic structure Fig. 3. Morphotectonic scheme of the Kun-Manye ore region and adjacent areas based on the DEM visualized as a shaded relief map (taking into account the data of V.A. 1 - epicenters of earthquakes; 2 - contour of the ore field; 3 - hydraulic network; 4 - discharges; 5 - line of separation of the hanging side from the recumbent reverce thrust; 6 - rear edge of the reverce thrust hanging side; 7 - fronts of thrust faults; 8 - currently active tectonic disturbances of unclear kinematics; 9 - contours of rigid tectonic massivs y g Fig. 3. Morphotectonic scheme of the Kun-Manye ore region and adjacent areas based on the DEM visualized as a shaded relief map (taking into account the data of V.A. 1 - epicenters of earthquakes; 2 - contour of the ore field; 3 - hydraulic network; 4 - discharges; 5 - line of separation of the hanging side from the recumbent reverce thrust; 6 - rear edge of the reverce thrust hanging side; 7 - fronts of thrust faults; 8 - currently active tectonic disturbances of unclear kinematics; 9 - contours of rigid tectonic massivs 3 3 3 Results and discussion Usually they have no connection with the composition of rocks: they are composed of both igneous and metamorphosed sedimentary, effusive formations. They are split by a system of rare tectonic fractures, which are usually active. Such massifs concentrate and accumulate within themselves and nearby horizontal stresses of a tectonic nature. During the development of deposits located within the limits of their influence, dangerous geodynamic events can be initiated [17]. In the area under consideration, in the northern part of the plate, rigid rock massifs are also distinguished, which are confidently identified by smoothed relief forms. In fig. 2 these are areas with a practical absence of contours. A small massif in the northeastern corner is composed of Proterozoic granites. A large massif located in the northern part of the plate has a complex composition of rocks: granitoids, gneisses, diafluorites, tuffs, metagabbro [3]. It is split by active faults 1 and 2 (in Fig. 3). From the south, during the first cycle, a tectonic cover with front 1 [3] was pulled over it. The cover is deformed by diagonal structures of higher ranks formed as a result of the second cycle. The largest structure of this cycle is indicated by a lineament, which is shown on the plan by oblique shading (Fig. 2). Analysis of the DEM allowed us to assume that the southwestern part of the structure is thrust 2, which along the strike in the NE direction, after the intersecting fault 1, is replaced by a reverse thrust. Such phenomena are observed in other areas of the tectonic flow [13]. Mineralization is probably concentrated in the zone of influence of a rigid massif. Therefore, it is logical to assess the probability of dangerous geodynamic phenomena during field development as potentially high. Perhaps the diagonal structure described above reduces the influence of the massif to the southeast and this probability decreases. 4 4 E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” https://doi.org/10.1051/e3sconf/202019204015 E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Pro E3S Web of Conferences 192, 04015 (2020) https://doi.org/10.1051/e3sconf/202019204015 VIII International Scientific Conference “Problems of Complex Development of Georesources” p g One more fact should be noted. The vertical spread of mineralization for different ore bodies varies widely. From 100 m ("Triangle", "Small Kurumkan") to more than 1500 m on the Yan-Khegde section (according to V.A. Guryanov). 3 Results and discussion The interval of relief heights is also significant: 700 - 1000 m, which can lead to the appearance of an uncompensated horizontal component of geostatic stress. 4 Conclusion The analysis and interpretation of the DEM in the area of the Kurumkansky ore field, which contains the Kun-Manyo copper-nickel deposit, made it possible to identify the main tectonic structures that affect the geodynamic regime of the territory. The field is located within the Amur tectonic flow, drifting in the SE direction. The main structural element that determines its geodynamics is a rigid tectonic massif. Such massifs usually concentrate and accumulate horizontal stresses of a tectonic nature within themselves and nearby. And the stress-strain state of rocks provokes dangerous geodynamic events during the development of mineral deposits. Therefore, the object should be classified as potentially hazardous. As a consequence, it is necessary to carry out a special cycle of work to assess this hazard. 17. I.YU. Rasskazov, V.I. Usikov, B.G. Saksin, Tektonika, glubinnoye stroyeniye i minerageniya Vostoka Azii. materialy Vserossiyskoy konferentsii s mezhdunarodnym uchastiyem. Federal'noye gosudarstvennoye byudzhetnoye uchrezhdeniye nauki Inststitut tektoniki i geofiziki im. YU.A. Kosygina DVO RAN Dal'nevostochnogo otdeleniya Rossiyskoy Akademii nauk. 300-302 (2019) E3S Web of Conferences 192, 04015 (2020) VIII International Scientific Conference “Problems of Complex Development of Georesources” https://doi.org/10.1051/e3sconf/202019204015 References 1. V.A. Gur'yanov, V.S. Prikhod'ko, A.N. Perestoronin, L.L. Petukhova , YU.P. Pototskiy, L.P. Sobolev, Doklady Akademii nauk, 425(4), 505 – 508 (2009) 2. V.A. Gurianov, V.S. Prikhodko, V.I. Gvosdev, A.N. Perestoronin, L.L. Petukhova, Platinum of Russia. Colltction of Transactions. VII, 435-448 (2011) 3 A.YU. Peskov , A.N. Didenko, V.A. Gur'yanov, Tikhookeanskaya geologiya, 37(5), 3–15 (2018) y ( ) 2. V.A. Gurianov, V.S. Prikhodko, V.I. Gvosdev, A.N. Perestoronin, L.L. Petukhova, Platinum of Russia. Colltction of Transactions. VII, 435-448 (2011) 3 A.YU. Peskov , A.N. Didenko, V.A. Gur'yanov, Tikhookeanskaya geologiya, 37(5), 3–15 (2018) 4 V. A. Gur'yanov, V. S. Prikhod'ko, A.YU. Peskov, L.L. Petukhova, A.N. Perestoronin, A.V. Kosynkin, Otechestvennaya geologiya, 6, 48 – 55 (2014) 5. [Elektronnyy resurs] https://dds.cr.usgs.gov/srtm/version2_1/ (srtm03 v1,2.1, srtm30) 6. [Elektronnyy resurs] 6. [Elektronnyy resurs] https://www.usna.edu/Users/oceano/pguth/website/microdem/microdem.htm [ yy ] https://www.usna.edu/Users/oceano/pguth/website/microdem/microdem.htm 7. [Elektronnyy resurs] http://www.hangsim.com/3dem/ Elektronnyy resurs] http://www.staff.city.ac.uk/~jwo 8. [Elektronnyy resurs] http://www.staff.city.ac.uk/~jwo/landserf/download/ 9 l k h k k l f l d 9. Lateral'nyye tektonicheskiye potoki v litosfere Zemli Otv. red. M.G. Leonov – M.: GEOS, 318 s. (Tr. GIN RAN; Vyp. 604, 2013) 10. Ye.I. Patalakha, A.M. Lukiyenko, V.V. Gonchar, Tektonicheskiye potoki kak osnova ponimaniya geologicheskikh struktur. Kiyev: Feniks, 160 р. (1995) 11. A.V. Smirnov, Geodinamika formirovaniya podvizhnykh poyasov Zemli. Materialy mezhdunarodnoy nauchnoy konferentsii. Yekaterinburg: Institut geologii i geokhimii UrO RAN, 285-289 (2007) 12. V.I. Usikov, Tektonika, magmatizm i geodinamika Vostoka Azii: VII Kosyginskiye chteniya: materialy vserossiyskoy konferentsii, 12–15 sentyabrya 2011, g. Khabarovsk/otv. red. A.N. Didenko, YU.F. Manilov. – Khabarovsk: ITiG im.YU.A. Kosygina DVO RAN, 328 — 331 (2011) 13. V.I. Usikov, Russian Journal of Pacific Geology. 5(6), 492-508 (2011) 13. V.I. Usikov, Russian Journal of Pacific Geology. 5(6), 492-508 (2011) 14. I.YU. Rasskazov, B.G. Saksin, M.I. Potapchuk, V.I. Usikov, Fiziko-tekhnicheskiye problemy razrabotki poleznykh iskopayemykh, 1, 13-22 (2014) , gy ( ), ( ) 14. I.YU. Rasskazov, B.G. Saksin, M.I. Potapchuk, V.I. Usikov, Fiziko-tekhnicheskiye problemy razrabotki poleznykh iskopayemykh, 1, 13-22 (2014) 15. I.YU. Rasskazov, B.G. Saksin, V.A. Petrov, B.F. Shevchenko, V.I.Usikov, G.Z. Gil'manova, Fizika Zemli, 3, 144-153 (2014) 15. I.YU. Rasskazov, B.G. Saksin, V.A. Petrov, B.F. Shevchenko, V.I.Usikov, G.Z. Gil'manova, Fizika Zemli, 3, 144-153 (2014) 16. S.V. Ashurkov, Geologiya i geofizika, 57(11), 2059—2070 (2016) , , , ( ) 16. S.V. Ashurkov, Geologiya i geofizika, 57(11), 2059—2070 (2016) 5 5 17. I.YU. Rasskazov, V.I. Usikov, B.G. Saksin, Tektonika, glubinnoye stroyeniye i minerageniya Vostoka Azii. materialy Vserossiyskoy konferentsii s mezhdunarodnym uchastiyem. References Federal'noye gosudarstvennoye byudzhetnoye uchrezhdeniye nauki Inststitut tektoniki i geofiziki im. YU.A. 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W4220746600.txt	https://www.frontiersin.org/articles/10.3389/fenrg.2022.819939/pdf	en		Simulation and Study of PEMFC System Directly Fueled by Ammonia Decomposition Gas	Frontiers in energy research	2,022	cc-by	8,886	ORIGINAL RESEARCH published: 22 March 2022 doi: 10.3389/fenrg.2022.819939 Simulation and Study of PEMFC System Directly Fueled by Ammonia Decomposition Gas Jian Feng Zhao , Qian Chao Liang and Yi Fan Liang College of Power Engineering, Naval University of Engineering, Wuhan, China Edited by: Jinliang Yuan, Ningbo University, China Reviewed by: Shanwen Tao, University of Warwick, United Kingdom Yusuf Bicer, Hamad bin Khalifa University, Qatar Correspondence: Jian Feng Zhao jianfeng62300_zhao@163.com Specialty section: This article was submitted to Fuel Cells, a section of the journal Frontiers in Energy Research Received: 22 November 2021 Accepted: 14 February 2022 Published: 22 March 2022 Citation: Zhao JF, Liang QC and Liang YF (2022) Simulation and Study of PEMFC System Directly Fueled by Ammonia Decomposition Gas. Front. Energy Res. 10:819939. doi: 10.3389/fenrg.2022.819939 Ammonia can be stored as a liquid under relatively easy conditions (Ambient temperature by applying 10 bar or Ambient pressure with the temperature of 239 K). At the same time, liquid ammonia has a high hydrogen storage density and is, therefore, a particularly promising carrier for hydrogen storage. At the same time, the current large-scale industrial synthesis of ammonia has long been mature, and in the future, it will be possible to achieve a zero-emission ammonia regeneration cycle system by replacing existing energy sources with renewable ones. Ammonia does not contain carbon, and its use in fuel cells can avoid NOx production during energy release. high temperature solid oxide fuel cells can be directly fueled by ammonia and obtain good output characteristics, but the challenges inherent in high temperature solid oxide fuel cells greatly limit the implementation of this option. Whereas PEMFC has gained initial commercial use, however, for PEMFC, ammonia is a toxic gas, so the general practice is to convert ammonia to pure hydrogen. Ammonia to hydrogen requires decomposition under high temperature and puriﬁcation, which increases the complexity of the fuel system. In contrast, PEMFC that can use ammonia decomposition gas directly can simplify the fuel system, and this option has already obtained preliminary experimental validation studies. The energy efﬁciency of the system obtained from the preliminary validation experiments is only 34–36%, which is much lower than expected. Therefore, this paper establishes a simulation model of PEMFC directly using ammonia decomposition gas as fuel to study the maximum efﬁciency of the system and the effect of the change of system parameters on the efﬁciency, and the results show that the system efﬁciency can reach up to 45% under the condition of considering certain heat loss. Increasing the ammonia decomposition reaction temperature decreases the system efﬁciency, but the effect is small, and the system efﬁciency can reach 44% even at a temperature of 850°C. The results of the study can provide a reference for a more scientiﬁc and quantitative assessment of the potential value of direct ammonia decomposition gas-fueled PEMFC. Keywords: ammonia, ammonia decomposition gas, PEMFC, efﬁciency, simulation Frontiers in Energy Research \| www.frontiersin.org 1 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas INTRODUCTION into pure hydrogen, so the whole power generation system contains the following subsystems: decomposition of ammonia, puriﬁcation of hydrogen, fuel cell, etc.(Araya et al., 2016). Alessandra Perna et al.(Perna et al., 2020)studied a distributed on-site hydrogenation system based on ammonia supply from an energy and economic point of view. The system’s core is a hydrogen production system containing an ammonia cracking reactor and its auxiliary equipment. The electrical energy demand (i.e., hydrogen compression and cooling) necessary for the power station’s operation is met by a PEMFC power module. The energy performance was evaluated based on the daily demand of hydrogen, and the balance cost of hydrogen was estimated. Kyunghwa Kim et al. (Kim et al., 2020) investigated the economics and environmental friendliness of ammonia-fueled PEMFC propulsion systems for a 2,500 Twenty-foot Equivalent Unit (TEU) container feeder ship demand. Li et al. developed a physicochemical system model of a 300 Nm3 h−1 on-site ammonia hydrogen production station based on experiments. They analyzed the efﬁciency and hydrogen production capacity of eight H2 puriﬁcation subsystems (Lin et al., 2020). At present, the research hotspots of ammonia-fueled PEMFC systems focus on the efﬁcient decomposition of ammonia and the on-site puriﬁcation of hydrogen. The research on the efﬁcient decomposition of ammonia includes the study of efﬁcient catalysts (Fujitani et al., 2020; Sayas et al., 2020), microﬂuidic reactors (Kim and Kwon, 2011), and process optimization (Badescu, 2020). For catalysts, Ni-loaded catalysts with Al2O3 are still the most stable, economical, and efﬁcient catalysts for ammonia decomposition. Although various combinations of Ru can obtain better results, they are less durable and cannot work for a long time, and Ru is a precious metal, which is not very economical. The microﬂuidic reactor can signiﬁcantly enhance the heat transfer between the reaction unit and the heating unit and improve the reactor’s volumetric hydrogen production rate and energy utilization efﬁciency. The process optimization study includes the study of the reaction mechanism and the optimization of the reactor boundary conditions (e.g., geometry conﬁguration, heating conditions, etc.), which can provide a reference for developing more efﬁcient catalysts and reactor structures. Research on hydrogen puriﬁcation includes variable pressure adsorption (Zhang et al., 2021), deep cold adsorption (Aasadnia et al., 2021), and membrane separation technology (Iulianelli et al., 2019). Compared with VPS and membrane separation technologies, deep-cooled adsorption requires a large amount of energy to maintain low temperatures and is only suitable for large-scale production. The hot spots of research on hydrogen separation in small-scale on-site hydrogen production are mainly variable pressure adsorption and membrane separation technologies. Currently, large-scale VPS equipment can achieve a 90% hydrogen recovery rate. Still, for small-scale onsite hydrogen production scenarios, there are problems of large equipment systems and high inlet pressure (Lin et al., 2020). Membrane separation technology can reduce the size of separation equipment and is, therefore, a potential solution for hydrogen separation in small-scale on-site hydrogen production systems. Current selective separation membranes can be divided Ammonia can be stored as a liquid under relatively easy conditions [Ambient temperature by applying 10 bar (Aziz et al., 2020) or Ambient pressure with the temperature of 239 K (Iverson et al., 2018)]. Liquid ammonia has a high hydrogen storage density, 17.8 wt% by mass and 121 Kg H2 m−3 by volume, so it is a particularly promising hydrogen storage carrier. The large-scale industrial synthesis of ammonia has long been mature, and in the future, it can also be used to achieve zero-emission renewable energy cycle storage systems through renewable energy sources (Cinti et al., 2020). Currently, facilities for ammonia transportation and storage exist in society, so it can be easily and economically transported to its destination (Mckinlay et al., 2021). Ammonia contains no elemental carbon and therefore has no carbon emissions. The synthesis of ammonia requires nitrogen and hydrogen. Nitrogen can be obtained in large quantities from the air, hydrogen can be obtained from the electrolysis of water, and ammonia is regenerated into nitrogen and water in the process of releasing energy, so the whole process can achieve harmless emissions (Yapicioglu and Dincer, 2019). The electrochemical synthesis of ammonia at low temperature and low pressure is also being studied in recent years (Lu et al., 2018; Garagounis et al., 2019). Therefore the construction of an ammonia-based zero-emission renewable energy system has a good prospect. Ammonia does not contain carbon and therefore has no carbon emissions, but the use of ammonia by direct combustion can lead to the generation of NOx, which can cause damage to the environment (Naha et al., 2019). In contrast, the use of ammonia in fuel cells avoids the generation of NOx. For indirect ammonia fuel cells, ammonia is ﬁrst converted to nitrogen and hydrogen under medium temperature conditions, thus avoiding the generation of NOx. For direct ammonia fuel cells, even for high-temperature solid oxide fuel cells (SOFC), the operating temperature is 700–800°C, which is far below the NOx generation temperature (Wu, et al., 2020), and relevant studies have demonstrated that SOFC can avoid NOx generation when using ammonia as fuel (Barelli et al., 2020; Ni et al., 2010). Although high temperature solid oxide fuel cells (SOFC) can directly use ammonia as fuel (Hashinokuchi et al., 2018; Okanishi et al., 2017; Zeng et al., 2020), SOFC is still in the research and development stage, with disadvantages such as start-up time, low lifetime, and high cost, and has not yet entered commercial applications. In contrast, the proton exchange membrane fuel cell (PEMFC) has already gained initial commercial application and has made signiﬁcant progress in terms of start-up time, lifetime, and cost (Wang et al., 2019). Therefore, the use of ammonia gas as the hydrogen storage medium for PEMFC is gradually attracting attention. Since ammonia can poison the acidic proton exchange membrane and the cathode catalyst (Imamura et al., 2011; Lopes et al., 2014), PEMFC is very sensitive to ammonia. The residual amount of ammonia in the fuel should be less than 0.1 ppm (Miyaoka et al., 2018), so ammonia cannot be used directly in PEMFC. The current method is to convert ammonia Frontiers in Energy Research \| www.frontiersin.org 2 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas FIGURE 1 \| System structure and procedure. into two categories: ﬁrst, metallic membranes, mainly palladium membranes, with high permeability and selectivity, suitable for high-purity hydrogen production but expensive. Dolan et al. have developed tubular palladium-coated vanadium membranes. The developed membranes exhibit high permeability (above 3.0 × 10−7 mol/ms·Pa0.5at temperatures above 320°C) and robustness. Furthermore, vanadium is a relatively inexpensive material, the separation cost of such membranes is lower compared to single Pd-based membranes (Dolan et al., 2018); secondly, polymer membranes are cheap but have lower permeability and selectivity and are not suitable for ultra-high purity hydrogen production (Galizia et al., 2017). In recent years, it has been shown that combining a variable pressure adsorption device with membrane separation is expected to obtain a high recovery and economical method for high-purity hydrogen production, which can solve the problem of high-purity hydrogen separation for small-scale on-site hydrogen production systems (Lin et al., 2020; Li et al., 2016). In conclusion, the puriﬁcation technique of hydrogen requires complex equipment, which is very unfavorable for small on-site hydrogen production scenarios. The strategy of feeding the ammonia decomposition gas, which has undergone residual ammonia removal, directly to the PEMFC will signiﬁcantly simplify the system (referred to as ADG-PEMFC in this paper). In this regard, some scholars have already started to conduct preliminary studies. Alagharu et al. (Alagharu et al., 2010) analyzed a 100 W ADG-PEMFC system with an electrically heated ammonia decomposer as a component. The system’s total output power was 142 W, the ammonia ﬂow rate was 0.0009 mol/s, and the efﬁciency was 35%. Hunter et al. established an ADG-PEMFC demonstration platform based on an ammonia decomposer heated by ammonia catalytic combustion. They demonstrated the feasibility of ADG-PEMFC, but the energy efﬁciency obtained was only 34% (Hunter et al., 2016). Cha et al. (Cha et al., 2018) established a 1 kW ADG-PEMFC demonstration system, and the heat required for the ammonia decomposer reaction was provided by isobutane combustion, and the system efﬁciency obtained was about 32–36%. The simulations showed that the system efﬁciency could reach 49% when the fuel cell anode tail gas reuse was considered. Frontiers in Energy Research \| www.frontiersin.org The efﬁciency of the existing experimental system is much lower than that of the fuel cell, and it is difﬁcult to reﬂect the advantages of ammonia, so it is necessary to conduct in-depth research and analysis on the ideal efﬁciency of the system and related factors. This paper establishes an ADG-PEMFC simulation model to study and analyze the maximum efﬁciency of the system and the effect of changes in system parameters on the efﬁciency so that the potential value of ADG-PEMF can be quantitatively evaluated more scientiﬁcally. SYSTEM STRUCTURE AND PROCEDURE The system structure and process are shown in Figure 1. Firstly, the ammonia gas is evaporated and preheated through the Evaporator and Recuperator 1. It enters Recuperator 2 to raise the required temperature for the decomposition reaction. It then enters the Cracker for catalytic decomposition, the decomposition gas is cooled through the Recuperator 1 and Evaporator, and at the same time, it provides energy for the gasiﬁcation and preheating of liquid ammonia. Because the latent heat of gasiﬁcation of liquid ammonia is large, theoretically, it can cool the hightemperature decomposition gas to the same temperature. After that, the decomposition gas enters the Absorber to remove the residual ammonia and obtain a pure hydrogennitrogen mixture (crude hydrogen), which is sent to the PEMFC for electrochemical reaction to generate electric energy, and then the anode exhaust gas enters the Combustor to generate high-temperature gas by catalytic combustion with air. The high-temperature gas is sent to the Cracker to provide the required energy for the ammonia decomposition reaction. It then ﬂows through Recuperator 2 and Recuperator 3 to heat the ammonia entering the Cracker and the air and fuel entering the Combustor, respectively. When the high-temperature gas generated by the anode tail gas alone cannot meet the heat demand of the ammonia decomposition reaction, additional energy will be added to the Combustor by adding additional ammonia gas and corresponding air. 3 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas SYSTEM MODELING TABLE 1 \| Speciﬁc heat of air (273 ~ 1800K). The links in the system can be categorized as the Evaporator module, Recuperator module, Cracker module, Absorption module, Combustor module, and the PEMFC module. Also, to simplify the model, the following assumptions are made: H2 N2 NH3 O2 H 2O 1) Ignore the pipeline heat loss and resistance loss, so the ﬂuid thermal parameters on the pipeline between modules do not change. In this paper, the pipelines between modules in the system are numbered to identify the gas status parameters of each module. 2) The heat loss of each module of the system is considered and expressed as the ratio of the heat transferred in the module to the max total heat that can be provided by the hightemperature heat source in the module. a0 a1102 a2105 a3109 29.11 28.90 27.568 25.48 32.24 −0.1916 −0.1571 2.5630 1.520 0.1923 0.4003 0.8081 0.99072 -0.7155 1.055 −0.8704 −2.873 −6.6909 1.312 −3.595 1.5xcH2 + 0.5xcN2 + (1 − x)cNH3 (T5 − T7 )ηex1 cNH3 (T3 − T1 ) + γ (6) At constant pressure, the approximate relationship between speciﬁc heat capacity and temperature can be expressed as follows: Cp a0 + a1 T + a2 T2 + a3 T3 Evaporator and Recuperator 1 Modeling (7) The role of the Evaporator and the Recuperator 1 is to use the waste heat of the high-temperature ammonia decomposition gas to gasify and preheat the liquid ammonia. Since this is a continuous process, the two modules are modeled together in this paper. According to the conservation of energy: The parameters corresponding to the group elements involved in this paper are shown in Table 1 (Kyle, 1984). Equation 7 shows that the constant pressure speciﬁc heat capacity is a function of temperature, so Eq. 6 needs to be changed to an integral form as follows: G5 c5 T5 − G7 c7 T7 )ηex1 G3 c3 T3 − G1 c1 T1 + G1 γ), G5 G7 , G3 ηex1 1.5xcH2 + 0.5xcN2 + (1 − x)cNH3 dT cNH3 dT + γ T5 T3 T7 T1 G1 (8) (1) Recuperator 2 Modeling Where, ci the constant pressure speciﬁc heat capacity of the gas (unit: J/(mol-K)), Gi is the gas ﬂow rate (unit: mol/s), γ is the latent heat of gasiﬁcation of liquid ammonia (unit: J/mol), ηex1 is the heat transfer efﬁciency of the Evaporator and the Recuperator 1, which is equal to the product of the heat transfer efﬁciency of the Evaporator and the Recuperator 1, i = 1,2,. . .,n. Since in the Recuperator 1, the substance of the lowtemperature ﬂuid is NH3, and the substances of the hightemperature ﬂuid include NH3, N2, and H2, the constant pressure speciﬁc heat capacity and ﬂow rate of different substances are not the same, so for Eq. 1 needs to be further modiﬁed as: The role of the Recuperator two is to use high-temperature gas to preheat ammonia to the temperature required for the decomposition reaction, the substance of the low-temperature ﬂuid is NH3, and the substances of the high-temperature ﬂuid include O2, N2, H2O. With reference to formula (2), the conservation of energy equation for the Recuperator two is shown below: T14 T4 ⎝ G14,m cm ⎞ ⎠dT G3 cNH dT , m ηex2 ⎛ 3 T15 m T3 N2 , O2 , H2 O, G14 G15 , G3 G4 (9) G5,j cj (T5 − T7 )ηex1 G3 c3 T3 − c1 T1 + γ, j NH3 , N2 , H2 where ηex2 is the heat transfer efﬁciency of the Recuperator 2. j Recuperator 3 Modeling (2) Due to the following relationship between the molar ﬂow of substances for low-temperature ﬂuids and high-temperature ﬂuids: G5,NH3 (1 − x)G3 G5,N2 0.5xG3 G5,H2 1.5xG3 The role of Recuperator 3 is to use the waste heat of hightemperature gas to heat the fuel and air entering the combustor, which is the same as the role of micro gas turbine’s Recuperator (McDonald, 2000). “Regenerator effectiveness” is used to calculate the thermal parameters of Recuperator 3 refer to the thermal calculation of micro gas turbine’s Recuperator. Regenerator effectiveness The ratio of the actual heat absorbed by the low-temperature ﬂuid in the recuperator to the maximum heat released to the low-temperature ﬂuid from the hightemperature ﬂuid under ideal conditions in the recuperator. (3) (4) (5) Where x is the decomposition rate of ammonia, substituting Eqs 3–5 into Eq. 2 yields: Frontiers in Energy Research \| www.frontiersin.org 4 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas α cp,c Tc,out − Tc,in cp,h Th,in − Tc,in TABLE 2 \| Molar ﬂow rate values for each component during the decomposition reaction. (10) Component Where cp,c is the heat capacity at a constant pressure of lowtemperature ﬂuid (SI unit: J/(molk)), cp,h is the heat capacity at a constant pressure of high-temperature ﬂuid (SI unit: J/(molk)), Tc,out, Tc,in is the outlet and inlet temperature of the lowtemperature ﬂuid (SI unit: k), Th,out, Th,in is the outlet and inlet temperature of the high-temperature ﬂuid (SI unit: k). Assumption cp,c = cp,h (McDonald, 2000), Eq. 1 can be rewritten as: α Tc,out − Tc,in Th,in − Tc,in NH3 H2 N2 Total molar ﬂow rate α0 + (1 − α0 ) qr q0 0.2 (12) 1−x P 1+x 1.5x P 1+x 0.5x P 1+x PNH3 (16) PH2 (17) (13) PN2 (18) This leads to the equilibrium constant of the decomposition reaction Keq: (14) Keq Cracker Modeling The ammonia cracker contains two critical links: the decomposition of ammonia and the energy transfer between the heating gas and the reaction gas. And the two links are coupled with each other. The decomposition of ammonia gas involves chemical reaction kinetics, molecular diffusion kinetics, and ﬂuid mechanics, while the energy transfer involves heat transfer and ﬂuid mechanics. Since the goal of the study in this paper is the overall ideal performance of the system, therefore, assuming that the reactor can ensure that the ammonia decomposition reaction reaches the equilibrium state, ignore the inﬂuence of molecular diffusion as well as ﬂuid ﬂow on the decomposition reaction, and only consider the chemical reaction kinetics and heat transfer. Then the cracker model can be separated into two submodules, one for the decomposition reaction of ammonia gas and one for the heat transfer between hot and cold ﬂuids. The two submodules are linked together by energy conservation. Frontiers in Energy Research \| www.frontiersin.org (15) The initial reactants are only NH3, and the pressure in the Plug ﬂow reactor is a constant value P. The reaction rate of ammonia gas decomposition is x. Table 2 shows the component changes during the decomposition reaction. The state point I is the reactor inlet and the state point II is the reactor outlet. Because it is a plug ﬂow reaction, it can be regarded as a constant pressure reaction, the equilibrium partial pressure of each component at state point II is as follows: From Eq. 13, the inlet and outlet temperature model of heat exchanger 3 can be obtained as: T12 [(T15 − T11 )α + T11 ](1 − σ) (1-x) G4 1.5x G4 0.5x G4 (1 + x) G4 2NH3 ↔ N2 + 3H2 , ΔH298K 46kJmol Where α0 is the regenerator effectiveness of rated condition, qr is the actual ﬂow rate, q0 is the rated ﬂow rate (SI unit: L/min). Since the recuperator is not ideally adiabatic in the heat transfer process, the recuperator also exchanges heat with the external environment. This loss is mainly determined by the external environment. It can be assumed that the heat loss from the recuperator to the external environment remains constant and independent of the operating conditions. The heat loss rate δ at the rated state can be taken as 2% in a standard environment. The outlet temperature of low-temperature ﬂuid: Tc,out Th,in − Tc,in α + Tc,in (1 − σ) G4 0 0 G4 The decomposition reaction of ammonia will absorb a lot of heat, so increasing the reaction temperature will signiﬁcantly promote the decomposition reaction of ammonia. The reaction of ammonia decomposition is as follows: Under the rated condition, the regenerator effectiveness can generally be set as 0.9. For counter-ﬂow heat exchanger, the regenerator effectiveness under partial operating conditions is α Ⅱ The meaning of the italic values is explained in the “Evaporator and Recuperator 1 Modeling”section. (11) α0 Ⅰ P3H2 PN2 1.6875x4 P2 2 PNH3 (1 − x)2 (1 + x)2 (19) The equilibrium constant is a function of temperature and pressure. Since the pressure in the system is atmospheric, the effect of pressure is not signiﬁcant, so the effect of pressure on the equilibrium constant is ignored, and according to the law of thermodynamics, the equilibrium constant can be expressed as: lnKeq − ΔG0T RT (20) Where ΔG0T is the Gibbs free energy of the decomposition reaction. ΔG0T ΔH0T − T × ΔS0T ΔH0T vi H0i,T , i ΔS0T vi S0i,T i (21) (22) T H0i,T H0i,T0 + ci dT, T0 298.13K, i NH3 , N2 , H2 . (23) T0 5 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas Where w is the regeneration energy consumption constant [SI unit:J/(sL)], which equal to the ratio of the energy consumed by the regeneration of the adsorbent to the mass of absorbed ammonia in one cycle. TABLE 3 \| Enthalpy and entropy (T0 = 298.13 K). NH3 H2 N2 S0i,T S0i,T0 + T T0 H0i,T0 /J/mol S0i,T0 /(J/(mol · K)) −46190 0 0 192.33 130.57 191.50 PEMFC Moduling The conventional PEMFC modeling approach is based on the fuel cell electrochemical reaction model, ﬂow, diffusion, and heat transfer theory. The empirical parameters in the model are then corrected based on experimental data to achieve better modeling accuracy. However, the output characteristics of the cell are affected by both current and fuel ﬂow when crude hydrogen is used as fuel, which leads to difﬁculties in the above modeling method. Therefore, this paper uses interpolation through experimental data to obtain the PEMFC module power, efﬁciency, and relevant output parameters. Figure 2 shows the experimental data of PEMFC obtained by the experiment. ci dT, T0 298.13K, i NH3 , N2 , H2 . (24) T The enthalpy and entropy values of the gas at temperature T0 are shown in Table 3 (Wark, 1977). In the case of constant pressure, the approximation of speciﬁc heat capacity versus temperature can be expressed as Eq. 7, The parameters corresponding to the components involved in this paper are shown in Table 1. The energy absorbed by the decomposition reaction is Q xG4 × ΔH0T (25) Combustor Moduling For high-temperature gases that provide heat for the decomposition reaction, the heat released is calculated as follows: In this paper, since only the system characteristics in the ideal state are discussed, the complex mass transfer process, heat transfer process, and chemical reaction process involved in catalytic combustion are not considered, and the gas temperature is theoretically related only to the state of the inlet and outlet substances, independent of other factors. Therefore the exit temperature of the combustion chamber can be obtained by the energy conservation of the inlet and outlet substances in the catalytic combustion chamber. In the combustion chamber, the following reactions mainly occur. T13 ⎠dT, m N2 , O2 , H2 O, G14 G13 ⎛ G14,m cm ⎞ Q ηex3 ⎝ T14 m (26) Where ηex3 is the heat transfer efﬁciency of the decomposer. According to the second law of thermodynamics, the outlet temperature of the heat source ﬂuid must not be lower than the inlet temperature of the reactants. If it is not satisﬁed, the inlet temperature of the heat source ﬂuid is increased by adding supplementary combustion ammonia and recalibrating the calculation. 2H2 + O2 2H2 O 4NH3 + 3O2 6H2 O + 2N2 2NH3 + 2O2 3H2 O + N2 O (29) (30) (31) Ammonia Absorption Moduling In this paper, porous material adsorption is used to absorb residual ammonia, so the main effects on the absorption process are ﬂow resistance loss, mass loss, and energy consumption for adsorbent regeneration. 1) Mass loss G8 G7 − λG7,NH3 (27) Where λ is the absorption rate of ammonia, because after puriﬁcation, the content of ammonia is extremely low and can be approximated as λ = 1. 2) Energy consumption The energy loss in the separation process of ammonia is mainly due to the additional energy consumed for the regeneration of the adsorbent, which can be averaged over a complete working cycle. Therefore the energy loss can be simply expressed as a linear relationship. Elost wpG7,NH3 Frontiers in Energy Research \| www.frontiersin.org FIGURE 2 \| Voltage-current characteristic curve of PEMFC under different crude hydrogen ﬂow rates. (28) 6 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas RESULTS AND DISCUSSION TABLE 4 \| System parameters. Parameters Unit Value Ammonia ﬂow rate (Standard state) Ammonia decomposition reaction temperature, T4 Temperature of liquid ammonia, T1 Regenerator effectiveness of Recuperator 3, α0 Energy efﬁciency of Recuperator 1, ηex1 Energy efﬁciency of evaporator, ηev Energy efﬁciency of Recuperator 2, ηex2 Energy efﬁciency of cracker, ηex3 Combustion efﬁciency of combustor, ηcom Energy efﬁciency of combustor, ηie The regeneration energy consumption constant, w excess air coefﬁcient PEMFC operating temperature, T8 L/min °C °C 6.5 275–800 25 0.9 0.9 0.9 0.9 0.9 0.99 0.9 108 1.3 50 J/(s*L) 4NH3 + 5O2 6H2 O + 4NO °C Analysis of the Inﬂuence of Ammonia Decomposition Temperature and PEMFC Current on the System Table 4 shows the initial system parameters. The ammonia decomposition temperature and the current of the PEMFC are critical operating parameters of the system, and both affect the system’s efﬁciency. Figure 3 shows the efﬁciency of the system at different temperatures and currents. According to the current of PEMFC, the value of system efﬁciency with temperature can be divided into three intervals. When the current is in the interval of 0–19 A, the temperature has little effect on the system efﬁciency. When the current is in the interval of 19–26.5 A, the system efﬁciency increases and then decreases with temperature, but the decrease is tiny. The system efﬁciency decreases sharply at the low-temperature end when the current is greater than 26.5 A. The reason for this phenomenon can be further analyzed by Figure 4. In the low current interval, the remaining hydrogen in the anode is more, which can provide sufﬁcient energy for the ammonia decomposer, as shown in curves 4/5/6 of Figure 4, so the ammonia ﬂow rate of the supplemental combustion is zero. And at this time, the fuel in the PEMFC is sufﬁcient, and the PMEFC working current is in the ohmic polarization interval. Hence, the PEMFC output power corresponding to different ammonia decomposition temperatures is the same under the same current, as shown in curve 1/2/3 of Figure 4. Therefore, the system efﬁciency does not change with the reaction temperature. Increasing the current of PEMFC, different phenomena appear at the two ends of the reaction temperature, ﬁrstly, the lowtemperature end, at the lower decomposition temperature due to the signiﬁcant reduction of ammonia decomposition rate, the residual undecomposed ammonia greatly increased, resulting in the ﬁnal total amount of fuel into the PEMFC is reduced, according to the experimental results in Cracker Modeling, it is known that the current of PEMFC has entered the concentration polarization inﬂuence region at this time. Therefore, the output power of PEMFC decreases signiﬁcantly, as shown in curve 7. At the same time, because the tail gas of the anode cannot provide enough energy for the decomposer, it has to supplement the ammonia gas for the combustion chamber, which further leads to the decrease in the efﬁciency of the system, as shown in the curve eight of Figure 4. And at the high-temperature end, more supplemental combustion ammonia gas is also required because of the higher reactor decomposition temperature. Meanwhile, in the high-temperature end region, the decomposition rate of ammonia is high at this time, and the change with temperature is yet small, the fuel in the PEMFC is still sufﬁcient, the PMEFC operating current is still in the ohmic polarization region, and the output power of the PEMFC is basically the same, so the system efﬁciency decreases slowly due to the increase of the additional fuel. Finally, when the operating current of PEMFC is further increased, at this time, at all temperatures, the operating current of PEMFC enters the region affected by the dense differential polarization, resulting in (32) The chemical reaction Equations 31, 32 are unfavorable side reactions of ammonia combustion because NOx is generated. Li et al. (Li et al., 2014) investigated the generation of NOx during the eruption of ammonia. It showed that when the NH3-H2 fuel mixture containing 50% ammonia was combusted at an excess air coefﬁcient of 1.25, the detected amount of NOx in the resulting gas was 1,200–1300 PPM, while the amount of NOx detected in the gas generated from pure hydrogen fuel at the same excess air coefﬁcient is 650 PPM. It means that some ammonia undergoes chemical reactions (31) and (32), but its reaction amount is not high. The results of also showed that increasing the excess air coefﬁcient and ammonia content would reduce the total NOx production. In this paper, the excess air coefﬁcient is set to 1.3, so the amount of ammonia involved in the chemical reactions 31 and 32 can be considered negligible for the thermal calculations. Therefore, to simplify the model, the following assumptions are made. 1) The degree of reaction of combustible gases in the combustion chamber is close to 100%, the values of which are shown in the following table of simulation parameters, ignoring the inﬂuence of unreacted gas on the thermal parameters of the mainstream gas. 2) Neglecting the side reactions of ammonia combustion. 3) The heat loss transferred from the combustion chamber to the environment is reﬂected by the insulation efﬁciency of the combustion chamber. According to the conservation of energy ηcom ηen G12,n ΔHn G13,m ΔHm , n H2 , NH3 , N2 , O2 n m (33) Where ηcom is the combustion efﬁciency of combustibles and ηie is the insulation efﬁciency of the combustion chamber. The enthalpy of gas ΔHi can be calculated by Eq. 23. Frontiers in Energy Research \| www.frontiersin.org 7 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas FIGURE 3 \| The efﬁciency of the system at different temperature and current. FIGURE 4 \| Variation of PEMFC power and amount of additional fuel with temperature at different current. Frontiers in Energy Research \| www.frontiersin.org 8 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas FIGURE 5 \| The system’s maximum efﬁciency of different reaction temperatures and the corresponding fuel utilization rate, working current value. FIGURE 7 \| System energy distribution diagram. achieve a high rate of ammonia decomposition. The above simulation results show that a signiﬁcant increase in the reaction temperature has less impact on the system efﬁciency. Still, of course, this conclusion is without considering the impact of the increase in reaction temperature on the radiation heat loss. Effect of Current on System Characteristics The operating temperature of the Cracker in the system is set to 650°C in this paper. Figure 6 shows the variation of system efﬁciency, additional fuel quantity, and PEMFC output power with the current. The system efﬁciency increases linearly with the change of current at the initial stage, and the system reaches the highest efﬁciency point when the current is 20.9 A. After that, the system efﬁciency starts to decrease slowly with the increase of current, and ﬁnally, the system efﬁciency drops sharply when the current exceeds 26.5 A. The change of the power curve of PEMFC and the change of the charge fuel ﬂow curve correspond to the analysis results in 4.1. From the fuel replenishment curve in the ﬁgure, it can also be seen that the current corresponds to the maximum efﬁciency (referred to as critical current in this paper) is at the point where the fuel in the tail gas of the PEMFC is just enough to provide the energy required for ammonia reforming. Figure 6 also shows that the highest system efﬁciency point a is not at the same location as the highest PEMFC efﬁciency point b. The output power of the PEMFC at point a is less than that at point b, which means that more fuel is reserved to supply the combustor. FIGURE 6 \| The variation of system efﬁciency, additional fuel quantity, and PEMFC output power with current. a sharp drop in the output power of PEMFC and a signiﬁcant decrease in system efﬁciency. Figure 5 shows the system’s maximum efﬁciency of different reaction temperatures and the corresponding fuel utilization rate, working current value. From the Fuel utilization curve and the Current curve in the ﬁgure, it can be seen that both the working current and the fuel utilization rate vary very little, with the variation range of the current being 20–21 A and the variation range of the fuel utilization rate being between 0.72 and 0.77. The efﬁciency curve shows that although the system efﬁciency varies greatly at the low reaction temperature end when the reaction temperature of the system exceeds 340°C, the highest efﬁciency is obtained at a temperature of 418°C with a value of 45.02%, and the lowest efﬁciency is obtained at a temperature of 650°C with a value of 44.53%, which is only a 0.5% decrease inefﬁciency. This is a magniﬁcent phenomenon for the ADG-PEMFC system. Because in the actual system, the rate of ammonia decomposition and the volume of the decomposer are limited, a reaction temperature higher than 600°C is often required to Frontiers in Energy Research \| www.frontiersin.org Energy Flow Distribution at the Highest Efﬁciency Point The ﬁnal distribution of the energy input into the system is mainly the heat loss from the individual modules, the electrical energy generated by the PEMFC, and the exhaust gas. Figure 7 shows the percentage of energy lost from each module in the system and the electrical power generated by the PEMFC to the 9 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas TABLE 5 \| Output characteristics of the system at different PEMFC operating temperatures. Temperature/°C System efﬁciency (%) PEMFC power/W Critical current/A 44.53 44.66 44.69 44.73 44.78 44.95 684.2 684.3 684.4 684.7 686.7 688.4 20.94 20.97 21.00 21.02 21.11 21.17 50 60 70 80 100 150 total input energy at the ammonia decomposition reaction temperature of 650°C and the PEMFC current of 20.9, respectively. From the ﬁgure, it can be seen that the main energy distribution in the system is the electrical energy generated by PEMFC (44.52%), the PEMFC heat loss (41%), and the waste heat loss of exhaust gas (6.9%). For the waste heat loss of the exhaust gas, the exit temperature of the exhaust gas is only 365.2 K at this time, so the quality of the remaining heat is also low, and there is a serious difﬁculty in continuing to improve the efﬁciency of waste heat utilization. Improving the power generation efﬁciency of PEMFC and reducing the heat loss of PEMFC is an effective way to improve the system efﬁciency. A three-dimensional simulation study of a PEMFC fueled by a nitrogen-hydrogen mixture shows that the efﬁciency of the PEMFC can be improved by 3–5% through geometry optimization. For PEMFC heat loss, the key factor is the maximum operating temperature of PEMFC, so this loss can be reduced by improving the high-temperature resistance of PEMFC. FIGURE 8 \| Output characteristics of the system under different ηie. Effect of PEMFC Operating Temperature on System Efﬁciency gas, and in fact, most of the waste heat from the tail gas is taken away by the cathode gas, so that increasing the operating temperature of the PEMFC has little effect on the efﬁciency of the system. The effect of PEMFC efﬁciency on system efﬁciency is a linear and explicit effect, and the amount of system efﬁciency improvement is equal to the amount of PEMFC efﬁciency increase while keeping other conditions constant. However, the increase in PEMFC operating temperature has an implicit effect on the system efﬁciency. The impact of the increase in operating temperature on the efﬁciency is twofold: ﬁrst, the anode tail gas contains higher thermal energy, which reduces the heat loss of the PEMFC; second, the higher thermal energy in the anode tail gas increases the critical current value, which makes the PEMFC generate more power. Table 5 shows the assumed maximum system efﬁciency, corresponding PEMFC power, and current values at different PEMFC operating temperatures. It should be emphasized that the maximum operating temperature of the current PEMFC is generally not higher than 80°C, and the simulation data in the table with an operating temperature greater than 80°C is for the mediumtemperature PEMFC that may gain application in the future. It can be seen from Table 5 that the signiﬁcant increase in the operating temperature of the PEMFC does not result in a signiﬁcant increase in the efﬁciency of the system, probably because the ADG-PEMF system in this paper only considers the anode when recovering the waste heat from the PEMFC tail Frontiers in Energy Research \| www.frontiersin.org FIGURE 9 \| Experimental data of ammonia decomposition. 10 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas FIGURE 10 \| Output characteristics of the system based on experimental data of ammonia decomposition. corresponding current values, and additional fuel ﬂow rates obtained based on the ammonia decomposition experimental data. From the efﬁciency curves, it can be seen that the system obtains a maximum efﬁciency value of 44.05% at the point of 830°C and 20.59 A, which is a small decrease in efﬁciency compared to the results of 4.1 and 4.2. And it can be seen from the current curve versus the additional fuel ﬂow curve that the critical current disappears when the decomposition rate of ammonia is low. The reason is that the decomposition rate of ammonia is insufﬁcient, which leads to the need for re-fueling to obtain a higher system efﬁciency, which corresponds to a lower limit of the decomposition rate of 0.97 under the parameters set in this paper. The efﬁciency curve of the system shows that the system with a critical current will have a higher system efﬁciency. Also there is a deep in current at 790°C, the reason is that inﬂuenced by the ammonia decomposition rate, the fuel entering the PEMFC is signiﬁcantly reduced before 790°C due to the low decomposition rate of ammonia. And as we can see from the ammonia decomposition rate curve, the slope of the curve at 790°C is also large, so the efﬁciency of the system at this time is affected by both the PEMFC power and the additional fuel, and the effect of the additional fuel is greater than the effect of the PEMFC power, and the only way to reduce the additional fuel is to increase the amount of fuel in the PEMFC anode tail gas, so a certain amount of PEMFC power output needs to be sacriﬁced (i.e. lower operating current) to ensure that the system obtains higher efﬁciency. After 790°C, the decomposition rate of ammonia is high enough that it is no longer a major inﬂuence, and the system enters a state with critical current at this time, so the current corresponding to the highest efﬁciency is the critical current. Effect of Anode Tail Gas Energy Utilization on System Efﬁciency The analysis results in Effect of Current on System Characteristics show that when the system works at the maximum efﬁciency point, the heat required for the ammonia decomposition reaction is completely provided by the fuel of the anode tail gas, so the anode tail gas energy utilization must have a signiﬁcant inﬂuence on the system efﬁciency, so this paper investigates the inﬂuence of the anode tail gas energy utilization on the maximum efﬁciency of the system, and the anode tail gas energy utilization is expressed by the insulation efﬁciency ηie of the combustion chamber. The simulation results shown in Figure 8 show that ηie has an important inﬂuence on the system coefﬁcient, and the maximum efﬁciency of the system is only 40% when ηie is 0, while the system efﬁciency is 44.5% when ηie is 0.9. And the ﬁgure also shows that the system critical current is also inﬂuenced by ηie. When ηie is low, there is no critical current. As ηie increases, the critical current appears. And the critical current value increases as ηie increases. Study of System Efﬁciency at an Experimental Ammonia Decomposition Rate Figure 9 shows the results of ammonia decomposition rate with decomposition temperature obtained experimentally in this paper, the experimental device is a tubular reactor with external electric heating, 800 mm in length and 20 mm in inner diameter, ﬁlled with NiO-AL2O3 catalyst particles (NiO≥14.0wt%), the porosity of catalyst is 0.47, and the ﬁlling amount of catalyst is 250 g. It can be seen from the ﬁgure that the experimental ammonia reaction temperature needs to be greater than 760°C to obtain greater than 90% ammonia decomposition rate, so the temperature of the ammonia decomposition reaction was set to be 760–800°C. Figure 10 shows the maximum efﬁciency of the ADGPEMF at different ammonia decomposition reaction temperatures, Frontiers in Energy Research \| www.frontiersin.org CONCLUSION In this paper, a thermal simulation model of a PEMFC system directly fueled by ammonia decomposition gas is developed to study and analyze the maximum efﬁciency of the system and the 11 March 2022 \| Volume 10 \| Article 819939 Zhao et al. PEMFC and Ammonia Decomposition Gas key factors affecting the system efﬁciency. It enables a more scientiﬁc and quantitative assessment of the potential value of ADG-PEMFC. The following conclusions were obtained from the simulation analysis. reuse of anode tail gas are the two main ways to improve the system efﬁciency. 3) In order to ensure that the system can obtain high system efﬁciency, the actual system should meet the decomposition rate of ammonia to make the system have a critical current. 4) At the ammonia decomposition temperature of 650°C, the reuse of anode tail gas energy can increase the efﬁciency of the system from 40 to 44.05%. 1) The ideal efﬁciency of the system based on the current commercial PEMFC (not optimized for mixed gas fuel) can reach 45.02% at the ammonia decomposition temperature of 418°C. Although increasing the ammonia decomposition reaction temperature will lead to a decrease in system efﬁciency, the decrease is small, so the system can obtain relatively good efﬁciency at higher ammonia decomposition reaction temperatures. The efﬁciency of the system obtained from the experimental data of ammonia decomposition in this paper is 44.05%, which shows that it has a high potential for development. 2) The main energy loss in the system is the heat loss of the fuel cell, but due to the defect of exhaust gas waste heat recovery, the improvement of the system efﬁciency by increasing the operating temperature of the fuel cell is small. Optimizing the PEMFC using ammonia decomposition gas and ensuring the DATA AVAILABILITY STATEMENT The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS JZ commonly ﬁnished the manuscript. YL conduct the experiment. All authors have read and approved the ﬁnal manuscript. NH3-fueled Solid Oxide Fuel Cells. Solid State Ionics 319, 180–185. doi:10.1016/j.ssi. 2018.02.015 Hunter, H. M. A., Makepeace, J. W., Wood, T. J., Mylius, O. S., Kibble, M. G., Nutter, J. B., et al. (2016). 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Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Zhao, Liang and Liang. 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. 13 March 2022 \| Volume 10 \| Article 819939
https://openalex.org/W4387886712	https://online-journal.unja.ac.id/kal/article/download/20295/14067	Indonesian	null	Ungkapan Pujian Bahasa Melayu Jambi di Masyarakat Melayu Kota Jambi	Kajian Linguistik dan Sastra	2,022	cc-by-sa	8,798	P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-I j g Volume 1, No. 2, September 2022 Expressions of Praise the Jambi Malay in the Melay Community of Jambi City Lerisa Ramadhea1, Ade Kusmana2, Julisah Izar3 1,2,3Program Studi Sastra Indonesia, Fakultas Keguruan dan Ilmu Pendidikan, Universitas Jambi Lerisaramadhea0512@gmail.com , ade.kusmana@unja.ac , julisahizar@unja.ac.id Abstrak Penelitian ini bertujuan untuk mengklasifikasikan dan mendeskripsikan jenis-jenis ungkapan pujian dan responnya, sekaligus fungsi dari pujian. Penelitian ini merupakan penelitian deskriptif kualitatif. Data dalam penelitian ini adalah ungkapan pujian pujian dan responnya dalam bahasa melayu Jambi. Sumber data dalam penelitian ini adalah masyarakat melayu kota Jambi. Pengumpulan data dalam penelitian ini menggunakan teknik observasi dan dokumentasi. Hasil dari penelitian ini adalah jenis ungkapan pujian yang tergolong dalam Kemampuan (Ability) ditemukan ada sebanyak 18 ungkapan pujian dan respon pujian sebanyak 9 jenis, Kepemilikan (Possession) ditemukan ada sebanyak 19 ungkapan pujian dan respon pujian sebanyak 8 jenis, Penampilan Fisik (Physical Appearance) ditemukan ada sebanyak 20 ungkapan pujian dan respon pujian sebanyak 5 jenis, Kepribadian (Personality) ditemukan ada sebanyak 20 ungkapan pujian dan respon pujian sebanyak 5 jenis, dan Kualitas (Quality) ditemukan ada sebanyak 10 ungkapan pujian dan respon pujian sebanyak 6 jenis. Adapun fungsi dari memberikan pujian pada orang lain yakni untuk mengekspresikan rasa kagum maupun rasa takjub yang disampaikan secara tulus atas pencapaian orang lain, penghargaan orang lain, untuk menjaga agar komunikasi verbal antara penutur dan mitra tutur dapat berjalan harmonis, untuk mengekspresikan rasa cemburu ataupun rasa iri terhadap pencapaian seseorang, penampilan seseorang, dan terhadap barang yang miliki orang lain. g y g g Dari hasil penelitian disimpulkan bahwa ungkapan pujian masih sering digunakan oleh masyarakat melayu Jambi, dari kelima jenis pujian tersebut yang paling sering digunakan adalah jenis pujian Penampilan Fisik (Physical Appearance), hal ini menujukkan masyarakat melayu Jambi sering memperhatikan penampilan fisik satu sama lainnya Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX n Linguistik dan Sastra P-ISSN XXXX-XXXX No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 The function of giving praise to others is to express a sense of admiration or amazement that is conveyed sincerely for the achievements of others, appreciation of others, to keep verbal communication between the speaker and the speech partner running harmoniously, to express jealousy or envy. to one's accomplishments, to one's appearance, and to the possessions of others. From the results of the study it was concluded that the expression of praise is still often used by the Jambi Malay community, of the five types of praise the most frequently used is the Physical Appearance type, this shows that the Jambi Malay community often pays attention to each other's physical appearance. Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. Abstract Riwayat Artikel Diterima: 2 Juli 2022 Direvisi: 28 Juli 2022 Disetujui: 15 Agustus 2022 Riwayat Artikel Diterima: 2 Juli 2022 Direvisi: 28 Juli 2022 Disetujui: 15 Agustus 2022 This study aims to classify and describe the types of expressions of praise and their responses, as well as the function of praise. This research is a qualitative descriptive study. The data in this study are expressions of praise and response in the Jambi Malay language. The source of data in this study is the Malay community in Jambi. Collecting data in this study using observation and documentation techniques. The results of this study are the types of expressions of praise belonging to Ability (Ability) found there were 18 expressions of praise and 9 types of praise responses, Possession found there were 19 expressions of praise and praise responses as many as 8 types, Physical Appearance (Physical Appearance) ) it was found that there were 20 expressions of praise and 5 types of praise responses, Personality (Personality) was found to be as many as 20 expressions of praise and 5 types of praise responses, and Quality (Quality) was found to be as many as 10 expressions of praise and 6 types of praise responses. Keywords Expressions of Praise Types of Compliments Language Jambi Malay 129 erisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. 1. Pendahuluan Bahasa memiliki kaitan yang sangat erat dengan manusia, karena manusia merupakan mahkluk sosial yang tidak bisa hidup tanpa orang lain. Sehingga bahasa dijadikan sebagai sarana dalam berkomunikasi dan berinteraksi antar satu sama lainnya. Sejalan dengan pendapat Achmad (2012:10) bahwa bahasa umumnya digunakan oleh manusia untuk saling berinteraksi, berkerja sama, dan lain sebagainya. Kemudian didukung oleh Martinet (1987:22) yang 130 Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 j g Volume 1, No. 2, September 2022 menyatakan bahasa merupakan sesuatu yang dipergunakan hingga kini oleh masyarakat untuk saling berkomunikasi antar satu sama lain. Bahasa kerap dijadikan sebagai identitas diri, sehingga orang lain bisa dengan mudah mengenali dari mana kita berasal melalui bahasa yang digunakan. Dengan adanya bahasa dalam masyarakat membuat komunikasi antar masyarakat terjalin menjadi lebih baik. Dalam mempelajari bahasa secara mendetail, dapat mempelajarinya dalam linguistik. Linguistik merupakan ilmu ilmiah yang mengkaji tentang bahasa (Lyons,1975:1). Linguistik memiliki banyak cabang dan bagian didalamnya salah satunya Pragmatik. Leech (1983:3) berkata pragmatik ialah ilmu yang memepelajari mengenai makna dan kaitannya dengan situasi tutur. Tarigan (2009:32-34) mengatakan bahwa pragmatik merupakan kajian yang membahas mengenai bagaimana cara konteks dapat mempengaruhi cara kita dalam menafsirkan suatu kalimat. Sedangkan menurut Yule (2014:3) pragmatik ialah ilmu tentang penafsiran makna ujaran oleh pendengar atau pembaca, yang kemudian disampaikan oleh penutur atau penulis. Sehingga dapat dikatakan pragmatik adalah ilmu yang mengkaji mengenai tuturan tertentu pada situasi- situasi tertentu yang memfokuskan pada konteks sosial bahasa yang bisa mempengaruhi makna atau tafsiran si mitra tutur. Pragmatik bisa digunakan dalam menelaah tentang makna yang disampaikan oleh penutur dan diartikan sipendengar ataupun sipembaca. Sehingga dapat dikatakan, pragmatik adalah ilmu yang tak terlepas dari bahasa dan konteks, oleh sebab itu bisa dikatakan bahwa pragmatik merupakan bagian dari linguistik membahas mengenai susunan bahasa dan konteksnya yang dijadikan sebagai alat agar komunikasi bisa berjalan baik antar sipenutur dan mitra tutur, sekaligus lebih menekankan pada makna dan sutuasi ujaran. Dalam Pragmatik, ilmu ini mengkaji mengenai deiksis, praanggapan, implikatur percakapan, dan tindak tutur. Peneliti lebih menfokuskan pada tindak tutur, tindak tutur dapat membantu dalam menafsirkan pesan karena manusia umumnya dapat mengenali tindakan yang ditunjukkan oleh pembicara melalui tuturan. Dalam menjaga hubungan baik dalam masyarakat umumnya menggunakan perbuatan ataupun kelakuan, dengan maksud untuk menghasilkan kesejahteraaan pada masyarakat. Tindakan ini tak hanya tampak dari perilaku fisik, tapi dari perilaku tutur yang positif. Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. 1. Pendahuluan Perilaku tutur positif merupakan suatu bentuk tuturan atau ujaran yang sejalan dengan norma kesopanan, maksudnya menciptakan rasa kenyaman antar pembicara agar terhindar dari kesalah pahaman dan menciptakan hubungan yang baik. Menurut Wolfson dan Manes (dalam Narjes Ziaei, 2012) menyatakan bahwa dengan memberikan pujian pada orang lain merupakan suatu bentuk perilaku atau sikap yang berfungsi untuk membangun dan menguatkan solidaritas pada kehidupan bermasyarakat. Pemberian pujian bisa dijadikan sebagai salah satu strategi kesopanan positif, yang masuk kedalam kategori menyadari akan keinginan ataupun kekaguman pada si lawan tutur. Sehingga dapat dikatakan bahwa pemberian pujian termasuk kedalam bagian penting dalam menjalin suatu keharmonisan antar masyarakat. Pujian biasanya 131 Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra j g Volume 1, No. 2, September 2022 digunakan dalam mengekspresikan suatu kekaguman kepada personal, yang umumnya disampaikan secara langsung, namun bisa juga melalui perantara. Menurut Holmes (2003:183) pujian merupakan bentuk tuturan yang menandakan adanya kesantunan positif didalamnya dan pujian sering dipakai oleh para wanita, karena umumnya para wanita lebih sering mendapatkan pujian sekaligus sering melontarkan pujian dikarenakan mereka lebih menghargai yang namanya pujian. Hal ini menunjukkan bahwa pujian menunjukkan sisi kesopanan seseorang yang digunakan untuk tujuan baik yaitu menghargai seseorang dengan cara mempererat jalinan melalui pujian antar satu sama lainnya. Holmes (2003:177) mengatakan bahwa pemberian pujian merupakan suatu tindak tutur yang secara langsung maupun tidak langsung menyumbangkan pengakuan pada seorang selain penutur, umumnya sejumlah “kelebihan” yang dipunyainya, misalnya kepemilikan, karakteristik, keahlian, dan lainnya sebegainya yang dinilai secara positif oleh penutur dan petutur. Kemudian, Holmes (dalam Paulston dan Tucker, 2003:187) mengelompokkan pujian kedalam lima jenis yakni, pujian tentang Kemampuan (ability), Kepemilikan (Possession), Penampilan Fisik (Physical Appearance), Kepribadian (Personality), Kualitas (Quality). Faktanya dilapangan kelima jenis ungkapan pujian ini masih sering terdengar dan terlihat digunakan oleh masyarakat sekitar, kelima jenis ungkapan ini bermaksud untuk menjalin hubungan baik antra masyarakat agar terciptanya nuansa yang aman, tentram, sekaligus nyaman. Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. 1. Pendahuluan B ik t t h j ji Berikut contoh ujaran pujian : Berikut contoh ujaran pujian : (a): elok nian tas kau, jadi pengen aku (a): elok nian tas kau, jadi pengen aku (cantik sekali tas milikmu, jadi ingin (a): elok nian tas kau, jadi pengen aku (cantik sekali tas milikmu, jadi ingin saya memiliknya) (cantik sekali tas milikmu, jadi ingin saya memiliknya) (b): mokaseh (terima kasih) Berdasarkan kutipan diatas menurut teori holmes termasuk kedalam jenis pujian Kepemilikan (Possession), karena didalamnya terdapat ujaran yang menunjukkan suatu kekaguman atas apa yang dimiliki oleh si lawan tutur yang berbentuk benda, pada kutipan percakapan diatas yang menjadi fokus pujian adalah “Tas” milik si lawan tutur. Berdasarkan penjabaran sebelumnya bahwasannya teori holmes ini terdapat beberapa jenis pujian yaitu pujian tentang penampilan petutur (apperance compliment), pujian tentang kemampuan, prestasi atau perbuatan baik petutur (ability/performance compliment), pujian tentang benda yang dimiliki petutur (possesion compliment), dan pujian tentang kepribadian atau keramahan petutur (personality/friendliness). Menurut Sri Wahyuni Sepriani Daromes (2019) dalam penelitiannya yang berjudul “Ungkapan Pujian Dalam Film Me Before You Karya Jojo Moyes: Suatu Analisis Pragmatik”. Dalam penelitiannya terdapat 10 pujian yang tergolong kedalam pujian kepemilikian dan beberapa jenis pujian lainnya. Sehingga dapat dikatakan yang membedakan penelitian ini dibandingkan penelitian yang sedang dikaji ini yaitu berada pada objek penelitian, jika penelitian ini yang 132 132 Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 j g Volume 1, No. 2, September 2022 dijadikan sebagai objek yakni film sedangkan penelitian yang akan dikaji yaitu ujaran pujian dalam bahasa daerah. Berdasarkan Summer Institute Of Linguistic (2006), Indonesia berada di posisi kedua yang mempunyai bahasa daerah terbanyak di dunia yaitu sebanyak 741 bahasa (dalam Nursiwan, 2012). Hal ini menunjukkan bahwa indonesia memiliki bahasa yang sangat beragam yang memiliki keunikannya dan ciri khasnya tersendiri disetiap daerahnya, oleh sebab itu bahasa menjadi salah satu kekayaan dan aset negara yang perlu dijaga dan dilestarikan agar tidak musnah termakan waktu. Salah satu bahasa daerah yang masih digunakan sampai sekarang adalah bahasa Melayu Jambi. sehingga objek dalam peneltian ini yakni bentuk ujaran pujian dalam bahasa Melayu Jambi. p y j p j y Adapun urgansi dalam penelitian ini yakni, untuk melihat jenis-jenis ujaran pujian apa yang masih digunakan hingga sekarang dan melihat bagaimana respon dari pujian tersebut, sekaligus fungsi dari pujian tersebut guna membantu mengurangi kesalahpahaman melalui pendekatan seperti seringnya memberi pujian untuk mempererat tali persaudaraan dan keakraban antar si penutur dan mitra tutur agar terciptanya suasana yang nyaman antar keduanya. Adapun tujuan tujuan dalam penelitian ini adalah untuk mengidentifikasi sekaligus mengklasifikasikan jenis-jenis ungkapan pujian dalam bahasa Melayu Jambi, untuk meneliti dan mendeskripsikan fungsi dari masing-masing jenis pujian dalam bahasa Melayu Jambi, dan untuk mengidentifikasi respon terhadap pujian dalam bahasa Melayu Jambi. Penelitian serupa yang berhubungan dengan penelitian ini adalah Warni, dkk (2019, 2020), dan Kusmana, dkk., (2018). Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. 2. Metode Dalam penelitian ini adapun jenis penelitian yang dipakai yaitu penelitian kulitatif. Penelitian kualitatif merupakan penelitian yang memahami mengenai makna mendalam dari sekolompok atau sejumlah orang yang sedang mengalami masalah sosial, umumnya penelitian kualitatif ini digunakan dalam meneliti mengenai kehidupan bermasyarakat, perilaku, kejadian atau fenomena, sejarah, dan lain sebagainya. j g y Bisa dikatakan bahwa penelitian kualitatif adalah suatu aturan dalam penelitian yang lebih memfokuskan pada kualitas penelitian yang bengacu pada konsep, teori, karakteristik, dan lain sebagainya. Penelitian ini dilakukan berdasarkan pada pengamatan peneliti mengenai lingkungan sosial sekitarnya sehingga menghasilkan data deskriptif. Metode deskriptif hanya berdasarkan fakta, sehingga data yang didapatkan adalah data yang sebenarnya dan bukan berupa angka-angka (Moleong, 1998: 3). Data deskriptif didapatkan dalam sebuah penelitian kualitatif yang kemudian hasilnya akan dideskripsikan berdasarkan tujuan dari penelitian Dalam penelitian ini terdapat tiga jalur analisis data kualitatif, yaitu reduksi data, penyajian data, dan penarikan kesimpulan (Miles dan Huberman, 1992: 16). Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX Menurut Wolfson dan Manes (dalam Paulston dan Tucker, 2003:181) menyatakan bahwa dengan memberikan pujian pada orang lain merupakan suatu bentuk perilaku atau sikap yang berfungsi untuk untuk mengekspresikan rasa kagum maupun rasa takjub yang disampaikan secara tulus atas pencapaian orang lain, penghargaan orang lain, dan lain sebagainya yang patut diapresiasi dengan pujian, untuk menjaga agar komunikasi verbal antara penutur dan mitra tutur dapat berjalan harmonis dengan lancar tanpa hambatan, untuk mengekspresikan rasa hormat terhadap seseorang yang sedang diajak berbicara untuk menghindari kesalahpahaman antar satu sama lainnya, untuk mengekspresikan rasa cemburu ataupun rasa iri terhadap pencapaian seseorang, penampilan seseorang, dan terhadap barang yang miliki orang lain. Pemberian pujian merupakan suatu tindak tutur yang secara langsung maupun tidak langsung menyumbangkan pengakuan pada seorang selain penutur, umumnya sejumlah “kelebihan” yang dimilikinya. Holmes (dalam Paulston dan Tucker, 2003:187) mengelompokkan pujian kedalam lima jenis yakni, pujian tentang Kemampuan (ability), Kepemilikan (Possession), Penampilan Fisik (Physical Appearance), Kepribadian (Personality), Kualitas (Quality). Menurut Wolfson dan Manes (dalam Paulston dan Tucker, 2003:181) menyatakan bahwa dengan memberikan pujian pada orang lain merupakan suatu bentuk perilaku atau sikap yang berfungsi untuk untuk mengekspresikan rasa kagum maupun rasa takjub yang disampaikan secara tulus atas pencapaian orang lain, penghargaan orang lain, dan lain sebagainya yang patut diapresiasi dengan pujian, untuk menjaga agar komunikasi verbal antara penutur dan mitra tutur dapat berjalan harmonis dengan lancar tanpa hambatan, untuk mengekspresikan rasa hormat terhadap seseorang yang sedang diajak berbicara untuk menghindari kesalahpahaman antar satu sama lainnya, untuk mengekspresikan rasa cemburu ataupun rasa iri terhadap pencapaian seseorang, penampilan seseorang, dan terhadap barang yang miliki orang lain. Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Kalistra: Kajian Linguistik dan Sastra j g Volume 1, No. 2, September 2022 a. Reduksi Data adalah suatu analisis data yang membantu dalam menajamkan, penyederhanaan, mengarahkan, menggolongkan, dan lain sebagainya. Pada tahapan ini data yang didapatkan di lapangan akan melalui tahapan reduksi, maksudnya data tersebut akan dipilah mana yang tergolong kedalam ungkapan pujian. Lalu membuat ringkasan yang berisi inti-inti pembahasan yang akan dimasukkan kedalam penelitian, kemudian menandai bagian- bagian yang penting untuk mempermudah proses penelitian. b. Penyajian data adalah suatu kegiatan analisis dalam proses pembuatan laporan dari hasil penelitian yang dilakukan sebelumnya agar data yang telah didapat lebih mudah dipahami dan dianalisis sesuai kajian yang diinginkan oleh peneliti. Miles dan Huberman (1992:18) membatasi suatu penyajian sebagai sekumpulan informasi tersusun yang memberikan kemungkinan adanya penarikan kesimpulan dan pengambilan tindakan. Data yang telah melalui tahap direduksi nantinya akan disajikan berdasarkan bagan-bagan tertentu, hal ini berguna untuk mempermudah agar data yang didapatkan akan lebih mudah dipahami. c. Penarikan kesimpulan adalah tahap akhir yang ada pada analisis data, berupa deskripsi ataupun gambaran suatu objek yang sebelumnya masih belum jelas atau abu-abu menjadi lebih jelas, dan dapat berupa hubungan kausal atau interaktif, hipotesis atau teori (Sugiyono, 2008:253). Penarikan kesimpulan akan terus diteliti untuk memastikan kebenarannya mulai dari pengumpulan data, mencari arti benda, teori- teori, kejelasan – kejelasan, dan lain sebagainya. 2. Hasil dan Pembahasan Pemberian pujian merupakan suatu tindak tutur yang secara langsung maupun tidak langsung menyumbangkan pengakuan pada seorang selain penutur, umumnya sejumlah “kelebihan” yang dimilikinya. Holmes (dalam Paulston dan Tucker, 2003:187) mengelompokkan pujian kedalam lima jenis yakni, pujian tentang Kemampuan (ability), Kepemilikan (Possession), Penampilan Fisik (Physical Appearance), Kepribadian (Personality), Kualitas (Quality). 133 133 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra j g Volume 1, No. 2, September 2022 Saat dua orang saling berinteraksi tentunya akan menimbulkan efek timbal balik, begitu pula dengan pujian saat orang lain menuturkan pujian, maka pujian tersebut akan mendapatkan respon yang bermacam-macam pula. Herbert, 1990 (dalam Nur Mukminatien dan Andhuna Wisnu Patriana, 2005) telah merumuskan 12 macam respons dari pujian yakni, Menerima Pujian, Mengomentari Pujian, Meningkatkan Pujian, Menjelaskan Sejarah, Mengalihkan Pujian, Pengembalian Pujian, Merendahkan Diri, bertanya, Ditentang, Tidak Sepakat, Tidak Ada Respon, dan Permintaan. Setelah melalui beberapa tahapan sebelumnya diperolehlah hasil dari penelitian ungkapan pujian jenis Kemampuan (Ability) ditemukan ada sebanyak 18 ungkapan pujian dan respon pujian sebanyak 9 jenis, Kepemilikan (Possession) ditemukan ada sebanyak 19 ungkapan pujian dan respon pujian sebanyak 8 jenis, Penampilan Fisik (Physical Appearance) ditemukan ada sebanyak 20 ungkapan pujian dan respon pujian sebanyak 6 jenis, Kepribadian (Personality) ditemukan ada sebanyak 20 ungkapan pujian dan respon pujian sebanyak 5 jenis, dan Kualitas (Quality) ditemukan ada sebanyak 10 ungkapan pujian dan respon pujian sebanyak 5 jenis. A Kemampuan (Ability) Pujian ini merupakan bentuk kekaguman akan kemampuan yang dimiliki si lawan tutur, seperti prestasi, perbuatan baik pada orang lain, kemampuan yang diperoleh secara natural, dan lain sebagainya. Pujian jenis ini bisa dihubungkan dengan kemampuan dari penerima pujian atau kinerja sipenerima pujian. A: fotoin dulu aku bentar (fotokan dulu saya sebentar) B: yolah (baiklah) A: mokaseh e, wih ini hasil fotoan awak tadi? keren, bebakat awak ni (terima kasih ya, waw ini hasil potret kamu tadi? Keren, berbakat kamu ini) B: mokaseh yuk (terima kasih kak) A: iyo (iya) konteks tuturan: perbincangan diatas terjadi saat acara pernikahan, A menuturkan pujian kepada B dengan memberitahukan bahwa hasil potretnya sangat mengagumkan untuknya, dan merasa bahwa B memiliki bakal dalam bidang tersebut. Analisis: dari kutipan bercetak tebal diatas merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur terlihat sedang bebincang-bincang kecil yang didalamnya terdapat ungkapan pujian. Pujian tersebut berfungsi untuk memgekpresikan rasa kagum dalam diri A terhadap hasil potret dari si B, karena itulah A melontarkan pujian terhadap si B secara langsung. A: anak siapo tu yang ngaji? A: anak siapo tu yang ngaji? A: anak siapo tu yang ngaji? Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. 134 134 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. (iya besok anak saya mau dimasukkan ke pesantren lah biar bisa hebat seperti itu) Situasi tutur : percakapan diatas terjadi saat syukuran, A menuturkan pujian kepada B dengan mengatakan bahwa suara anaknya saat membacakan al-qur’an sangat merdu dan mengagumkan. Analisis : dari kutipan bercetak tebal diatas merupakan suatu penuturan pujian dan respon dari pujian tersebut, A selaku penutur dan B selaku mitra tutur sedang membicarakan seorang anak kecil yang memiliki suaranya yang indah dan mengagumkan. Pujian ini timbul karena A merasa bahwa hal tersebut mengagumkan dan memang pantas untuk mendapatkan pujian, dengan adanya pujian tersebut membuat B merasa tersanjung dan bangga akan kemampuan yang dimiliki oleh anaknya. Situasi tutur: percakapan diatas terjadi saat acara kumpul keluarga. A menuturkan pujian kepada B dengan mengatakan rasa senang atas kemampuan B karena telah mendapatkan peringkat pertama dikelasnya. Analisis : dari kutipan bercetak tebal diatas merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur terlihat sedang berinterkasi satu sama lain membicarakan mengenai peringkat kelas. Pujian tersebut berfungsi untuk mengekspresikan rasa kagum A terhadp B karena dengan kerja kerasnya dirinya bisa mendapatkan peringkat teratas, dan terlontarlah ungkapan pujian karena A merasa hal tersebut perlu di apresiasi agar B merasa termotivasi dan lebih semangat dalam belajar Situasi tutur: percakapan diatas terjadi saat acara kumpul keluarga. A menuturkan pujian kepada B dengan mengatakan rasa senang atas kemampuan B karena telah mendapatkan peringkat pertama dikelasnya. g j A: lagi masak apo? wangi nian sampe kedepan bauknyo A: lagi masak apo? wangi nian sampe kedepan bauknyo 135 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2 Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. A: harum nian minyak wangi awak sampe kesini bauknyo Situasi tutur: perbincangan diatas terjadi saat acara kumpul keluarga. Dari perbincangan diatas terlihat bahwa A melontarkan pujian kepada B karena memiliki parfum yang sangat harum. Analisis : berdasarkan kutipan percakapan diatas bagian yang bercetak tebal diatas merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur sedang membicarakan mengenai parfum yang dimiliki B karena baunya harum sehingga 136 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra j g Volume 1, No. 2, September 2022 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. (lagi masak apa? Harum sekali sampai kedepan baunya) B: masak biasolah, ayam dicabein (masak biasalah, ayam dicabaikan) A: nak makan dululah kalo kektu, boleh dak? (mau makan dululah jika seperti itu, boleh tidak?) B: makanlah, pakek betanyo pulo (makanlah, pakai bertanya pula) A: wis enak nian, padek awak masak (wah enak sekali, pandai kamu masak) B: makanlah banyak-banyak kalo enak (makanlah banyak-banyak kalau enak) A: ok Situasi tutur : perbincangan diatas terjadi saat acara kumpul keluarga. A menuturkan pujian secara langsung kepada B karena masakannya sangat menggugah selera dari bentuk dan wanginya. gg g g y Analisis : berdasarkan percakapan diatas pada bagian yang bercetat tebal merupakan ungkapan pujian sekaligus respon dari pujian tersebut. A sebagai penutur dan B sebagai mitra tutur saling beinteraksi satu sama lain dan terdapat ungkapan pujian dalam percakapan antar keduanya yang membahasa mengenai kepandaian B dalam hal memasak yang dirasa pantas untuk dipuji. Pujian ini berfungsi untuk mengekspresikan rasa kagum dan senang dengan kemampuan A khususnya dalam hal memasak. Analisis : berdasarkan percakapan diatas pada bagian yang bercetat tebal merupakan ungkapan pujian sekaligus respon dari pujian tersebut. A sebagai penutur dan B sebagai mitra tutur saling beinteraksi satu sama lain dan terdapat ungkapan pujian dalam percakapan antar keduanya yang membahasa mengenai kepandaian B dalam hal memasak yang dirasa pantas untuk dipuji. Pujian ini berfungsi untuk mengekspresikan rasa kagum dan senang dengan kemampuan A khususnya dalam hal memasak. B. Kepemilikan (Possession) Pujian jenis ini merupakan pujian yang dilontarkan si penutur kepada lawan tutur atas benda ataupun barang yang dimiliki si lawan tutur berujung sebagai rasa kekaguman. Pujian jenis ini dihubungkan dengan hal-hal kepemilikan dari penerima pujian dan semuanya bersifat nyata atau terlihat. A: harum nian minyak wangi awak sampe kesini bauknyo (harum sekali parfum kamu sampai kesini baunya) B: iyopo? (benarkah) A: iyo merek apo tu? (iya merk apa itu?) B: pengen tau be awakni (pengen tau saja kamu ini) Sit asi t t pe bincangan diatas te jadi saat aca a k mp l kel a ga B. Kepemilikan (Possession) Pujian jenis ini merupakan pujian yang dilontarkan si penutur kepada lawan tutur atas benda ataupun barang yang dimiliki si lawan tutur berujung sebagai rasa kekaguman. Pujian jenis ini dihubungkan dengan hal-hal kepemilikan dari penerima pujian dan semuanya bersifat nyata atau terlihat. B. Kepemilikan (Possession) Pujian jenis ini merupakan pujian yang dilontarkan si penutur kepada lawan tutur atas benda ataupun barang yang dimiliki si lawan tutur berujung sebagai rasa kekaguman. Pujian jenis ini dihubungkan dengan hal-hal kepemilikan dari penerima pujian dan semuanya bersifat nyata atau terlihat. 137 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra j g Volume 1, No. 2, September 2022 menimbulkan rasa penasaran dalam di B mengenai merek tersebut. Pujian ini berfungsi untuk mengekspresikan rasa penasaran akan barang yang dimiliki oleh B yakni “parfum” yang memiliki bau yang harum sehingga munculah ungkapan pujian tersebut. A: awak baru datang yo? (kamu baru datang ya?) B : iyo, nunggu motor tadi lamo (iya, tunggu kendaraan tadi lama) A: ih tas awak elok nian, warnonyo jugo (ih tas kamu bagus nian, warnanya juga) B: mokaseh (terimakasih) Situasi tutur : percakapan diatas terjadi saat acara pernikahan. A menuturkan pujian kepada B dikarenakan barang yang dikenannya cukup menarik perhatiannya yakni “tas” yang memiliki bentuk dan warna yang bagus. Analisis : berdasarkan percakapan diatas yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur sedang membicarakan mengenai barang yang dikenakan oleh B, karena A merasa bahwa barang tersebut sangat cantik dan menarik dimatanya sehingga keluarlah kata-kata pujian yang disampaikan secara tulus sebagai bentuk kagum sekaligus rasa iri akan barang tersebut dan ingin memilikinya juga. A: jilbab baru kayaknyo ni, cantek warnony samoan pulo dengan baju warnonyo (hijab baru sepertinya ini, cantik warnanya samaan pula dengan baju warnanya) B: ayuk jugo keren harini (kakak juga keren hari ini) A: ah idaklah aku biasobe (ah tidaklah saya biasa saja) B: ayuk keren jugo serius (kakak keren juga serius) A: jilbab baru kayaknyo ni, cantek warnony samoan pulo dengan baju warnonyo (hijab baru sepertinya ini, cantik warnanya samaan pula dengan baju warnanya) B: ayuk jugo keren harini (kakak juga keren hari ini) A: ah idaklah aku biasobe (ah tidaklah saya biasa saja) B: ayuk keren jugo serius (kakak keren juga serius) Situasi tutur : kutipan percakapan diatas terjadi saat acara yasinan. A menuturkan pujian kepada B karena barang miliknya terlihat menarik dari segi bentuk dan warna. Analisis: terlihat dari percakapan diatas yang bercetak tebal merupakan suatu ungkapan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur tampak sedang berbincang kecil saling melontarkan pujian antara satu sama lainnya. Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. 137 Ungkapan Pujian ini berfungsi untuk mengekspresikan rasa kagum A terhadap barang yang dimiliki B yang terlihat sangat cantik dari segi bentuk maupun warnanya. A: rok beli maren yo? (rok beli kemarin ya?) B: iyo bagos dak? (iya bagus tidak?) A: bagos, pas nian rok tu samo awak (bagus, cocok sekali rok itu dengan kamu) A: mokaseh (terima kasih) B: iyo samo-samo (iya sama-sama) A: bagos, pas nian rok tu samo awak (bagus, cocok sekali rok itu dengan kamu) A: mokaseh (terima kasih) B: iyo samo-samo (iya sama-sama) Situasi tutur : dialog tersebut terjadi saat acara yasinan, A melontarkan pujian kepada B sebagai bentuk rasa dukungan kepada A agar dirinya lebih percaya diri akan barang yang digunakannya saat itu. Analisis : berdasarkan percakapan diatas bagian yang bercetak tebal merupakan suatu tuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur sedang berbicara satu sama lain membahas mengenai rok yang dibaru dibeli dan dikenakan oleh si A. Pujian tersebut berfungsi untuk mengekpresikan rasa kagum sekaligus dukungan agar B lebih percaya diri dalam hal apapun terutama barang yang dimilikinya. Situasi tutur : dialog tersebut terjadi saat acara yasinan, A melontarkan pujian kepada B sebagai bentuk rasa dukungan kepada A agar dirinya lebih percaya diri akan barang yang digunakannya saat itu. eb pe caya d a a ba a g ya g d gu a a ya saat tu Analisis : berdasarkan percakapan diatas bagian yang bercetak tebal merupakan suatu tuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur sedang berbicara satu sama lain membahas mengenai rok yang dibaru dibeli dan dikenakan oleh si A. Pujian tersebut berfungsi untuk mengekpresikan rasa kagum sekaligus dukungan agar B lebih percaya diri dalam hal apapun terutama barang yang dimilikinya. C. Penampilan Fisik (Physical Appearance) Penutur memberikan pujian pada penerima pujian terhadap penampilan fisiknya, dan lebih memfokuskan pada apa yang dikenakan oleh si penutur. Penampilan fisiknya meliputi tentang fisik seperti wajah, kecantikan, bibir, hidung, mata, telinga, rambut, badan, bahu, alis, tangan, kulit, dan lain sebagainya. C. Penampilan Fisik (Physical Appearance) Penutur memberikan pujian pada penerima pujian terhadap penampilan fisiknya, dan lebih memfokuskan pada apa yang dikenakan oleh si penutur. Penampilan fisiknya meliputi tentang fisik seperti wajah, kecantikan, bibir, hidung, mata, telinga, rambut, badan, bahu, alis, tangan, kulit, dan lain sebagainya. C. Penampilan Fisik (Physical Appearance) Penutur memberikan pujian pada penerima pujian terhadap penampilan fisiknya, dan lebih memfokuskan pada apa yang dikenakan oleh si penutur. Penampilan fisiknya meliputi tentang fisik seperti wajah, kecantikan, bibir, hidung, mata, telinga, rambut, badan, bahu, alis, tangan, kulit, dan lain sebagainya. C. Penampilan Fisik (Physical Appearance) Penutur memberikan pujian pada penerima pujian terhad penampilan fisiknya, dan lebih memfokuskan pada apa yang dikena oleh si penutur. Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Kalistra: Kajian Linguistik dan Sastra j g Volume 1, No. 2, September 2022 A: bagos, pas nian rok tu samo awak (bagus, cocok sekali rok itu dengan kamu) A: mokaseh (terima kasih) B: iyo samo-samo (iya sama-sama) Penampilan fisiknya meliputi tentang fisik seperti waj kecantikan, bibir, hidung, mata, telinga, rambut, badan, bahu, a tangan, kulit, dan lain sebagainya. A: muko awak mulos nian,pake bedak apo? (wajah kamu mulus sekali, pakai skincare apa?) B: biso bae awakni (bisa saja kamu ini) A: pakek bedak apo? (pakai skincare apa?) B: agekla kukasih tau (nantilah saya kasih tau) A:yolah kalo kektu (iyalah kalau seperti itu) Situasi tutur : dialog diatas terjadi saat acara pernikahan. A menuturkan pujian kepada B karena memiliki kulit yang mulus, sehingga menimbulkan pertanyaan mengenai skincare apa yang digunakan oleh B Analisis : berdasarkan percakapan diatas yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur saling beinteraksi satu sama lain Situasi tutur : dialog diatas terjadi saat acara pernikahan. A menuturkan pujian kepada B karena memiliki kulit yang mulus, sehingga menimbulkan pertanyaan mengenai skincare apa yang digunakan oleh B A li i b d k k di t b t k t b l Situasi tutur : dialog diatas terjadi saat acara pernikahan. A menuturkan pujian kepada B karena memiliki kulit yang mulus, sehingga menimbulkan pertanyaan mengenai skincare apa yang digunakan oleh B Analisis : berdasarkan percakapan diatas yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur saling beinteraksi satu sama lain membahas mengenai penampilan fisik si B yakni “wajah” yang terlihat mulus. Pujian ini berfungsi untuk mengekspresikan rasa kecemburuan pujian kepada B karena memiliki kulit yang mulus, sehingga menimbulkan pertanyaan mengenai skincare apa yang digunakan oleh B Analisis : berdasarkan percakapan diatas yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur saling beinteraksi satu sama lain membahas mengenai penampilan fisik si B yakni “wajah” yang terlihat mulus. Pujian ini berfungsi untuk mengekspresikan rasa kecemburuan sekaligus rasa penasaran terhadap B tentang skincare apa yang digunakan oleh dia. 139 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Kalistra: Kajian Linguistik dan Sastra j g Volume 1, No. Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. A: bagos, pas nian rok tu samo awak (bagus, cocok sekali rok itu dengan kamu) A: mokaseh (terima kasih) B: iyo samo-samo (iya sama-sama) 2, September 2022 B: turunannyo bagos, awak tengoklah idong emaknyo (keturunannya bagus, kamu lihatlah hidung ibunya) A: iyo jugosih awak jugo mancung (iya juga sih kamu juga mancung) B: iyo tau kau (iya kamu tau) A: youdah (ya sudah) Situasi tutur: percakapan diatas terjadi saat acara syukuran. A menuturkan pujian kepada B karena anaknya memiliki hidung yang mancung yang membuatnya merasakan sedikit rasa iri. Analisis: terlihat dari percakapan diatas pada bagian yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur terlihat sedang berbincang- bincang mengenai penampilan fisik anak B yang mancung. Pujian ini berfungsi untuk mengekpresikan rasa kagum dan rasa cemburu yang dialami oleh A terhadap hidung anak B. Situasi tutur: percakapan diatas terjadi saat acara syukuran. A menuturkan pujian kepada B karena anaknya memiliki hidung yang mancung yang membuatnya merasakan sedikit rasa iri. g y g y Analisis: terlihat dari percakapan diatas pada bagian yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur terlihat sedang berbincang- bincang mengenai penampilan fisik anak B yang mancung. Pujian ini berfungsi untuk mengekpresikan rasa kagum dan rasa cemburu yang dialami oleh A terhadap hidung anak B. p g A: kekmano caro lebarin bahu (bagaimana cara lebarin bahu) B: tuk apo? (untuk apa?) A: bahu awak lebar nian, jadi bagos badan nampaknyo (bahu kamu lebar sekali, jadi bagus badan kelihatannya) B: mokaseh (terimakasih) A: kekmano? (bagaimana) B: banyain olahraga be awaktu, jangan tedok teros gawenyo (perbanyak olahraga saja kamu itu, jangan tidur terus kerjanya) A: kekmano caro lebarin bahu (bagaimana cara lebarin bahu) B: tuk apo? (untuk apa?) A: bahu awak lebar nian, jadi bagos badan nampaknyo (bahu kamu lebar sekali, jadi bagus badan kelihatannya) B: mokaseh (terimakasih) A: kekmano? (bagaimana) B: banyain olahraga be awaktu, jangan tedok teros gawenyo (perbanyak olahraga saja kamu itu, jangan tidur terus kerjanya) Situasi tutur : percakapan diatas terjadi saat acara kumpul keluarga, A memberikan pujian kepada B karena memiliki bahu yang lebar, hal ini dikarenakan kebanyaan orang khususnya laki-laki ingin memiliki bahu yang lebar. Analisis : berdasarkan percakapan diatas pada bagian yang bercetak tebal merupakan ungkapan pujian dan respon dari pujian tersebut. A sebagai menutur dan B sebagai mitra tutur sadang berbincang-bincang membahas mengenai penampilan fisik yang dimiliki oleh B yakni “bahu” yang terlihat lebar, pujian ini berfungsi untuk mengekspresikan rasa penasaran sekaligus rasa iri terhadap penampilan fisik B. A: iri nian aku dengan awakni (iri sekali saya dengan kamu ini) B: iri ngapo awak? (iri kenapa kamu?) A: badan awak enak tinggi, jadi make baju apobe bagos A: iri nian aku dengan awakni (iri sekali saya dengan kamu ini) B: iri ngapo awak? (iri kenapa kamu?) A: badan awak enak tinggi, jadi make baju apobe bagos A: iri nian aku dengan awakni (iri sekali saya dengan kamu ini) B: iri ngapo awak? (iri kenapa kamu?) A: badan awak enak tinggi jad 140 P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 j g Volume 1, No. 2, September 2022 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. (badan kamu enak tinggi, jadi pakai baju apa saja bagus) g ) B: mokaseh pujiannyo (terimakasih pujiannya) Situasi tutur : percakapan diatas terjadi saat acara kumpul keluarga, terlihat A menuturkan pujian kepada B karena memiliki penampilan fisik khususnya “tinggi badan” yang terbilang tinggi. Analisis : terlihat melalui percakapan diatas pada bagian yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B mitra tutur saling berkomunikasi antar satu sam lainnya, mereka berdua membahas mengenai penampilan fisik yang cukup menarik perhatiannya yakni “hidung”. A memberi pujian kepada B karena anaknya memiliki hidung yang terbilang mancung, pujian ini muncul sebagai bentuk pengekspresian dari rasa kagum dan kecemburuannya mengenai hidung anak B yang terlihat mancung. D K ib di (P lit ) B: mokaseh pujiannyo (terimakasih pujiannya) Situasi tutur : percakapan diatas terjadi saat acara kumpul keluarga, terlihat A menuturkan pujian kepada B karena memiliki penampilan fisik khususnya “tinggi badan” yang terbilang tinggi. Analisis : terlihat melalui percakapan diatas pada bagian yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B mitra tutur saling berkomunikasi antar satu sam lainnya, mereka berdua membahas mengenai penampilan fisik yang cukup menarik perhatiannya yakni “hidung”. A memberi pujian kepada B karena anaknya memiliki hidung yang terbilang mancung, pujian ini muncul sebagai bentuk pengekspresian dari rasa kagum dan kecemburuannya mengenai hidung anak B yang terlihat mancung. D. Kepribadian (Personality) Pujian ini mengacu pada diri si lawan tutur atas perlakuan yang dan sikap ramah yang dimilikinya, kepribadian biasanya ialah ten penampilan non-fisik dari penerima pujian atau berupa karakteristik keramahan. A: rajen nian awak ngerapiin bungo didepan tu (rajin sekali kamu merapikan bunga didepan itu) B: iyolah, kalo dak mati dio (iyalah, kalau tidak mati dia) A: iyo jugo sih (iya juga sih) B: kalo mati sayang (kalau mati sayang) A: youdah (ya sudah) Situasi tutur : percakapan ini di terjadi saat acara kumpul keluarga. A menuturkan pujian kepada B karena dirinya sangat rajin dalam hal menata dan merapikan tanaman. p Analisis: pada bagian yang bercetak tebal diatas merupakan ungkapan pujian dan respon dari pujian tersebut, A sebagai penutur dan B sebagai mitra tutur. A memberikan pujian kepada B karena memiliki kepribadian yang rajin dalam hal merapikan tanaman, pujian tersebut berfungsi untuk mengekspresikan rasa kagum A terhadap B yang terbilang sangat rajin. Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. (badan kamu enak tinggi, jadi pakai baju apa saja bagus) A: nambah banyak be kucing dirumah ni (tambah banyak saja kucing dirumah ini) B: iyo bibik, maren beranak lagi dio (iya tante, kemarin melahirkan lagi dia) A: bagi be ke orang dari pada banyak kekni (bagi saja ke orang dari pada banyak seperti ini) B: sayang, gek dak dirawat dio (sayang, nanti tidak dirawat dia) A: awak ni orangnyo penyayang kucing nian (kamu ini orangnya penyayang kucing nian) B: iyo bik, soalnyo lucu jugo kadang (iya te, soalnya lucu juga kadang) A: nambah banyak be kucing dirumah ni (tambah banyak saja kucing dirumah ini) B: iyo bibik, maren beranak lagi dio (iya tante, kemarin melahirkan lagi dia) A: bagi be ke orang dari pada banyak kekni (bagi saja ke orang dari pada banyak seperti ini) B: sayang, gek dak dirawat dio (sayang, nanti tidak dirawat dia) A: awak ni orangnyo penyayang kucing nian (kamu ini orangnya penyayang kucing nian) B: iyo bik, soalnyo lucu jugo kadang (iya te, soalnya lucu juga kadang) Situasi tutur : percakapan diatas terjadi saat acara kumpul keluarga. Terlihat A menuturkan pujian kepada B karena merasa bahwa B adalah orang yang penyayang khususnya pada kucing, terlihat dari semakin banyaknya kucing yang berkembang biak menunjukkan bahwa dirinya orang yang penyayang sekaligus teliti dalam hal merawat kucing. g y g p y y g g g Analisis: berdasarkan pada kutipan percakapan diatas pada bagain yang bercetak tebal merupakan salah satu ungkapan pujian dan respon dari 142 (badan kamu enak tinggi, jadi pakai baju apa saja bagus) A: anak awak rajen nian begawe kutengok (anak kamu rajin sekali bekerja kulihat) B: iyolah, dari sekarang diajarin biak dak malas (iyalah, dari sekarang diajarkan biar tidak malas) A: iyosih bagos jugo (iyasih bagus juga) B: iyolah harus (iyalah harus) 141 Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 j g Volume 1, No. 2, September 2022 Situasi tutur : perbincangan diatas terjadi saat acara yasinan. A menuturkan pujian kepada B karena anaknya sangat rajin dalam mengerjakan sesuatu. Situasi tutur : perbincangan diatas terjadi saat acara yasinan. A menuturkan pujian kepada B karena anaknya sangat rajin dalam mengerjakan sesuatu. g j Analisis : dari kutipan percakapan diatas pada bagian yang bercetak tebal merupakan ungkapan pujian dan respon pujian. A menuturkan pujian karena timbulnya rasa kagum terhadap anak B yang sangat rajin bekerja, dan B mengatakan bahwa itu hal yang wajib diajarkan sedari sekarang agar terbiasa untuk kedepannnya. A: abangni lucu nian orangnyo (abang ini lucu sekali orangnya) B: biso bae awak ni (bisa saja kamu ini) A: iyo serius abangni enak diajak maen (iya serius abang ini enak diajak main) B: besok kito pegi maen kalo kektu (besok kita pergi main kalau seperti itu) A: ok kalo kektu (oke kalau seperti itu) A: abangni lucu nian orangnyo (abang ini lucu sekali orangnya) B: biso bae awak ni (bisa saja kamu ini) A: iyo serius abangni enak diajak maen (iya serius abang ini enak diajak main) B: besok kito pegi maen kalo kektu (besok kita pergi main kalau seperti itu) A: ok kalo kektu (oke kalau seperti itu) Situasi tutur: percakapan tersebut terjadi saat acara pernikahan. A menuturkan pujian kepada B karena merasa bahwa B orang yang memiliki kepribadian yang lucu sekaligus ramah. Analisis: berdasarkan kutipan percakapan diatas pada bagian yang bercetak tebal berisikan pujian dan respon pujian. A menuturkan pujian kepada B karena dirinya memiliki kepribadian yang lucu sehingga bisa membuat suasana yang saling nyaman antara satu sama lainnya. Pujian ini berfungsi untuk mengekspresikan rasa senang karena lawan tutur memiliki kepribadian yang lucu. 142 Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra Kalistra: Kajian Linguistik dan Sastra Volume 1 No 2 September 20 j g Volume 1, No. 2, September 2022 pujian tersebut. A sebagai penutur dan B sebagai mitra tutur terlihat sedang berkomunikasi antar satu sama lainnya membahas mengenai hewan peliharaan yakni “kucing” yang semakin bertambah banyak, hal ini menunjukkan bahwa B memiliki kepribadian penyayang terhadap mahkluk lain. Pujian ini berfungsi untuk mengekspresikan rasa kagum terhadap B karena memiliki kepribadian yang penyayang terutama terhadap hewan yakni kucing. E. E. Kualitas (Quality) Pujian ini lebih mengacu pada kualitas tingkat baik dan buruknya si mitra tutur atau lawan bicara. A: agek aku nak jaet tempat awak be (nanti saya mau jahit tempat kamu saja) B: iyo bole E. Kualitas (Quality) Pujian ini lebih mengacu pada kualitas tingkat baik dan buruknya si mitra tutur atau lawan bicara. A: agek aku nak jaet tempat awak be (nanti saya mau jahit tempat kamu saja) B: iyo bole (iya boleh) A: emang jaetan baju awak dak diraguin lagi kualitasnyo (memang jahitan baju kamu tidak diragukan lagi kualitasnya) B: daklah, biso bae ayuk ni (tidaklah, bisa saja kakak ini) (Q y) Pujian ini lebih mengacu pada kualitas tingkat baik dan buruknya si mitra tutur atau lawan bicara. Pujian ini lebih mengacu pada kualitas tingkat baik dan buru mitra tutur atau lawan bicara. A: agek aku nak jaet tempat awak be (nanti saya mau jahit tempat kamu saja) B: iyo bole (iya boleh) A: emang jaetan baju awak dak diraguin lagi kualitasnyo (memang jahitan baju kamu tidak diragukan lagi kualitasnya) B: daklah, biso bae ayuk ni (tidaklah, bisa saja kakak ini) Situasi tutur : perbincangan diatas terjadi saat acara yasinan. A menuturkan pujian kepada B karena merasa bahwa dirinya bisa dipercaya menuturkan pujian kepada B karena merasa bahwa dirinya bisa dipercaya dalam menjahit baju dalam segi jahitan, model, dan lain sebagainya. Analisis: berdasarkan percakapan diatas pada bagian yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A selaku penutur dan B selaku mitra tutur sedang berbincang membahas mengenai jahitan bajunya yang rapi sehingga menimbulkan rasa percaya akan kualitas jahitan yang tidak perlu diragukan lagi. 142 Pujian tersebut berfungsi untuk mengekspresikan rasa kagum A terhadap B karena kualitasnya saat menjahit baju sangat rapi. Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. y j j g p A: awak suda baco buku ini dk? (kamu sudah baca buku ini tidak?) B: suda, bagos nian beda emng kualitas tulisannyo (sudah, sekali beda memang kualitas tulisannya) A: diotu penulis terkenal, wajarbe isi bukunyo bekulitas galo (dia itu penulis terkenal, wajar saja isi bukunya berkualitas semua) B: iyo jugo sih (iya juga sih) y j j g p A: awak suda baco buku ini dk? (kamu sudah baca buku ini tidak?) B: suda, bagos nian beda emng kualitas tulisannyo (sudah, sekali beda memang kualitas tulisannya) A: diotu penulis terkenal, wajarbe isi bukunyo bekulitas galo (dia itu penulis terkenal, wajar saja isi bukunya berkualitas semua) B: iyo jugo sih (iya juga sih) Situasi tutur : percakapan ddiatas terjadi saat acara kumpul keluarga. A memberi pujian yang disetujui oleh B karena sama-sama merasa bahwa penulis ini memang berkuliatas dalam segi penulisan, isi, dan lain sebagainya. Analisis : berdasarkan percakapan diatas pada bagian yang bercetak tebal merupakan suatu penuturan pujian dan respon dari pujian tersebut A sebagai penutur dan B sebagai mitra tutur saling berbincnag antar satu sama lain membahas mengenai buku salah satu penulis yang cukup 4. Simpulan p Ungkapan pujian masih sering digunakan oleh masyarakat melayu Jambi, dari kelima jenis pujian tersebut yang paling sering digunakan adalah jenis pujian Penampilan Fisik (Physical Appearance), hal ini menujukkan masyarakat melayu Jambi sering memperhatikan penampilan fisik satu sama lainnya. Berdasarkan empat fungsi pujian menurut Wolfson dan Manes (dalam Paulston dan Tucker, 2003:181), pada hasil penelitian terdapat satu fungsi yang tidak ditunjukkan yakni fungsi yang menujukkan rasa hormat. Sehingga bisa disimpulkan bahwa orang melayu Jambi tidak menunjukkan ungkapan pujian yang berfungsi untuk menunjukkan rasa hormat. Dari hasil penelitian respon pujian (Herbert 1990, dalam Nur Mukminatien dan Andhuna Wisnu Patriana, 2005) yang paling sering digunakan yaitu menerima pujian, mengomentari pujian, dan menjelaskan sejarah. Pada Kemampuan (Ability) ditemukan respon pujian sebanyak 9 jenis, Kepemilikan (Possession) ditemukan respon pujian sebanyak 8 jenis, Penampilan Fisik (Physical Appearance) ditemukan respon pujian sebanyak 6 jenis, Kepribadian (Personality) ditemukan respon pujian sebanyak 6 jenis, dan Kualitas (Quality) ditemukan respon pujian sebanyak 6 jenis. Ungkapan pujian masih sering digunakan oleh masyarakat melayu Jambi, dari kelima jenis pujian tersebut yang paling sering digunakan adalah jenis pujian Penampilan Fisik (Physical Appearance), hal ini menujukkan masyarakat melayu Jambi sering memperhatikan penampilan fisik satu sama lainnya. Berdasarkan empat fungsi pujian menurut Wolfson dan Manes (dalam Paulston dan Tucker, 2003:181), pada hasil penelitian terdapat satu fungsi yang tidak ditunjukkan yakni fungsi yang menujukkan rasa hormat. Sehingga bisa disimpulkan bahwa orang melayu Jambi tidak menunjukkan ungkapan pujian yang berfungsi untuk menunjukkan rasa hormat. Dari hasil penelitian respon pujian (Herbert 1990, dalam Nur Mukminatien dan Andhuna Wisnu Patriana, 2005) yang paling sering digunakan yaitu menerima pujian, mengomentari pujian, dan menjelaskan sejarah. Pada Kemampuan (Ability) ditemukan respon pujian sebanyak 9 jenis, Kepemilikan (Possession) ditemukan respon pujian sebanyak 8 jenis, Penampilan Fisik (Physical Appearance) ditemukan respon pujian sebanyak 6 jenis, Kepribadian (Personality) ditemukan respon pujian sebanyak 6 jenis, dan Kualitas (Quality) ditemukan respon pujian sebanyak 6 jenis. 143 A sebagai penutur dan B sebagai mitra tutur sedang berinteraksi satu sama lain membahas mengenai kehebatan dalam hal berolahraga sehingga disarankan untuk menjadi atlit saja, ungkapan pujian ini berfungsi untuk mengekspresikan rasa kagum atas kehebatan B dan merasa besar kemungkinan bahwa dia bisa menjadi atlit berkualitas kedepannya. 143 143 Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX j g Volume 1, No. 2, September 2022 terkenal akan tulisannya sehingga tidak sedikit banyak penggemar yang turut membicarakannya. Pujian ini berfungsi untuk mengekspresikan rasa kagum atas kualitas isi buku penulis tersebut. kagum atas kualitas isi buku penulis tersebut. A: abang ni padek nian, besok jadi atlet be (abang ini hebat sekali, besok jadi atlit saja) B: iyopo? (benarkah?) A: iyo, terosin be kalo emang suko (iya, teruskan saja kalau memang suka) B: iyo gekla dicubo (iya nanti di coba) Situasi tutur : percakapan diatas terjadi saat acara kumpul keluarag. A memberikan pujian kepada B karena melihat kehebatannya khususnya dalam bidang berolahraga sehingga disarakan untuk menjadi atlit saja. Analisis : berdasarkan kutipan percakapan diatas pada bagian yang bercetak tebal tersebut merupakan ungkapan pujian dan respon dari pujian tersebut. A sebagai penutur dan B sebagai mitra tutur sedang berinteraksi satu sama lain membahas mengenai kehebatan dalam hal berolahraga sehingga disarankan untuk menjadi atlit saja, ungkapan pujian ini berfungsi untuk mengekspresikan rasa kagum atas kehebatan B dan merasa besar kemungkinan bahwa dia bisa menjadi atlit berkualitas kedepannya. kagum atas kualitas isi buku penulis tersebut. A: abang ni padek nian, besok jadi atlet be (abang ini hebat sekali, besok jadi atlit saja) B: iyopo? (benarkah?) A: iyo, terosin be kalo emang suko (iya, teruskan saja kalau memang suka) B: iyo gekla dicubo (iya nanti di coba) Situasi tutur : percakapan diatas terjadi saat acara kumpul keluarag. A memberikan pujian kepada B karena melihat kehebatannya khususnya dalam bidang berolahraga sehingga disarakan untuk menjadi atlit saja. Analisis : berdasarkan kutipan percakapan diatas pada bagian yang bercetak tebal tersebut merupakan ungkapan pujian dan respon dari pujian tersebut. A sebagai penutur dan B sebagai mitra tutur sedang berinteraksi satu sama lain membahas mengenai kehebatan dalam hal berolahraga sehingga disarankan untuk menjadi atlit saja, ungkapan pujian ini berfungsi untuk mengekspresikan rasa kagum atas kehebatan B dan merasa besar kemungkinan bahwa dia bisa menjadi atlit berkualitas kedepannya. g g gg j j Analisis : berdasarkan kutipan percakapan diatas pada bagian yang bercetak tebal tersebut merupakan ungkapan pujian dan respon dari pujian tersebut. Daftar Pustaka Achmad. (2012). Linguistik Umum. Jakarta: Erlangga. Chaer, A. (2012). Linguistik Umum. Yogyakarta: Rineka Cipta. Achmad. (2012). Linguistik Umum. Jakarta: Erlangga. Chaer, A. (2012). Linguistik Umum. Yogyakarta: Rineka Cipta. 144 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. 144 144 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX P-ISSN XXXX-XXXX E-ISSN XXXX-XXXX Kalistra: Kajian Linguistik dan Sastra Volume 1, No. 2, September 2022 Kalistra: Kajian Linguistik dan Sastra j g Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX j g Volume 1, No. 2, September 2022 Daromes, S, W. (2019). Ungkapan Pujian Dalam Film Me Before You Karya Jojo Moyes: Suatu Analisis Pragmatik. Skripsi. Universitas Sam Ratulangi, Manado. Departemen Pendidikan dan Kebudayaan. (1997). Kamus Besar Bahasa Indonesia. Jakarta: Balai Bahasa. Giri, N, L., Aryani, M, R. (2019). Variasi Pujian Terhadap Tayangan Komedi Berbahasa Bali. Seminar Internasional Riksa Bahasa XIII.Bali. Herbert, R. (1990). Language in Society.Cambridge University Press. Holmes, J. (1986). Compliment And Compliment and Responsese in New Zealand English. Journal Of Pragmatics. Holmes, J., Brown. (1987). Teacher And Student Learning About Compliment. TESOL Quarterly, 21(3). Q y, ( ) Holmes, J. (1992) An Introdunction to Sociolinguistics. London: Blackwell. Ismi, F., Ermanto. (2020). Ungkapan Marah Dan Ungkapan Pujian Masyarakat Nias Di Kaki Gunung Padang. Jurnal Bahasa Dan Sastra, 8(1). Keraf, G. (1997). Komposisi. Jakarta: Ikrar Media Mandiri. g g Keraf, G. (1997). Komposisi. Jakarta: Ikrar Media Mandiri. ( ) p Kridalaksana, Harimurti. (2008). Kamus Linguistik.Jakarta: Gramedia Pustaka Utama. Kusmana, A., & Afria, R. (2018). Analisis Ungkapan Makian dalam Bahasa Kerinci: Studi Sosiolinguistik. Titian: Jurnal Ilmu Humaniora, 2(02), 173 -. https://doi.org/10.22437/titian.v2i02.6090 Lyons, J. (1975). Penghantar Teori Lingu istik. Jakarta: Gramedia Pustaka Utama. Miles., Huberman. (1992). Analisis Data Kualitatif. Jakarta: Universitas Indonesia Press. Moleong, Lexy J. (2006). Metodologi Penelitian Kualitatif. Bandung: PT Remaja Rosdakarya. Mukminatien, N., Patriana, A, W. (2005). Respon Pujian Dalam Bahasa Indonesia Oleh Dwibasawan Indonesia- Inggris.Jurnal Bahasa dan Seni. Paulston, Tucker. (2003). Sociolinguistics: The Essential Readings. Australia: Blackwell Publishing. Purwo, B, K. (1990). Paragmatik dan Pengajaran Bahasa. Yogyakarta: Kanisius Warni, W., & Afria, R. (2019). Menelisik Kearifan Lokal Masyarakat Melayu Jambi Berbasis Cerita Rakyat dalam Membangun Peradaban. Titian: Jurnal Ilmu Humaniora, 3(2), 295-313. https://doi.org/10.22437/titian.v3i2.8222 Warni, W., Afria, R. (2020). Analisis Ungkapan Tradisional Melayu Jambi: Kajian Hermeneutik. Sosial Budaya, 17(2), 83-94, http://dx.doi.org/10.24014/sb.v17i2.10585 Wijana., Dewi, I., Muhammad, R. (2011). Analisis Wacana Pragmatik, Kajian Teori dan Analisis. Daftar Pustaka Surakarta: Yuma Pustaka. Wolfson, N., Manes, J. (1980). Pujian Sebagai Strategi Sosial. Makalah Dalam Linguistik,13:391- 410. Yule, George. (2010). The Study of Language. Cambridge: Cambridge University. Yule, George. (2014). Pragmatik.Jilid 2. Yogyakarta: Pustaka Pelajar. 145 Lerisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di ….. Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX Zainal, Arifin. (2011). Evaluasi Pembelajaran Prinsip, Teknik, Prosedur. Bandung: PT Remaja Rosdakarya. Ziaei, N. (2012). Tanggapan Pujian Dan Pujian.Jurnal Terjemahan Hak Cipta Dan Penulis, 16(3). Kalistra: Kajian Linguistik dan Sastra P-ISSN XXXX-XXXX Volume 1, No. 2, September 2022 https://online-journal.unja.ac.id/kal E-ISSN XXXX-XXXX Zainal, Arifin. (2011). Evaluasi Pembelajaran Prinsip, Teknik, Prosedur. Bandung: PT Remaja Rosdakarya. Ziaei, N. (2012). Tanggapan Pujian Dan Pujian.Jurnal Terjemahan Hak Cipta Dan Penulis, 16(3). j g Volume 1, No. 2, September 2022 Zainal, Arifin. (2011). Evaluasi Pembelajaran Prinsip, Teknik, Prosedur. Bandung: PT Remaja Rosdakarya. Ziaei, N. (2012). Tanggapan Pujian Dan Pujian.Jurnal Terjemahan Hak Cipta Dan Penulis, 16(3). 146 erisa Ramadhea, Ade Kusmana, Julisah Izar: Ungkapan Pujian Bahasa Melayu Jambi di …..
W4205842143.txt	https://enforcement.omsu.ru/jour/article/download/566/628	en		Tax obligation fulfillment	Pravoprimenenie	2,022	cc-by	7,195	УДК 347.73 DOI 10.52468/2542-1514.2021.5(4).135-147 ИСПОЛНЕНИЕ НАЛОГОВОГО ОБЯЗАТЕЛЬСТВА А.В. Красюков Воронежский государственный университет, г. Воронеж, Россия Информация о статье Дата поступления – 17 февраля 2021 г. Дата принятия в печать – 10 октября 2021 г. Дата онлайн-размещения – 24 декабря 2021 г. Ключевые слова Налоговое обязательство, исполнение обязательства, субъективные права и обязанности, стадии исполнения обязательства, критерии надлежащего исполнения обязательства, сделка, добросовестность Изучается механизм исполнения налогового обязательства. Ставится задача определить сущность исполнения налогового обязательства. В процессе исследования установлено, что механизм исполнения налогового обязательства представляет собой определенный законом набор алгоритмов поведения его субъектов, использующих конкретные правовые средства (например, налоговую декларацию, требование об уплате налога и др.). Выделяются пять критериев надлежащего исполнения налогового обязательства: 1) надлежащие субъекты исполнения; 2) надлежащее место; 3) надлежащее время; 4) надлежащий объект; 5) надлежащий способ. Как итог проведенного исследования делается вывод, что сущность исполнения налогового обязательства может быть охарактеризована как сделка его сторон, направленная на взаимное прекращение и возникновение прав его сторон. TAX OBLIGATION FULFILLMENT Andrey V. Krasyukov Voronezh State University, Voronezh, Russia Article info Received – 2021 February 17 Accepted – 2021 October 10 Available online – 2021 December 24 Keywords Tax obligation, obligation fulfillment, subjective rights and obligations, stages of an obligation fulfillment, criteria for the proper obligation fulfillment, a transaction, good faith The subject. The article is devoted to the study of the mechanism of tax obligation fulfillment. The author established that there are several points of view in understanding the legal essence of the fulfillment of an obligation in Russia and abroad: 1) contractual theories (the general contractual theory, the theory of a real contract, the limited contractual theory); 2) the theory of target impact; 3) the theory of real impact. The purpose of the article is to determine the legal essence of tax obligation fulfillment, to study the concept of the tax obligation fulfillment, the mechanism for exercising subjective rights and obligations, and the criteria for the proper fulfillment of a tax obligation. The methodology. The author uses general and specific scientific methods of scientific research: observation, systemic-structural, dialectical, analysis, comparative jurisprudence and others. The main results, scope of application. There are two sides of the tax obligation fulfillment: legal and factual. From an actual point of view, the tax obligation fulfillment is a set of operations by its parties with the object of the obligation. As a result, the object of the obligation must pass from one owner to another. From a legal point of view, the tax obligation fulfillment always represents the realization of its content through the exercise of rights and the fulfillment of obligations. The author believes that the tax obligation fulfillment should not be equated with the fulfillment of the obligation to pay tax, since not every obligation is executed through the payment of tax. In this regard, a situation may arise when the obligation is properly fulfilled by the debtor, and the creditor does not receive the property grant (for example, if a loss is received at the end of the tax period). The mechanism of the implementation of subjective tax rights and the fulfillment of obligations is a certain system of legal means and algorithms of behavior that allow to determine the appropriate size of the claim of a public law entity on the taxpayer's property and ensuring the transfer of the monetary is equivalent to this economic benefit to the budget 135 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2542-1514 (Print) system. This mechanism is a set of algorithms for the behavior of subjects of tax liability, defined by law, using specific legal means. As such means, depending on the party of the tax obligation, the law provides, for example, a tax return, a requirement to pay tax, etc. The mechanism of exercising subjective rights and fulfilling obligations includes the following stages: 1) pre-implementation; 2) procedural implementation; 3) actual implementation; 4) protection of the violated right. The author identifies five criteria for the proper fulfillment of a tax obligation: 1) the proper subjects of execution; 2) the proper place; 3) the proper time; 4) the proper object; 5) the proper way. Conclusions. The legal essence of the tax obligation fulfillment can be characterized as a transaction between its parties, aimed to mutual termination and the emergence of the rights of its parties. In order to protect the rights of bona fide taxpayers the author proposes to enshrine in tax legislation a ban on contradictory behavior in the process of fulfilling a tax obligation. ____________________________________________ 1. Введение Правовая природа исполнения обязательства давно интересует ученых-правоведов не только в России, но и за рубежом. Так, в Европе сложилось несколько точек зрения в понимании правовой сущности исполнения обязательства: 1. Договорные теории (общая договорная теория, теория реального договора, ограниченная договорная теория) видят в исполнении договор или соглашение об исполнении. Исполняя обязательство, по мнению сторонников данных теорий, должник и кредитор заключают некий договор или соглашение, опосредующее их волю на прекращение обязательства. Данный договор заключается посредством предоставления должником исполнения и принятия кредитором этого исполнения, что расценивается в качестве распоряжения им своим требованием, которое в результате этого распоряжения прекращается. При этом ограниченная договорная теория рассматривает исполнение в качестве сделки только в отношении тех обязательств, результаты которых передаются по акту приема-передачи. Если составление такого акта не требуется, то для исполнения обязательства сделка не требуется, достаточно самого предоставления. Теория реального договора выделяет в составе договора о прекращении обязательства исполнением два элемента: 1) совершение задолженного предоставления (объективный элемент состава) и 2) соглашение о цели предоставления (субъективный элемент состава). 2. В соответствии с теорией целевого воздействия исполнение обязательства не является сделкой. Обязательство исполняется путем совершения обусловленного предоставления, которое должно сопровождаться односторонним заявлением испол- нителя о том, что он действовал с целью исполнить определенное обязательство. 3. Приверженцы теории реального воздействия предоставления считают, что обязательство прекращается, если удовлетворены интересы кредитора. В большинстве случаев для этого достаточно активности должника по предоставлению необходимого блага. Однако в некоторых случаях необходимо и более активное участие самого кредитора, например для принятия исполнения (по: [1, с. 140]). Среди российских ученых также довольно распространена позиция о том, что исполнение обязательства является сделкой, направленной на прекращение обязательства (правосделочные теории). Однако есть представители научного сообщества, которые считают, что прекращение обязательства посредством исполнения происходит в силу предписания закона и, следовательно, не зависит от наличия соответствующей воли сторон (теории легального освобождения) [1, с. 144]. Сторонников «правосделочной теории» можно разделить на тех, кто рассматривает исполнение обязательства как совокупность двух односторонних сделок должника и кредитора, направленных на прекращение обязательства и состоящих в односторонних актах предложения и принятия исполнения [2, с. 23–25], и тех, кто придерживается дифференцированного подхода, в силу которого, если для исполнения обязательства требуется взаимная и согласованная воля двух сторон, то исполнение представляет собой двустороннюю сделку, а когда достаточно только активности должника – одностороннюю [3, с. 880]. В правовой науке даже выделяется особая разновидность сделок – ремиссионных, к числу которых ученые относят исполнение обязательства [4, с. 75–83]. 136 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2658-4050 (Online) В связи с этим учеными предлагается рассматривать исполнение обязанности как индивидуальную юридическую процедуру, прекращающую вспомогательную сделку [5, с. 78], направленную на обеспечение достижения определенного экономического блага [6, с. 34]. По мнению других ученых, исполнение обязательства является не сделкой, а юридическим поступком, иными словами – правомерным юридическим действием, правовые последствия которого наступают независимо от субъективного момента, т. е. направленности действий должника [7, с. 471; 8, с. 360–361; 9, с. 8]. Третьи полагают, что исполнение обязательства представляет собой правореализационный акт, состоящий в признании того субъективного права, которому корреспондирует исполняемая обязанность [10, с. 66]. В рамках настоящей работы нам на основе анализа механизма исполнения налогового обязательства предстоит разобраться, какой из вышеуказанных подходов к пониманию исполнения обязательства является верным применительно к налоговому обязательству. 2. Понятие исполнения налогового обязательства По мнению некоторых ученых, суть обязательства шире и разностороннее и имеет более объемное социальное содержание, нежели конкретные право и обязанность. Поэтому они предлагают разделять термины «исполнение обязанности» и «реализация обязательства» [11, с. 40]. Представляется, что данное предложение требует дополнительного обоснования, поэтому в данной работе термины «исполнение» и «реализация» будут использоваться как синонимы. В юридической литературе под исполнением обязательства в той или иной интерпретации обычно понимается совершение или воздержание от совершения действий, составляющих предмет обязательства (напр., [8, с. 417; 4, с. 10; 12, с. 7]) или содержание прав и обязанностей его сторон [13, с. 37; 4, с. 10]. Данный подход не вызывает дискуссий и является устоявшимся в российской правовой науке. Следовательно, в исполнении налогового обязательства следует выделять две стороны: юридическую и фактическую. С фактической точки зрения исполнение налогового обязательства представляет собой совокупность операций его сторон с объектом обязательства, в результате которых последний должен перейти от одного обладателя к другому. С юридической точки зрения исполнение налогового обязательства всегда представляет собой реализацию его содержания посредством осуществления прав и исполнения обязанностей. Под осуществлением субъективного права в правовой доктрине традиционно понимается реализация его содержания, совершение управомоченным лицом тех действий, возможность которых предусматривается содержанием соответствующего субъективного права в целях удовлетворения своих законных интересов, потребностей [14, с. 7]. Тем самым осуществление права связано с превращением составляющей содержания субъективного права из возможности в действительность [15, с. 44]. Результатом осуществления права кредитора в налоговом обязательстве, как правило, является присвоение денежных средств, передаваемых должником в качестве исполнения. Передача данного имущества приводит к прекращению обязательства вследствие удовлетворения имущественного интереса кредитора. Тем самым субъективное право в налоговом обязательстве может быть реализовано исключительно за счет исполнения (добровольного и принудительного) обязанности налогоплательщика, а надлежащее исполнения обязанности с необходимостью приводит к осуществлению субъективного права. По мнению ученых, осуществление субъективного права и исполнение обязанности можно назвать средством реализации обязательства, выражающееся в достижении его конечной цели – удовлетворении имущественных интересов управомоченного лица [16, с. 94]. Применительно к налоговому обязательству в качестве конечной цели подразумевается удовлетворение публичного фискального интереса1. 1 лизовано неосновное правомочие в содержании субъективного права [17, с. 55]. В соответствии с данным подходом вместо правомочия по истребованию суммы налога реализуется правомочие по списанию безнадежного налогового долга. При этом мы можем говорить о том, что право на взимание налога реализовано, поскольку отказ от осуществления права – составная часть этого права (правомочие). Существует мнение, что в объективной реальности отсутствует правовое состояние, когда субъективные права не реализуются или субъективные обязанности не исполняются. Недостижение идеальной основной цели субъективного права (удовлетворение имущественного интереса), которая была заложена позитивным правом, демонстрирует лишь то, что оно было осуществлено по дополнительной (факультативной) составляющей, а именно было реа- 137 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2542-1514 (Print) Удовлетворение публичного фискального интереса производится путем перераспределения экономических благ в установленной законом доле в пользу публично-правовых образований. Данное перераспределение не предполагает установление конкретных размеров платежей, а только – определенную долю от имеющегося или полученного налогоплательщиком экономического блага. В связи с этим размер имущественных притязаний налоговых органов определяется в процессе исполнения налогового обязательства. Следовательно, не следует отождествлять исполнение налогового обязательства и исполнение обязанности по уплате налога, поскольку не каждое обязательство реализуется посредством уплаты налога. В связи с этим может возникнуть ситуация, когда обязательство будет исполнено должником надлежащим образом, а кредитор не получит имущественного предоставления (например, при получении убытка по итогам налогового периода). Не следует также считать целью налогового обязательства обеспечение участия налогового должника в покрытии публичных расходов [18, с. 301], поскольку в этом случае обязанности налогоплательщиков разнились бы год от года в зависимости от показателей бюджета. Например, при профиците бюджета объем налогового обязательства должен сокращаться, а при дефиците – соответственно увеличиваться. Однако, в действительности этого не происходит. Нехватка у государства средств для исполнения бюджета по доходам не является основанием для предъявления дополнительных имущественных притязаний в рамках исполняемых или исполненных налоговых обязательств, даже если эти притязания замаскированы под изменение практики применения норм налогового права или их толкования. Тем более не допустимы прямое изменение действующих налоговых обязательств под предлогом получения налогоплательщиком в текущем налоговом периоде так называемых «сверхдоходов»2. Однако не следует принижать роль удовлетворения публичного фискального интереса как цели исполнения налогового обязательства. Именно данным интересом продиктованы некоторые изменения налогового законодательства, сближающие налоговое обязательство с гражданско-правовым. Например, нормативное закрепление возможности уплаты налога третьим лицом (ст. 45 Налогового кодекса (далее – НК) РФ) вполне соответствует данному интересу, поскольку государству важно получить свою часть имущества из гражданского оборота, а личностью плательщика можно и пренебречь. Субъективное право предоставляет управомоченному лицу возможность выбора определенного правомерного поведения с целью достижения желаемого результата (блага), возможность потенциальную, лишь зафиксированную в законе [19, с. 127– 128]. Субъект по общему правилу своим усмотрением может как осуществлять право, так и отказаться от его осуществления. Из этого правила имеются исключения, когда закон обязывает осуществлять права либо когда отказ от осуществления права признается злоупотреблением правом [17, с. 56]. Когда этого выбора нет, то поведение лица становится необходимостью, т. е. обязанностью. В этом случае субъективное право «смыкается» с обязанностью лица: совершение того или иного действия становится одновременно и правом лица, и его обязанностью [20, с. 37]. Налоговое обязательство многими приводится в качестве примера такого «смыкания» в части субъективных прав налогового кредитора [21, с. 15; 22, с. 30; 23]. Однако, на наш взгляд, следует разделять права налогового кредитора, коим является публично-правовое образование, и права уполномоченных государством органов (подробнее о финансовой правосубъектности уполномоченных государством органов см.: [24, с. 110]). Налоговый кредитор свободен в осуществлении своего права: он может как реализовать его, так и отказаться от этого. При этом отказ от осуществления права (но не от самого права) на взимание налога допускается только в форме закона (нормативного акта представительного органа местного самоуправления). Этот отказ может быть реализован напрямую (например, отмена налога или освобождение от налогообложения каких-то операций) либо посредством закрепления в законе специальных механизмов (например, списание безнадежных долгов). Уполномоченные же государством органы не вправе отказываться от осуществления прав, предоставленных им законом, при наличии всех необходимых юридических фактов, поэтому эти права – 2 www.rbc.ru/business/09/08/2018/5b6c5ee59a7947e3df055 4a3 (дата обращения: 01.01.2021). Бурмистрова С., Маринин В. Изъять ₽500 млрд: где Белоусов нашел сверхдоходы на майские указы / при участии: П. Химшиашвили, Т. Дзядко // РБК. 9 авг. 2018. URL: https:// 138 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2658-4050 (Online) обязанности, предлагается именовать полномочиями. Полномочия следует рассматривать как отдельные правомочия, составляющие субъективное право налогового кредитора, реализация которых налоговыми органами должна быть подробно регламентирована и лишена, насколько это возможно, свободы усмотрения с их стороны. Исполнение обязанностей, в свою очередь, представляет собой проведение их в жизнь путем превращения в действительность необходимости определенного поведения сторон правоотношения [25, с. 103–104; 16, с. 95]. 3. Механизм осуществления прав и обязанностей в налоговом обязательстве Механизмы осуществление прав и обязанностей имеют определенное сходство, по сути, представляя собой единое целое, поскольку исполнение обязанности играет ключевую роль при осуществлении субъективного права и является одной из гарантий его осуществления [26, с. 8]. Механизм осуществления субъективных гражданских прав и исполнения обязанностей представляет собой такую организацию правовых средств и создание условий, позволяющих достичь результата, заложенного в матрицу юридической связи права и обязанности [20, с. 40]. В полной мере данные слова могут быть отнесены и к налоговому обязательству. Тем самым механизм осуществления субъективных налоговых прав и исполнения обязанностей представляет собой определенную систему правовых средств и алгоритмов поведения, позволяющих определить надлежащий размер притязания публичноправового образования на имущество налогоплательщика и обеспечить переход денежного эквивалента этого экономического блага в бюджетную систему. Поскольку субъективные налоговые права и обязанности, как мы установили выше, могут быть осуществлены только через активное поведение сторон налогового обязательства, то вышеуказанный механизм представляет собой определенный законом набор алгоритмов поведения субъектов налогового обязательства, использующих конкретные правовые средства. В качестве таких средств в зависимости от стороны налогового обязательства закон предусматривает, например, налоговую декларацию (и иные формы налоговой отчетности), требование об уплате налога и др. Например, механизм осуществления субъективного права и исполнения обязанности по уплате налога на имущество физических лиц состоит из следующих алгоритмов поведения: Первый вариант: – исчислить налог и направить налоговое уведомление (налоговый орган); – получить налоговое уведомление (налогоплательщик); – заплатить налог предусмотренным законом способом (налогоплательщик); – отразить налог в качестве уплаченного в бюджетную систему (налоговый орган). Второй вариант: – заплатить единый налоговый платеж физического лица (далее – ЕНП) в достаточном размере (налогоплательщик); – исчислить налог и направить налоговое уведомление (налоговый орган); – произвести зачет ЕНП в счет уплаты налога и отразить налог в качестве уплаченного в бюджетную систему (налоговый орган). В качестве правовых средств вышеуказанного механизма выступают налоговое уведомление и платежные документы. Представляется допустимым в некоторых случаях в качестве средства осуществления субъективного права или исполнения обязанности рассматривать обеспечительные налоговые обязательства (поручительство, залог, банковская гарантия), а также обеспечительные меры не обязательственного характера (например, приостановление операций по счетам в банке). Механизм осуществления субъективного права и исполнения обязанности в широком смысле, по мнению некоторых ученых, включает в себя следующие стадии: – предреализационная: формирование и установление прав и обязанностей, – процедурная реализация, – фактическая реализация, – защита нарушенного права [27, с. 3]. Применительно к налоговым правам и обязанностям мы также можем выделить предреализационную, или подготовительную, стадию, направленную на определение объема прав и обязанностей, которая заключается в постановке на учет в налоговом органе налогоплательщиков (регистрации предметов налогообложения), а также аккумулирование информации о его предметах налогообложения из иных источников. Процедурная реализация заключается в ведении бухгалтерского и налогового учета, подготовке и подаче налоговой отчетности. В некоторых случаях на данной стадии требуется принятие решения о взыскании недоимки, задолженности по пеням и 139 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2542-1514 (Print) штрафам. Фактическая реализация заключается в уплате налога или его принудительном взыскании. Защита нарушенного права в налоговых отношениях осуществляется в двух формах: юрисдикционная и самозащита. Юрисдикционная защита подразделяется на судебную и внесудебную. Самозащита реализуется путем отказа от исполнения незаконных требований контрагента [28, с. 24]. В юридической литературе различают режимы реализации правоотношения как системы методов и способов правового регулирования поведения лица, включая особый порядок возникновения и формирования содержания прав и обязанностей, процедуры их осуществления, специфику санкций и способов их реализации. Выделяют нормативно-правовой, организационно-правовой, договорно-правовой и правоохранительный режимы реализации права [29, с. 152; 30, с. 78; 31, с. 101]. Применительно к налоговому обязательству основным является нормативно-правовой режим, поскольку порядок его исполнения практически полностью определяется законом. Однако в отношении налоговых обязательств, основанных на соглашении (например, инвестиционный налоговый кредит) мы можем говорить о смешанном режиме реализации прав и обязанностей, поскольку порядок реализации такого обязательства установлен и законом, и договором. 4. Надлежащее исполнение налогового обязательства Несмотря на режим реализации прав и обязанностей в обязательстве в его основе всегда заложен принцип надлежащего исполнения. Принцип надлежащего исполнения обязательства может рассматриваться как организованная совокупность элементов, каждый из которых имеет относительно самостоятельное и в то же время необходимое для существования всей системы значение [32, с. 116]. При этом выделяется пять критериев надлежащего исполнения обязательства: – надлежащие субъекты исполнения; – надлежащее место; – надлежащее время; – надлежащий объект; – надлежащий способ [33, с. 534; 13, с. 37; 32, с. 116]. Следует отметить, что некоторые авторы выделяют более краткий перечень признаков надлежащего исполнения [34, с. 43]. Данные критерии вполне применимы при определении надлежащего исполнения налогового обязательства. 1) Субъекты исполнения налогового обязательства представлены в лице налогового кредитора и налогового должника. Надлежащий должник. Долгое время закрепленный в ст. 45 НК РФ принцип самостоятельности исполнения налогового обязательства и учеными, и практиками трактовался как свидетельство личного характера налогового обязательства 3 и, тем самым, невозможности его исполнения третьим лицом, если иное не предусмотрено налоговым законодательством. Так, Конституционный Суд РФ в своем определении № 41-О от 22 января 2004 г.4 указал, что налог считается уплаченным самостоятельно, если он уплачен от имени налогоплательщика и за счет его собственных средств. В виде исключения исполнение налогового обязательства третьим лицом допускалось в следующих случаях: – законным или уполномоченным представителем налогоплательщика (ст. 26–29 НК РФ); – ликвидационной комиссией (ликвидатором) или учредителями ликвидируемой организации (ст. 49 НК РФ); – наследниками или правопреемниками налогоплательщика (ст. 44, 50 НК РФ); – поручителем или банком, выдавшим гарантию, а также залогодателем (ст. 73–74.1 НК РФ). Подобное регулирование исполнения налогового обязательства порождало определенный диссонанс, логическую ловушку. Во-первых, если налоговое обязательство неразрывно связано с личностью налогоплательщика и требует самостоятельного исполнения им обязанности по уплате налога, то почему вообще допускается уплата налога третьим лицом, даже в виде исключения? 3 лобы открытого акционерного общества “Сибирский Тяжпромэлектропроект” и гражданки Тарасовой Галины Михайловны на нарушение конституционных прав и свобод абзацем первым пункта 1 статьи 45 Налогового кодекса Российской Федерации» // Экономика и жизнь. 2004. № 20. Под обязательством личного характера мы понимаем обязательство неразрывно, связанное с личностью кредитора или должника. 4 Определение Конституционного Суда РФ от 22 января 2004 г. № 41-О «Об отказе в принятии к рассмотрению жа- 140 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2658-4050 (Online) Во-вторых, в чем личность налогоплательщика оказывает влияние на существо налогового обязательства, т. е. обязанность передать денежные средства (вещи, определенные родовыми признаками) в бюджетную систему, не понятно. Представляется, что денежное обязательство не может быть настолько связано с личностью должника, что это будет препятствием для кредитора в принятии исполнения, предложенного третьим лицом за должника. В связи с вышеуказанным диссонансом или в силу возрастающей потребности в финансовых ресурсах законодателем правовое регулирование исполнения налогового обязательство было изменено5, и в настоящее время оно может быть исполнено любым третьим лицом. Таким образом, налоговое обязательство по общему правилу должно быть исполнено налогоплательщиком, а также иными лицами, к которым объект налогового обязательства (налог) перешел в ходе его исполнения (налоговый агент, банк и др.), либо на иных законных основаниях (представители, правопреемники и др.). При этом любое третье лицо вправе исполнить обязанность по уплате налога за налогоплательщика в добровольном порядке либо посредством предоставления обеспечительных мер (залог или поручительство), либо непосредственно путем уплаты налога. Не следует путать исполнение налогового обязательства третьим лицом и перевод долга. Последний предполагает полное прекращение статуса налогового должника у одного лица с одновременным возникновением его у другого лица. Такой переход статуса в налоговых отношениях возможен только в порядке универсального правопреемства (реорганизация юридических лиц или наследование за физическим лицом). Договорные формы перевода долга в налоговом праве не применимы. А существование права у любого третьего лица исполнить налоговое обязательство за налогоплательщика при наличии у налогового кредитора обязанности принять такое испол- нение лишает данный вопрос в налоговом праве какой-либо актуальности. Надлежащий кредитор. Публично-правовые образования как налоговый кредитор представлены в налоговых отношениях единым уполномоченным органом – Федеральным казначейством и его территориальными органами, которое осуществляет кассовое обслуживание всех бюджетов бюджетной системы, включая проведение и учет всех операций по поступлениям в бюджеты6, а также распределение налоговых доходов между бюджетами бюджетной системы7. Иными словами, вышеуказанный орган уполномочен на получение всех налогов в бюджетную систему. Поэтому, несмотря на множественность лиц на стороне налогового кредитора (Российская Федерация, ее субъекты и муниципальные образования), у должника практически отсутствует проблема выбора надлежащего налогового кредитора. Со стороны налогоплательщика для исполнения налогового обязательства требуется правильно определить объект (указать код бюджетной классификации – КБК) и место исполнения налогового обязательства (указать код общероссийского классификатора территорий муниципальных образований – ОКТМО), а также указать банковские реквизиты органа Федерального казначейства. Неправильное указание КБК и (или) кода ОКТМО налоговым должником может привести к исполнению налогового обязательства ненадлежащему кредитору. Например, при указании вместо КБК транспортного налога КБК земельного налога, налог будет зачислен не в бюджет субъекта Российской Федераци, а в бюджет муниципального образования. Или неправильное указание кода ОКТМО может привести к поступлению местного налога в бюджет ненадлежащего муниципального образования. Однако попадание налогового платежа в бюджетную систему даже при зачислении в ненадлежащий бюджет оставляет налоговому должнику возможность уточнить назначение платежа в порядке, предусмотренном ст. 45 НК РФ, и исполнить налоговое обязательство надлежащим образом. 5 брание законодательства Российской Федерации. 2004. № 49. Ст. 4908). 7 См.: Приказ Минфина России от 18 декабря 2013 г. № 125н «Об утверждении Порядка учета Федеральным казначейством поступлений в бюджетную систему Российской Федерации и их распределения между бюджетами бюджетной системы Российской Федерации» // Российская газета. 2014. 28 февр. Федеральный закон от 30 ноября 2016 г. № 401-ФЗ «О внесении изменений в части первую и вторую Налогового кодекса Российской Федерации и отдельные законодательные акты Российской Федерации» // Собрание законодательства Российской Федерации. 2016. № 49. Ст. 6844. 6 См. п. 5.12 Положения о Федеральном казначействе, утвержденного постановлением Правительства РФ от 1 декабря 2004 г. № 703 «О Федеральном казначействе» (Со- 141 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2542-1514 (Print) Если допущенные нарушения устраняются посредством процедуры уточнения назначения платежа, то такое ненадлежащее исполнение налогового обязательства не может быть основанием для привлечения налогового должника к юридической ответственности и начисления пени8. Только неправильное указание банковских реквизитов органа федерального казначейства при исполнении налогового обязательства, в результате которого налог не поступает в бюджетную систему, приводит к неисполнению налогового обязательства налоговым должником. 2) Место возникновения налогового обязательства. Место возникновения налогового обязательства имеет принципиальное значение для определения надлежащего налогового кредитора при исполнении обязанности по уплате региональных и местных налогов, а также федеральных налогов, в отношении которых законодательством установлены нормативы отчисления в нижестоящие бюджеты бюджетной системы. Место возникновения налогового обязательства предопределяется местом возникновения (нахождения) предмета налогообложения (реализация товара, недвижимое имущество и др.) и определяет место исполнения налогового обязательства: региональные и местные налоги подлежат зачислению в бюджет по месту возникновения (нахождения) предмета налогообложения. Для обеспечения надлежащего исполнения налогового обязательства в надлежащем месте налогоплательщик (налоговый агент) в реквизитах платежа обязаны указать код ОКТМО того муниципального образования, на территории которого возник предмет налогообложения. На основании данной информации органы федерального казначейства обеспечивают зачисление налога в соответствующий бюджет бюджетной системы. 3) Надлежащее время. По общему правилу, налог должен быть уплачен в срок, установленный законодательством о налогах и сборах. Следует отметить, что срок уплаты налога, предусмотренный действующим законодательством, может быть изменен на более поздний путем предоставления отсрочки (рассрочки) или инвестиционного налогового кредита. В этом случае требование своевременности уплаты будет соблюдено, несмотря на нарушение установленного законом срока уплаты налога. Срок уплаты налога не подлежит изменению на более ранний, поскольку налогоплательщик изначально имеет право исполнить свою обязанность досрочно. При проведении государством налоговой амнистии зачастую требование своевременности исполнения налогового обязательства преодолевается с помощью юридической конструкции – фикции. Например, в соответствии с Федеральным законом № 269-ФЗ от 22 декабря 2006 г. «Об упрощенном порядке декларирования доходов физическими лицами»9 в случае уплаты декларационного платежа, налоги за прошлые налоговый периоды считаются уплаченными своевременно. 4) Надлежащий объект. Объектом налогового обязательства является экономическое благо в форме налога, переходящее от налогового должника к налоговому кредитору. По общему правилу налоговое обязательство исполняется в валюте Российской Федерации. В случае если налоговая база выражена в иностранной валюте, то сумма налога (сбора), подлежащая оплате, переводится в рубли по курсу Центрального банка РФ на день уплаты налога. В НК РФ могут быть предусмотрены исключительные случаи, когда налог (сбор) может быть уплачен в иностранной валюте. В настоящее время такое исключение предусмотрено п. 5 ст. 333.29, согласно которому государственная пошлина за выдачу, продление срока действия и восстановление в исключительных случаях виз иностранным гражданам и лицам без гражданства представительствами федерального органа исполнительной власти, ведающего вопросами иностранных дел, находящимися в пунктах пропуска через Государственную границу РФ, может быть уплачена в иностранной валюте по курсу, установленному Центральным банком ЦБ РФ на дату ее уплаты. В силу периодического характера уплаты налога и наличия различных налогов и систем налогообложения объект налогового обязательства должен быть индивидуализирован применительно к виду налога и периоду платежа. Индивидуализация налогового платежа с целью последующего зачисления в конкретный бюджет бюджетной системы Российской Федерации 8 9 См.: Постановление ФАС Северо-Кавказского округа по делу № А32-26703/2018 от 22 мая 2019 г. // СПС «КонсультантПлюс». Собрание законодательства Российской Федерации. 2007. № 1. Ст. 32. 142 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2658-4050 (Online) производится посредством указания кода бюджетной классификации (КБК) и налогового периода. 5) Надлежащий способ. Налоговое законодательство в ст. 45 НК РФ предусматривает следующие способы исполнения налогового обязательства: 1) посредством уплаты наличных денежных средств – данный способ предназначен для исполнения налогового обязательства физическими лицами; 2) посредством уплаты безналичных денежных средств – универсальный способ, но является основным для организаций и индивидуальных предпринимателей10; 3) посредством внутрибюджетных расчетов – способ, используемый организациями, которым открыт лицевой счет, при уплате налога в тот же бюджет, что финансирует налогоплательщика; 4) посредством зачета встречных требований при излишней уплате (взыскании) налоговых платежей – универсальный способ, предназначенный для всех налогоплательщиков, являющихся кредиторами в налоговых обязательствах из неосновательного обогащения, или физических лиц, заплативших единый налоговый платеж; 5) посредством электронных денежных средств – данный способ предназначен для исполнения налогового обязательства физическими лицами; 6) посредством передачи имущества в натуральной форме – способ, используемый в отдельных случаях для поддержки инвестиционной деятельности организаций (в настоящее время используется только в соглашениях о разделе продукции); 7) посредством удержания денежных средств – при уплате налога через налогового агента; 8) посредством зачет единого налогового платежа физического лица – способ, предназначенный для упрощенной уплаты имущественных налогов физических лиц и НДФЛ. Поскольку налоговое обязательство, как было установлено выше, является средством реализации публичного фискального интереса, то моментом его надлежащего исполнения следует считать момент поступления налогового платежа в распоряжение уполномоченного государством органа – территориального органа Федерального казначейства (или прекращения встречного требования зачетом). В момент зачисления денежных средств на счет казначейства налоговые отношения завершаются (ст. 40 БК РФ) и завершается исполнение налогового обязательства11. Соответственно, неисполнением налогового обязательства будет такое нарушение, которое привело к непоступлению денежных средств в бюджетную систему. 5. Вывод В результате проведенного исследования механизма исполнения налогового обязательства мы приходим к выводу, что для исполнения налогового обязательства практически всегда требуется взаимная и согласованная воля двух сторон для надлежащего определения его объекта, места исполнения и других условий. Например, такой законодательный механизм согласования волеизъявления сторон предусмотрен ст. 45 НК РФ при уточнении назначения налогового платежа, а также в ст. 78 НК РФ при возврате и зачете излишне уплаченных налогов. Кроме того, для исполнения налогового обязательства важнейшим является факт изменения (прекращения) прав и обязанностей его сторон в отношении экономического блага, являющегося объектом такого обязательства. На основании вышеизложенного можно прийти к выводу, что правовая сущность исполнения налогового обязательства может быть охарактеризована как сделка его сторон, направленная на взаимное прекращение и возникновение прав его сторон. Как любая сделка исполнение налогового обязательства будет наиболее эффективным в условиях правовой определенности и непротиворечивости поведения сторон. В гражданском обязательстве для того, чтобы установить, надлежащим ли является его исполнение, необходимо сопоставить его с условиями самого обязательства (справедливо для договорных форм) и требованиями законодательства или обычаев делового 10 зательства (кроме исполнения зачетом), а к переходу исполнения налогового обязательства на следующую стадию (например, исполнение обязанности налогового агента или банка). Только такое исполнение налоговой обязанности, которое приводит к поступлению денежных средств в бюджетную систему или прекращению встречных требований возвратить денежные средства из бюджета, приводит к исполнению налогового обязательства в целом. Особенность данного способа исполнения налогового обязательства заключается в том, что налоговое обязательство в этом случае исполняется не только путем активных действий налогоплательщика, но и налогового органа. 11 Не следует путать момент исполнения обязанности по уплате налога с моментом исполнения налогового обязательства. Исполнение обязанности по уплате налога налогоплательщиком приводит не к исполнению налогового обя- 143 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147 Правоприменение 2021. Т. 5, № 4. С. 135–147 ISSN 2542-1514 (Print) оборота. В налоговом же обязательстве в большинстве случаев имеется только законодательный критерий (п. 3 ст. 45 НК РФ) и только в отношении редких форм договорных налоговых обязательств (например, инвестиционный налоговый кредит) применяются условия соответствующего договора. При этом законодательное регулирование налогового обязательства сосредоточено на обязанностях налогоплательщика и полномочиях налогового органа, не уделяя должного внимания правам и другим формам дозволенного поведения налогового должника, что нередко порождает ситуации правовой неопределенности в процессе исполнения обязательства. Уплата дополнительной суммы налога даже без пеней и штрафа может быть для налогоплательщика обременительной обязанностью, зная о которой заранее он вполне мог бы и не вступать в соответствующие отношения. И финансовый орган, и фискальный орган в налоговом обязательстве, по сути, являются уполномоченными представителями налогового кредитора, противоречивое поведение которых при исполнении обязательства недопустимо. Определенной гарантией от такого поведения для налогоплательщиков может быть налоговый мониторинг, который расширяет возможности налогоплательщика по взаимодействию с налоговым органом в целях устранения неопределенности налогово-правового регулирования. Однако данный механизм является весьма обременительным для налогоплательщика в силу чего, а также из-за законодательных ограничений, не доступен для большинства из них. Неопределенность границ между надлежащим и ненадлежащим исполнением налогового обязательства объективно предопределяет потребность в иных механизмах, позволяющих добросовестному налогоплательщику получить гарантии того, чтобы его действия не будут в дальнейшем квалифицированы налоговой администрацией как злоупотребление правом. В западных государствах для этих целей используется процедура фискального рескрипта или private ruling в США [35, с. 62]. В Германии фискальный рескрипт существует в виде консультаций по отдельным налогам с органом фискального контроля. В Швеции он имеет форму предварительного заключения о налоговых последствиях сделки, которое дается специальным органом – Комиссией по налоговому праву. Предварительное заключение может быть запрошено как налогоплательщиком, так и налоговой администрацией [36, s. 38–44]. В принципе данные механизмы представляют собой трансформированный механизм эстопель – запрет на противоречивое поведение, давно известный гражданскому праву. 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Воронеж, Университетская пл., 1 E-mail: kav@law.vsu.ru SPIN-код РИНЦ: 2065-6411; AuthorID: 356148 Krasyukov Andrey Vladimirovich – PhD in Law, Associate Professor, Department of Financial Law Voronezh State University 1, Universitetskaya pl., Voronezh, 394006, Russia E-mail: kav@law.vsu.ru RSCI SPIN-код: 2065-6411; AuthorID: 356148 БИБЛИОГРАФИЧЕСКОЕ ОПИСАНИЕ СТАТЬИ Красюков А.В. Исполнение налогового обязательства / А.В. Красюков // Правоприменение. – 2021. – Т. 5, № 4. – С. 135–147. – DOI: 10.52468/ 2542-1514.2021.5(4).135-147. BIBLIOGRAPHIC DESCRIPTION Krasyukov A.V. Tax obligation fulfillment. Pravoprimenenie = Law Enforcement Review, 2021, vol. 5, no. 4, pp. 135–147. DOI: 10.52468/25421514.2021.5(4).135-147. (In Russ.). 147 Law Enforcement Review 2021, vol. 5, no. 4, pp. 135–147
https://openalex.org/W2075027215	https://europepmc.org/articles/pmc3223165?pdf=render	English	null	Firm-Level Perspectives on Public Sector Engagement with Private Healthcare Providers: Survey Evidence from Ghana and Kenya	PloS one	2,011	cc-by	9,220	Abstract This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by a grant from the World Bank Group. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. All views expressed are those of the authors. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nsood@sppd.usc.edu organizations and donors have begun to work more with the private health sector since the 1980s [7]. For developing country policymakers, given the size and contributions of the private health sector, a policy of engagement might no longer be just an option but a necessary step towards achieving large-scale improvements in public health. The central question is therefore not whether to interact with the private health sector, but how to best do so in a manner that produces desirable outcomes. Lagomarsino et al. (2009) [13] argue that it is important for policymakers to remain focused on effective public stewardship—i.e. setting and enforcing the rules and incentives that define the environment and guide the behaviors of health-system players, an issue on which there is relatively strong consensus and hence opportunity for progress. However, rigorous evaluations of different approaches to engage with the private sector remain sparse, and are confined largely to case studies [7,14] [15]. Firm-Level Perspectives on Public Sector Engagement with Private Healthcare Providers: Survey Evidence from Ghana and Kenya Neeraj Sood1, Nicholas Burger2, Joanne Yoong2, Dan Kopf2, Connor Spreng3 1 University of Southern California, Los Angeles, California, United States of America, 2 RAND Corporation, Arlington, Virginia, United States of America, 3 World Bank, Washington, D.C., United States of America Neeraj Sood1, Nicholas Burger2, Joanne Yoong2, Dan Kopf2, Connor Spreng3 1 University of Southern California, Los Angeles, California, United States of America, 2 RAND Corporation, Arlington, Virginia, United States of America, 3 World Bank, Washington, D.C., United States of America PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 Abstract Background: Health systems in Sub-Saharan Africa (SSA) are in urgent need of improvement. The private health sector is a major provider of care in the region and it will remain a significant actor in the future. Any efforts by SSA governments to improve health systems performance therefore has to account for the private health sector. Regional and international actors increasingly recognize importance of effectively engaging with the private health sector, and initiatives to improve engagement are underway in several countries. However, there is little systematic analysis of private health providers’ view and experience with engagement. Methodology/Principal Findings: In this study we surveyed private health facilities in Kenya and Ghana to understand the extent to which and how governments interact and engage with these facilities. The results suggest that government engagement with private health facilities is quite limited. The primary focus of this engagement is ‘‘command-and-control’’ type regulations to improve the quality of care. There is little attention paid to building the capacity of health care businesses through either technical or financial assistance. The vast majority of these facilities also receive no government assistance in meeting public health and social goals. Finally, government engagement with private pharmacies is often neglected and clinics receive a disproportionate share of government assistance. Conclusions/Significance: Overall, our findings suggest that there may be considerable untapped potential for greater engagement with private health facilities—particularly pharmacies. Improving engagement will likely help governments with limited resources to better take advantage of the private sector capacity to meet access and equity objectives and to accelerate the achievement of the Millennium Development Goals. Citation: Sood N, Burger N, Yoong J, Kopf D, Spreng C (2011) Firm-Level Perspectives on Public Sector Engagement with Private Healthcare Providers: Survey Evidence from Ghana and Kenya. PLoS ONE 6(11): e27194. doi:10.1371/journal.pone.0027194 itor: Abdisalan Mohamed Noor, Kenya Medical Research Institute - Wellcome Trust Research Programme, Kenya Received May 31, 2011; Accepted October 12, 2011; Published November 23, 2011 Received May 31, 2011; Accepted October 12, 2011; Published November 23, 2011 Copyright: 2011 Sood 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: 2011 Sood et al. Citation: Sood N, Burger N, Yoong J, Kopf D, Spreng C (2011) Firm-Level Perspectives on Public Sector Engagement with Private Healthcare Providers: Survey Evidence from Ghana and Kenya. PLoS ONE 6(11): e27194. doi:10.1371/journal.pone.0027194 Government Engagement and Private Health Providers While a majority of these experiences involve NGOs, a growing number also involve for- profit contractors. Sometimes financial assistance can be ‘‘performance based’’ in that it is tied to achievement of specific outcomes or provision of specific services. For example, private providers who report providing care to a target number of rural patients may receive a supplementary allowance or priority in receiving loans. Governments may also provide incentives to private facilities for the provision of public health goods such as health education, vaccination and other interventions to prevent infectious diseases. Financial Assistance: Subsidies, contracting and direct purchase. A more interventionist approach is providing direct financial assistance to the private health sector in the form of grants, subsidies or government contracts to purchase health care services. Examples of financial assistance include land grants for improving access or subsidies for purchasing or upgrading medical equipment to improve quality. Waters et al. (2003) [17] and Loevinsohn and Harding (2003) [19] note a number of examples of contracting-out for primary care and other child health services, from Senegal, Madagascar, El Salvador, Cambodia, Guatemala, South Africa and Zimbabwe. While a majority of these experiences involve NGOs, a growing number also involve for- profit contractors. Sometimes financial assistance can be ‘‘performance based’’ in that it is tied to achievement of specific outcomes or provision of specific services. For example, private providers who report providing care to a target number of rural patients may receive a supplementary allowance or priority in receiving loans. Governments may also provide incentives to private facilities for the provision of public health goods such as health education, vaccination and other interventions to prevent infectious diseases. Public-Private Engagement: Influencing Firms Prior studies of price controls in China document numerous other unintended consequences including the emergence of a black market, overprovision of profitable high-tech services, and overuse of prescription drugs [20]. Similarly, subsidies and financial assistance to providers might promote inefficiency by using public funds to bolster inefficient providers. As with most public programs, such Regulation. Government can choose to implement ‘‘command-and-control’’ style rules and penalties and thereby exercise its authority to require and enforce what it considers to be desirable practice. Regulation can apply to measures to protect the quality of care, for instance by establishing minimum cleanliness standards and penalties for violations. It can also be used to expand access and increase equity, for example by imposing price controls or requirements that private providers exempt certain types of patients from fees. Regulatory approaches are limited, however, in that they often treat all regulated entities the same and may not be economically efficient. Monitoring: Gathering information on the activities of the private sector. At the most fundamental level, governments seeking to engage with the private sector need to know the size and composition of the private health sector. They also need to have the ability to monitor its activity on an ongoing basis. Examples of this include administrative reporting on the number of providers and types/volume of patients they treat and routinely collecting and assessing information on adverse events or patient complaints. This is a crucial aspect of engagement for three reasons. First, such information is crucial for planning and implementing public health programs, especially in countries where private providers are an important access point for health care delivery. For example, it is difficult to establish effective disease surveillance programs without knowing the disease burden for patients being treated in private health facilities. Second, information on private sector size and composition is key for understanding how public policy and other macroeconomic factors are shaping the behavior of the private sector. Finally, without such information it is nearly impossible to evaluate the intended and unintended consequences of other forms of engagement with the private health sector. Monitoring: Gathering information on the activities of the private sector. At the most fundamental level, governments seeking to engage with the private sector need to know the size and composition of the private health sector. They also need to have the ability to monitor its activity on an ongoing basis. Government Engagement and Private Health Providers Simply providing information to the private sector can be a straightforward but important aspect of capacity building and can improve both access to care and quality of care. For example, lack of knowledge about formal credit markets and financial management can be a significant barrier to operating or expanding a health care facility. Government technical assistance programs can reduce such barriers by providing the necessary skills. Ultimately, improved facility performance and growth—induced by government technical assistance—will increase access to care. Similarly, governments can provide technical assistance to promote quality of care by disseminating information on standards of care or best practice guidelines and by providing continuing education workshops or classes that are open to both the public and private sector. istrative, and information constraints, systematic evidence on the many facets of government-provider interaction remains limited [13]. Much of the literature focuses primarily on the experience of government agencies and consumers, and none has clearly documented government engagement from the perspective of providers. In this paper, we provide new evidence about government engagement with private sector clinics and pharmacies in two countries, Ghana and Kenya, through the lens of survey data collected from these providers. Our sample of pharmacies and clinics captures the most prevalent providers of health services, though not the full variety of providers. For example, across the region at least one third of health services are provided by informal providers [13], which are largely absent in our sample. To structure the discussion, we first provide a general framework for conceptualizing government engagement. Next, we describe the context for our study and the providers included in the study. We then present the main findings of the paper and draw a number of conclusions about the specific setting of our study as well as more general implications and recommendations for research going forward. Financial Assistance: Subsidies, contracting and direct purchase. A more interventionist approach is providing direct financial assistance to the private health sector in the form of grants, subsidies or government contracts to purchase health care services. Examples of financial assistance include land grants for improving access or subsidies for purchasing or upgrading medical equipment to improve quality. Waters et al. (2003) [17] and Loevinsohn and Harding (2003) [19] note a number of examples of contracting-out for primary care and other child health services, from Senegal, Madagascar, El Salvador, Cambodia, Guatemala, South Africa and Zimbabwe. Introduction Across the developing world, the private sector has a well- documented presence in healthcare. In sub-Saharan Africa private providers account for as much as 50 percent of health care provision [1]. Yet the appropriate role for the private health sector remains a controversial issue. While users may prefer the private sector due to perceived quality, easier access and greater responsiveness, in some cases technical standards of care might be poor [2–6] and private sector provision may limit access for the poor [7], [8]. Others argue that budget-constrained national health systems may be best served by making the most of the private health sector [9,10] [11]. Recent evidence from 34 sub Saharan countries also suggests that increased private sector participation may be associated with greater access to services as well as greater equity [12]. Although the debate about the role of the private sector in health care is far from resolved, governments, international In spite of the perception that many governments fail to constructively engage the private sector due to political, admin- PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 1 Public-Private Engagement: Influencing Firms Prior studies have identified a number of key strategic tools that governments frequently employ under the scope of public-private engagement [16,17]. These can be thought of as falling into three categories: demand-side interactions that aim to influence consumer behavior (such as social marketing), supply-side interactions influencing firm behavior (such as direct purchase or training), and restructuring or reorganizing market interactions (such as the creating new public-private entities). This paper characterizes supply-side interactions that are intended to influence firm behavior in two primary domains: (1) increasing overall access and equity in access to health care, and (2) increasing the quality of health care services. We distinguish between the following approaches, which in practice may vary across domains as well as provider types. As an aside, We acknowledge that interventions and supply-side interactions in particular can be categorized differently. The breakdown offered here provides a chance to highlight the aspects our research focused on, while being largely compatible with other standard frameworks [18]. This paper characterizes supply-side interactions that are intended to influence firm behavior in two primary domains: (1) increasing overall access and equity in access to health care, and (2) increasing the quality of health care services. We distinguish between the following approaches, which in practice may vary across domains as well as provider types. As an aside, We acknowledge that interventions and supply-side interactions in particular can be categorized differently. The breakdown offered here provides a chance to highlight the aspects our research focused on, while being largely compatible with other standard frameworks [18]. Regulation. Government can choose to implement ‘‘command-and-control’’ style rules and penalties and thereby exercise its authority to require and enforce what it considers to be desirable practice. Regulation can apply to measures to protect the quality of care, for instance by establishing minimum cleanliness standards and penalties for violations. It can also be used to expand access and increase equity, for example by imposing price controls or requirements that private providers exempt certain types of patients from fees. Regulatory approaches are limited, however, in that they often treat all regulated entities the same and may not be economically efficient. While potentially more powerful than monitoring and informa- tion provision, financial assistance and regulation can have unintended consequences. For example, price controls to improve access might deter provider entry into health care markets and thus reduce access in the long run. Government Engagement and Private Health Providers sector. Firms may fail to maximize their potential scope of business or to comply with appropriate quality standards if they lack the requisite information or training. Simply providing information to the private sector can be a straightforward but important aspect of capacity building and can improve both access to care and quality of care. For example, lack of knowledge about formal credit markets and financial management can be a significant barrier to operating or expanding a health care facility. Government technical assistance programs can reduce such barriers by providing the necessary skills. Ultimately, improved facility performance and growth—induced by government technical assistance—will increase access to care. Similarly, governments can provide technical assistance to promote quality of care by disseminating information on standards of care or best practice guidelines and by providing continuing education workshops or classes that are open to both the public and private sector. These investments in improving the skills of private health providers are relatively uncontroversial in that the role of the government may be interpreted as simply increasing the flow or supply of information. sector. Firms may fail to maximize their potential scope of business or to comply with appropriate quality standards if they lack the requisite information or training. Simply providing information to the private sector can be a straightforward but important aspect of capacity building and can improve both access to care and quality of care. For example, lack of knowledge about formal credit markets and financial management can be a significant barrier to operating or expanding a health care facility. Government technical assistance programs can reduce such barriers by providing the necessary skills. Ultimately, improved facility performance and growth—induced by government technical assistance—will increase access to care. Similarly, governments can provide technical assistance to promote quality of care by disseminating information on standards of care or best practice guidelines and by providing continuing education workshops or classes that are open to both the public and private sector. These investments in improving the skills of private health providers are relatively uncontroversial in that the role of the government may be interpreted as simply increasing the flow or supply of information. sector. Firms may fail to maximize their potential scope of business or to comply with appropriate quality standards if they lack the requisite information or training. Sample description The data used for the study come from the Health Provider Assessment Survey, which was administered in Ghana and Kenya during 2010 by the study team. HPAS samples for each country were designed to capture a broad range of health facility types, focusing primarily on smaller, private sector firms. In Ghana, the sampling frame was based on a 2010 census of health facilities in seven districts purposively chosen to be geographically and economically diverse, carried out by the Results for Development Institute. We excluded laboratories and medical device manufac- turers and out of the remaining 647 facilities, we interviewed a random sample of 300 hospitals, clinics, nursing homes and pharmacies. Private hospitals and clinics were oversampled. In Kenya, we constructed a census of health facilities in five districts also reflective of geographic and economic diversity, by combining a list of 1920 hospitals, clinics, and nursing homes compiled by the Ministry of Health and KEMRI-Wellcome Trust with a list of 1948 pharmacies from a retail census collected by TNS Opinion. Government Engagement and Private Health Providers Table 1. HPAS sample composition by country. Kenya Ghana Public Private Public Private Hospital 1 10 8 21 Clinic 11 112 31 68 Pharmacy 1 145 0 92 Chemical Sellers 0 6 0 80 Nursing/maternity homes 0 7 0 0 Other 5 2 0 0 18 282 39 261 doi:10.1371/journal.pone.0027194.t001 Table 1. HPAS sample composition by country. Kenya Ghana Public Private Public Private Hospital 1 10 8 21 Clinic 11 112 31 68 Pharmacy 1 145 0 92 Chemical Sellers 0 6 0 80 Nursing/maternity homes 0 7 0 0 Other 5 2 0 0 18 282 39 261 doi:10.1371/journal.pone.0027194.t001 Table 1. HPAS sample composition by country. interventions may be prone to error, fraud and corruption, as well as susceptible to capture by special interest groups. Using performance based incentives can mitigate some of these concerns but may not offset others: for instance, targets may be inappropriately set, or measurement and compliance may be manipulated for gain. Given the tradeoffs across different types of engagement policies, the debate over the extent and nature of government intervention is domain and context specific. For instance, sensitivity to the issues above: may be greater when policies are aimed at providing financial incentives to private facilities to promote access rather than policies aimed at upholding quality standards; may depend on the type of provider; or may be linked to overall governance in the country. It is important to note that the approaches described above may be initiated or directed by entities that are not part of the government. Private health sector associations and third parties (e.g., accreditation agencies, insurance agencies) may engage in some or all of the approaches. In this paper, however, we leave the role of associations aside. The goal of this paper is to characterize the approaches taken by the governments of Ghana and Kenya, as reportedly experienced by private sector providers. doi:10.1371/journal.pone.0027194.t001 Public-Private Engagement: Influencing Firms Examples of this include administrative reporting on the number of providers and types/volume of patients they treat and routinely collecting and assessing information on adverse events or patient complaints. This is a crucial aspect of engagement for three reasons. First, such information is crucial for planning and implementing public health programs, especially in countries where private providers are an important access point for health care delivery. For example, it is difficult to establish effective disease surveillance programs without knowing the disease burden for patients being treated in private health facilities. Second, information on private sector size and composition is key for understanding how public policy and other macroeconomic factors are shaping the behavior of the private sector. Finally, without such information it is nearly impossible to evaluate the intended and unintended consequences of other forms of engagement with the private health sector. Information Provision/Technical Assistance: Supplying information to support the activities of the private PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 November 2011 \| Volume 6 \| Issue 11 \| e27194 2 Government Engagement and Private Health Providers HPAS Survey Questions HPAS survey questions are grouped into five core sections: basic facility characteristics, barriers and obstacles to operating a business, the policy environment, financial information, and business process management. In Ghana we included a supple- mental section regarding the national health insurance scheme, and in Kenya the survey contained a supplemental section specific to pharmacies. A final section asks enumerators to provide a basic assessment of the facility, including information on amenities and cleanliness. Regulation. In the case of regulation and its enforcement, the HPAS asks questions that cover two areas: access/equity and quality. Access/equity. Providers are asked if their facility is subject to laws and regulations that mandate (1) the need to provide free or subsidized care to poor patients, (2) inability to deny treatment based on cost/ability to pay, and (3) price caps or maximum price regulations that mandate that prices cannot exceed a certain threshold. With respect to interaction with the government, the HPAS instrument asks questions to providers about various aspects of regulation and about their experience with government assistance to build their human and financial capital. The survey also asks providers whether they have been exposed to financial incentive schemes and about the nature of partnerships with the government on certain public health activities. Quality. Providers are asked about whether they have had an inspection from the government in the past two years for monitoring the safety and quality of their services. With respect to enforcement, since facilities may be reluctant and unlikely to answer truthfully questions about failing inspections, they are also asked an indirect question about whether they know of any examples of facilities like theirs that have been penalized by the government for failing to meet quality standards (with examples given such as selling counterfeit drugs or causing adverse health events). Monitoring. The HPAS asks providers whether they report information about their activities to the government across a variety of domains related to health management systems reporting, epidemiological surveillance-oriented reporting, and business operations. In particular, the survey asks providers whether they send periodic reports to the government on each of the following: (1) service and drug utilization statistics on the number or types of patients seen and the number or types of drugs sold, (2) adverse events such as maternal or child deaths, (3) quality reporting on compliance with standards, and (4) financial reporting on operating information such as prices and revenues. Study sample and location The nature of health systems and the environment in which patients seek care and firms do business varies tremendously from country to country, across the developing world. Within any given country the private health sector encompasses a diverse set of providers, divided by many distinctions, including position on the supply-chain, for-profit status, religious/secular affiliation, degree of formality, and participation in allopathic or traditional medical practice [17,21]. Any discussion of engagement can be specific to a particular subset of this group. Figure 1. Periodic Reporting Type. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g001 Figure 1. Periodic Reporting Type. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g001 Figure 1. Periodic Reporting Type. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g001 November 2011 \| Volume 6 \| Issue 11 \| e27194 PLoS ONE \| www.plosone.org 3 Government Engagement and Private Health Providers Table 2. Facilities providing periodic reports to the government, by type. Kenya Ghana Clinics Pharmacies Clinics Pharmacies N % N % N % N % Service utilization (number and type of patients) 112 35.7% 151 8.6% 67 40.3% 90 3.3% Epidemiological reporting: adverse events 112 59.8% 151 39.1% 66 24.2% 89 10.1% Quality standards reporting 112 54.5% 151 41.7% 67 43.3% 89 31.5% Financial reporting 112 13.4% 150 13.3% 66 10.6% 86 12.8% Drug utilization (number and type of drugs) 112 19.6% 151 29.8% 66 7.6% 87 8.0% Any reporting 112 75.9% 151 70.2% 67 59.7% 91 42.9% Note: ‘‘Don’t know’’ and refusals are both treated as missing. doi:10.1371/journal.pone.0027194.t002 Note: ‘‘Don’t know’’ and refusals are both treated as missing. doi:10.1371/journal.pone.0027194.t002 questions about supportive resources received from the government, whether in the form of information, technical assistance or financial assistance. In particular, the survey asks providers about assistance (technical or financial) to support them in two types of activities, during the past three years: Similarly, we interviewed a random sample of 300 hospitals, clinics, nursing homes and pharmacies drawn from this census, oversampling private hospitals and clinics. Table 1 shows the final HPAS survey composition by provider type in each country. We note that response rates for the survey differed across countries - 90 percent in Ghana and 69 percent in Kenya - but we do not have any evidence of differential self- selection affecting the final sample composition. Skill and Technology Upgrades. Study sample and location Providers are asked about offers of assistance (technical or financial) to support them in five domains: (1) continuing education for existing providers, (2) training for future health providers, (3) information about clinical practice guidelines, (4) quality assurance practices, and (5) technology upgrades. A follow-up question for those offered assistance verified whether or not such assistance was actually received. Skill and Technology Upgrades. Providers are asked about offers of assistance (technical or financial) to support them in five domains: (1) continuing education for existing providers, (2) training for future health providers, (3) information about clinical practice guidelines, (4) quality assurance practices, and (5) technology upgrades. A follow-up question for those offered assistance verified whether or not such assistance was actually received. In this paper, we focus on private clinics and pharmacies. We note that because chemical sellers are not a separate class of providers for regulatory purposes in Kenya (but not in Ghana), we include the small number of Kenyan providers that identify as such under the category of pharmacies. In Ghana, the analytical sample therefore consisted of 68 clinics and 92 pharmacies. In Kenya, the sample consisted of 112 clinics and 151 pharmacies. Raising Capital. Providers are asked about whether they receive any technical assistance from the government to support them in improving their ability to apply for a bank loan, or whether they received direct financial assistance such as a loan, grant, subsidized interest, or bank guarantee. PLoS ONE \| www.plosone.org HPAS Survey Questions Technical and Financial Assistance for Improving Public Health and Reducing Disparities. As an alternative to mandates providers are also asked about government-provided incentives to achieve public health and broader social objectives. Access to Public Health Services. Providers are asked about whether in the past 3 years they have received any technical or financial assistance from the government to provide services related to public health, specifically: (1) childhood vaccinations, (2) HIV/AIDS control measures, (3) malaria or tuberculosis (TB) control measures, and (4) health education for consumers/patients. Technical and Financial Assistance for Capacity Building. Providers in both countries are also asked a series of PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 4 Government Engagement and Private Health Providers Figure 2. (top): Facilities Offered Technical Assistance. (bottom): Facilities Taking Up Technical Assist Grey: Kenya N Light Grey: Ghana. doi:10 1371/journal pone 0027194 g002 Figure 2. (top): Facilities Offered Technical Assistance. (bottom): Facilities Taking Up Technical Assistance if Offered. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g002 PLoS ONE \| www.plosone.org 5 November 2011 \| Volume 6 \| Issue 11 \| e27194 Figure 2. (top): Facilities Offered Technical Assistance. (bottom): Facilities Taking Up Technical Assistance if Offered. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g002 Figure 2. (top): Facilities Offered Technical Assistance. (bottom): Facilities Taking Up Technical Assistance if Offered. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g002 November 2011 \| Volume 6 \| Issue 11 \| e27194 November 2011 \| Volume 6 \| Issue 11 \| e27194 PLoS ONE \| www.plosone.org 5 Government Engagement and Private Health Providers Table 3. Facilities offered/receiving technical assistance for skill and technology upgrades from the government in last 3 years, by type. HPAS Survey Questions Kenya Ghana Clinics Pharmacies Clinics Pharmacies N % N % N % N % Facilities being offered technical support for Continuing Education 112 25.0% 151 7.3% 68 13.2% 90 6.7% Information on practice guidelines 112 40.2% 151 8.6% 68 20.6% 90 7.8% Quality assurance 112 32.1% 151 10.6% 68 22.1% 90 8.9% Technology upgrades 112 23.2% 151 4.6% 68 8.8% 90 1.1% Training for future health providers 112 16.1% 151 6.6% 68 8.8% 90 5.6% Facilities receiving technical support (as % offered) Continuing Education 28 85.7% 11 90.9% 9 77.8% 6 83.3% Information on practice guidelines 45 91.1% 13 84.6% 14 85.7% 7 100.0% Quality assurance 36 83.3% 16 87.5% 15 80.0% 8 100.0% Technology upgrades 26 88.5% 7 85.7% 6 50.0% 1 100.0% Training for future health providers 18 94.4% 10 90.0% 6 66.7% 5 100.0% Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t003 Table 3. Facilities offered/receiving technical assistance for skill and technology upgrades from the government in last 3 years, by type. Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t003 Ghana half of the facilities in the sample stated that they participated in none of the reporting activities described in the previous section—although results are not consistent across all categories. Interestingly, in both countries, facilities are most likely to report information to the government about quality standards and somewhat less likely to report on utilization, both in terms of patient and drug turnover. Finally, an even smaller number of facilities report any financial information to the government 213% in Kenya and 12% in Ghana. These findings suggest that the limited monitoring that does exist focuses on the medical aspects of these facilities but largely neglects their operation as businesses. Equity. Providers are asked about whether they know of any facilities that receive help from the government for (1) opening a facility in a rural area, (2) opening a facility in a poor urban area, and (3) providing treatment to poor patients. Monitoring Figure 1 below shows responses to the questions about monitoring in both countries, with the Kenyan sample showing slightly higher rates of overall contact. In Kenya, almost 30% and in Figure 3. Facilities Receiving Financial Assistance. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g003 Figure 3. Facilities Receiving Financial Assistance. Countries: N Dark Grey: Kenya N Light Grey: Ghana. Figure 3. Facilities Receiving Financial Assistance. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g003 Figure 3. Facilities Receiving Financial Assistance. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g003 PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 PLoS ONE \| www.plosone.org 6 Government Engagement and Private Health Providers eceiving financial assistance for operating activities from the government in last 3 years, by type. Table 4. Facilities receiving financial assistance for operating activities from the government in last 3 years, by type. Table 4. Facilities receiving financial assistance for operating activities from the government in last 3 years, by type. Kenya Ghana Clinics Pharmacies Clinics Pharmacies N % N % N % N % Facilities receiving financial assistance for Continuing Education 111 6.3% 151 0.7% 67 1.5% 92 0.0% Information on practice guidelines 111 6.3% 151 2.0% 67 3.0% 92 0.0% Quality assurance 111 6.3% 151 2.0% 67 3.0% 92 0.0% Technology upgrades 111 1.8% 151 0.7% 67 1.5% 92 0.0% Training for future health providers 111 3.6% 151 1.3% 67 1.5% 92 0.0% Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t004 From the top panel of Figure 2, we can see that surprisingly few facilities receive offers of support related to upgrading human capital whether in the form of continuing education or training of future providers (between 7–15% in either case for both countries). Similarly, less than 15% of facilities in either country report receiving offers of technology upgrades. Relatively more facilities are offered assistance related to maintaining quality and practice standards, although in each case this still reflects less than a quarter of all facilities. In Table 2, these figures are broken down by facility type in each country. In both countries clinics are more likely than pharmacies to report their activities to the government. One exception is reports related to drug inventory activity in Kenya, where pharmacies report higher levels of reporting. Monitoring In Ghana, pharmacies are only marginally more likely to report drug inventory relative to clinics. Few facilities—of either type and in either country—report financial information to the government. In summary, the level of government monitoring of private health facilities across a variety of domains is poor. Clinics are more likely to be monitored than pharmacies. Domains related to quality of care are more likely to be monitored than others, but even for these domains the majority of facilities are not monitored. The bottom panel of Figure 2 shows that the majority of facilities participate and take advantage of these services if offered. For facilities not taking up these services, it is not possible from the data to understand whether it is the result of refusing offered assistance, or whether it is due to the government’s failure to follow up. Technical Assistance for Capacity Building Table 3 shows an important divergence in these results by facility type. In both Ghana and Kenya, the offers of technical support across all dimensions are significantly skewed towards clinics; yet the take up of technical assistance among clinics and pharmacies is more comparable. Skill and Technology Upgrades. We first examine government interaction with private facilities regarding technical assistance related to improving provider skills and technology. The results can be found in Figure 2 below. Figure 4. Facilities Reporting Regulations. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g004 Figure 4. Facilities Reporting Regulations. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g004 PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 November 2011 \| Volume 6 \| Issue 11 \| e27194 7 7 Government Engagement and Private Health Providers Table 5. Facilities reporting being subject to access/equity-related regulation, by type. Kenya Ghana Clinics Pharmacies Clinics Pharmacies N % N % N % N % Facilities reportedly subject to regulation mandating Free/subsidized care for poor patients 112 24.1% 151 24.5% 67 11.9% 92 1.1% No denial of treatment due to cost/inability to pay 112 28.6% 151 22.5% 67 10.4% 91 2.2% Price ceilings 112 31.3% 151 37.7% 67 16.4% 90 18.9% Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t005 Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t005 Regulation Raising Capital. Virtually no facilities report receiving technical assistance to improve their ability to apply for bank loans. Specifically, 3.8% of facilities in Kenya and no facilities in Ghana report receiving such technical assistance (not reported in tables). Access/Equity. In both Ghana and Kenya, regulation with respect to equity-enhancing mandates is reported only by a minority of facilities (Figure 4). The results clearly point to the existence of an overall regulatory structure. However, in Kenya only a quarter to a third of all facilities report being subject to such mandates. In Ghana the percentages are even lower. Overall, though the levels differ, price controls are most commonly reported, while mandates related to the provision of care based on poverty and the inability to refuse care are much less frequently reported in both countries (Figure 4). Table 5 shows that in Kenya, a comparable if not equal amount of regulation appears to apply to clinics and pharmacies. This is in sharp contrast to Ghana, where 19% of pharmacies report being to be subject to price controls but otherwise the sector seems to be relatively untouched by regulations mandating free or subsidized care. In summary, the provision of technical assistance by the government to private health facilities is fairly uncommon with more than three quarters of these facilities reporting not receiving technical assistance. The provision of technical assistance for improving access to private credit markets is virtually non-existent. Clinics are more likely to receive technical assistance compared to pharmacies. Technical and Financial Assistance for Improving Public Health and Reducing Disparities The lack of monitoring of private health sector activity also suggests that the governments have an incomplete and dated view of the role the private sector is playing in these economies. Overall, it appears that there is a large fraction of the private sector that could potentially be further incentivized to help achieve public health goals; this is particularly salient given that three of the four measures above contribute directly to the achievement of the Millennium Development Goals. Equity. Figure 7 shows the percentage of facilities reporting that they have heard of another facility receiving financial support to promote service provision activities that address equity issues. Rates are considerably higher in Kenya rather than Ghana, particularly for promoting service in rural areas. In Kenya, nearly 40% of facilities reported knowing a facility that has received assistance to serve a rural area. About 30% reported knowing a facility that has received assistance to serve a poor urban area and a slightly higher number reported knowing a facility that has received assistance for treating the poor. In Ghana, these numbers are significantly lower, at 6–7% for all three categories. The nature of the interactions most commonly-reported in the survey suggests that, from the firm’s perspective, most govern- ment intervention in these countries still takes a cautious approach, viewing the private sector as a liability rather than an opportunity. Policies tend to focus more strongly on controlling the quality rather than fostering the private sector to meet access goals. Across the board, direct technical assistance is more prevalent across all types of capacity building than financial sponsorship of such activities, possibly due to logistical feasibility or ease of control. Many firms report receiving incentives to promote quality or being subject to regulation and inspection to ensure quality, but fewer report being involved with interventions to support expansion, even if this promotes access and equity outcomes. Table 8 shows that the difference between the two counties manifests not only as higher average response rates in Kenya overall, but also in a markedly different environment in the pharmacy sector. In Ghana, consistent with the previous results, pharmacies appear to be much less engaged than clinics. Technical and Financial Assistance for Improving Public Health and Reducing Disparities Technical and Financial Assistance for Improving Public Health and Reducing Disparities In this study we surveyed private health facilities in Kenya and Ghana to understand the extent to which and how governments interact and engage with these facilities. The main conclusion from this research is that government engagement with the private health facilities is quite limited. The primary focus of this engagement is command-and-control type regulations to improve the quality of care. There is little attention paid to capacity building for health care businesses through either technical or financial assistance. The vast majority of facilities also receive no government assistance in meeting public health and social goals. Finally, government engagement with private pharmacies is often neglected and clinics receive a disproportionate share of government assistance. For the private health sector to contribute more effectively to national health goals, increasing engagement with private facilities as both businesses and medical care providers will be critical. Supporting Access to Public Health Services. A visible but small minority of private sector providers are incentivized by the government in both countries to support them in providing health services that are public goods (Figure 6); the percentage of facilities involved however is no more than 10–20% for any type of service covered by the survey in either country. Comparing the top and bottom panel of Figure 6 shows that in terms of incentivizing service delivery, technical assistance is more widespread than financial assistance (similar to the case of capacity building). Overall, in both countries, the smallest number of facilities report being assisted in the provision of general health education, but interestingly, this fraction is not much smaller than the other categories. (We note, however, that our measure does not capture the magnitude of funding, and we thus miss an important dimension of the intensity with which the private sector is supported). Table 7 shows that clinics are more likely than pharmacies to receive services in this domain as well. A first order problem in both Kenya and Ghana is that the government does little to monitor the activity of the private sector in terms of the medical care provided and the finances of these facilities. Such a widespread lack of information about the private sector implies that governments in these countries have little ability to assess the effectiveness of public policies targeted towards the private health sector. Financial Assistance for Capacity Building Clinics Pharmacies Clinics Pharmacies N % N % N % N % Inspected for quality standards in last 2 years 112 91.1% 151 83.4% 68 79.4% 92 94.6% Know facility penalized for failing to meet quality standards 111 19.8% 151 46.4% 68 5.9% 91 13.2% Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t006 received assistance, relative to 30%, 18% and 21% for clinics respectively. received assistance, relative to 30%, 18% and 21% for clinics respectively. about penalties imposed. However, in Kenya, pharmacies are about 10% less likely to be inspected relative to clinics, but more than twice as likely to have heard of someone being penalized. Financial Assistance for Capacity Building Figure 3 shows that in both countries only a small minority of facilities (5% or fewer) report receiving direct financial support from the government for improving provider skills and technology upgrades. However, across all domains, financial support is more widely reported in Kenya than Ghana. Quality regulation. Almost 90% of firms in both countries report having been inspected in the last 2 years. 35% of Kenyan facilities but only 10% of Ghanaian facilities report having heard of a case in which a facility like theirs has been penalized for not meeting quality standards (Figure 5). The breakdown by facility type (Table 6) shows that in both countries, clinics and pharmacies report inspection rates of almost 80% or more. In Ghana, pharmacies are both more likely to be inspected, and more likely to have heard Quality regulation. Almost 90% of firms in both countries report having been inspected in the last 2 years. 35% of Kenyan facilities but only 10% of Ghanaian facilities report having heard of a case in which a facility like theirs has been penalized for not meeting quality standards (Figure 5). The breakdown by facility type (Table 6) shows that in both countries, clinics and pharmacies report inspection rates of almost 80% or more. In Ghana, pharmacies are both more likely to be inspected, and more likely to have heard Again, in Table 4, we observe that, as with technical assistance, what little financial assistance exists is skewed towards clinics rather than pharmacies. Virtually no pharmacies in Kenya and no pharmacies at all in Ghana report receiving financial assistance for continuing education, training or technology upgrades. Figure 5. Facilities Reporting Enforcement of Regulations. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g005 Figure 5. Facilities Reporting Enforcement of Regulations. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g005 Figure 5. Facilities Reporting Enforcement of Regulations. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g005 November 2011 \| Volume 6 \| Issue 11 \| e27194 PLoS ONE \| www.plosone.org 8 Government Engagement and Private Health Providers Table 6. Facilities reporting enforcement of quality-related regulation, by type. Table 6. Facilities reporting enforcement of quality-related regulation, by type. PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 Technical and Financial Assistance for Improving Public Health and Reducing Disparities However, in Kenya, pharmacies appear to be more frequently incentivized (or have better knowledge of incentives being provided) relative to clinics in the same country, and relative to pharmacies in Ghana, by a wide margin 245% report knowing a facility that has received assistance to serve a rural area, 36% reported knowing a facility that has received assistance to serve a poor urban area and 39% reported knowing a facility that has Finally, when contrasting engagement across provider types, it appears that across all types of interventions, in both countries, pharmacies are relatively excluded. In both Ghana and Kenya, government support for capacity building seems largely confined to clinics. Pharmacies reported virtually no financial support, and significantly less technical assistance, with some notable exceptions such as incentives to expand into underserved areas in Kenya. The PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 November 2011 \| Volume 6 \| Issue 11 \| e27194 9 Government Engagement and Private Health Providers relative lack of interaction with small retail pharmacies points to a need for policy reform in this area, given the large fraction of Some important limitations shou implications for the findings reported Figure 6. (top): Facilities Receiving Technical Assistance. (bottom): Facilities Receiving Financial Suppo N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g006 Figure 6. (top): Facilities Receiving Technical Assistance. (bottom): Facilities Receiving Financial Support. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g006 Figure 6. (top): Facilities Receiving Technical Assistance. (bottom): Facilities Receiving Financial Support. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g006 relative lack of interaction with small retail pharmacies points to a need for policy reform in this area, given the large fraction of consumers, especially the poor, that report receiving health services primarily or even exclusively from pharmacies. Some important limitations should be noted, as well as their implications for the findings reported here and for further research. The firms in our sample were largely formally registered, implying that the channels for communication between public and private PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 November 2011 \| Volume 6 \| Issue 11 \| e27194 PLoS ONE \| www.plosone.org 10 Government Engagement and Private Health Providers Table 7. Facilities receiving technical/financial assistance from the government to provide public health services in last 3 years, by type. Technical and Financial Assistance for Improving Public Health and Reducing Disparities Kenya Ghana Clinics Pharmacies Clinics Pharmacies N % N % N % N % Facilities receiving technical assistance for Providing childhood vaccinations 112 18.8% 151 4.0% 68 11.8% 92 1.1% Control of HIV/AIDS 112 34.8% 151 6.0% 68 14.7% 92 2.2% Control of malaria/TB 112 25.9% 151 4.6% 68 16.2% 92 5.4% Health education 112 17.0% 151 4.0% 68 10.3% 91 2.2% Facilities receiving financial support for Providing childhood vaccinations 111 8.1% 151 0.0% 68 1.5% 92 0.0% Control of HIV/AIDS 111 13.5% 151 1.3% 68 4.4% 92 0.0% Control of malaria/TB 111 11.7% 151 0.7% 68 4.4% 92 3.3% Health education 111 3.6% 151 0.0% 68 0.0% 92 0.0% Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t007 Table 7. Facilities receiving technical/financial assistance from the government to provide public health services in last 3 years, by type. Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t007 At the same time we acknowledge that lack of engagement could be a function of limited resources, either financial or in terms of technical capacity. Just as many governments struggle to provide public health care services, they may also struggle to act as effective monitors and engaged partners with the private sector. The results presented here raise difficult questions about how to allocate scarce resources: should resources be allocated to additional public provision or to better engagement with the private sector? Should additional monitoring resources be focused on small, ubiquitous facilities (i.e., pharmacies) or larger facilities providing more substantial care (i.e., clinics)? These challenges should be addressed by future research. sectors are at least in theory open and available. The survey did not investigate engagement with many informal providers. Insofar as levels of government interaction with informal providers are likely to be lower, our results may be interpreted as an upper-bound on engagement activity with the private sector as a whole. This study is descriptive, but further research with this dataset will allow us to understand variation in engagement within provider types—e.g., by experience, size—and the degree to which engagement is associated with outcomes for providers. Overall, our findings suggest that there may be considerable untapped potential for greater engagement with private health facilities, particularly pharmacies. Technical and Financial Assistance for Improving Public Health and Reducing Disparities Improving this engagement will likely help governments with limited resources to better take advantage of the capacity of the private sector to meet access and equity objectives, and to accelerate the achievement of the Millennium Development Goals. Author Contributions Author Contributions provided verbal informed consent. Verbal consent was approved by the RAND HSPC and required that (1) the interviewer follow an oral script approved by the RAND HSPC when administering the consent process, and (2) the interviewer provide an information letter to survey participants that explains the purpose of the study, the risks and benefits of the study, and provides contact information for the principal investigator of the study and local research team. Conceived and designed the experiments: NS NB JY DK CS. Performed the experiments: NS NB JY DK. Analyzed the data: NS NB JY DK. Wrote the paper: NS NB JY DK CS. References 11. Preker A, Harding A (2003) Private Participation in Health Services’’ World Bank. Washington DC: World Bank. 1. International Finance Corporation (2008) The Business of Health in Africa: Partnering with the Private Sector to Improve People’s Lives; Corporation IF, editor. Washington, DC: World Bank Publications. 1. International Finance Corporation (2008) The Business of Health in Africa: Partnering with the Private Sector to Improve People’s Lives; Corporation IF, editor. Washington, DC: World Bank Publications. g 12. Yoong J, Burger N, Spreng C, Sood N (2010) Private Sector Participation and Health System Performance in Sub-Saharan Africa. PLoS ONE 5: e13243. g 2. Brugha R, Zwi A (1998) Improving the quality of private sector delivery of public health services: challenges and strategies. Health Policy Plan 13: 107–120. y 13. Lagomarsino G, de Ferranti D, Pablos-Mendez A, Nachuk S, Nishtar S, et al. (2009) Public stewardship of mixed health systems. The Lancet 374: 1577–1578. (2009) Public stewardship of mixed health systems. The Lancet 374: 15 3. Kamat VR (2001) Private practitioners and their role in the resurgence of malaria in Mumbai (Bombay) and Navi Mumbai (New Bombay), India: serving the affected or aiding an epidemic? Social Science and Medicine 52: 885–909. 14. Bustreo F, Harding A, Axelsson H (2003) Can developing countries achieve adequate improvements in child health outcomes without engaging the private sector? Bull World Health Organ 81: 886–895. 4. Sauerborn R (2001) Low quality of care in low income countries: is the private sector the answer? International Journal for Quality in Health Care 13: 281–282. g 15. Bennett S, Hanson K, Kadama P, Montagu D (2005) Working with the Non- State Sector to Achieve Public Health Goals. Making Health Systems Work: World Health Organization. 5. Zwi AB, Brugha R, Smith E (2001) Private health care in developing countries. BMJ 323: 463–464. 16. Mills A, Palmer N, Gilson L, McIntyre D, Schneider H, et al. (2004) The performance of different models of primary care provision in Southern Africa. Soc Sci Med 59: 931–943. 6. Chakraborty S, Frick K (2002) Factors influencing private health providers’ technical quality of care for acute respiratory infections among under-five children in rural West Bengal, India. Social Science & Medicine 55: 1579–1587. 17. Waters H, Hatt L, Peters D (2003) Working with the private sector for child health. Health Policy Plan 18: 127–137. g 7. Ethics Statement This study was reviewed and approved by the RAND Human Subjects Protection Committee (HSPC). All survey participants Figure 7. Facilities Reporting Knowing of Facilities Receiving Support for Activities. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g007 Figure 7. Facilities Reporting Knowing of Facilities Receiving Support for Activities. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g007 Figure 7. Facilities Reporting Knowing of Facilities Receiving Support for Activities. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g007 Figure 7. Facilities Reporting Knowing of Facilities Receiving Support for Activities. Countries: N Dark Grey: Kenya N Light Grey: Ghana. doi:10.1371/journal.pone.0027194.g007 PLoS ONE \| www.plosone.org 11 November 2011 \| Volume 6 \| Issue 11 \| e27194 November 2011 \| Volume 6 \| Issue 11 \| e27194 11 Government Engagement and Private Health Providers Table 8. Facilities reporting knowing of another facility receiving support from the government to promote equity, by type. Kenya Ghana Clinics Pharmacies Clinics Pharmacies N % N % N % N % Facilities reporting knowing another facility receiving support for Opening a facility in a rural area 112 30.4% 151 45.0% 67 10.4% 92 3.3% Opening a facility in a poor urban area 112 17.9% 151 36.4% 67 11.9% 92 4.3% Providing treatment to the poor 112 21.4% 151 39.1% 67 10.4% 92 4.3% Note: ‘‘Don’t know’’ and refusals are both coded as missing values. doi:10.1371/journal.pone.0027194.t008 Table 8. Facilities reporting knowing of another facility receiving support from the government to promote equity, by type. References Mills A, Brugha R, Hanson K (2002) What can be done about the private health sector in low-income countries? Bull World Health Organ 80: 325–330. y 18. (2011) Healthy Partnerships: How Governments Can Engage the Private Sector to Improve Health in Africa. Washington, DC: World Bank Publications. 180 p. 8. Marriott A (2009) Blind Optimism: Challenging the Myths About Private Health Care in Poor Countries Oxfam International. p g p 19. Loevinsohn B, Harding A (2005) Buying results? Contracting for health service delivery in developing countries. The Lancet 366: 676–681. delivery in developing countries. The Lancet 366: 676–681. 9. Berman P (1998) Rethinking health care systems: private health care provision in India. World Development 26: 1463–1479. 20. Ma S, Sood N (2008) A Comparison of Health Systems in India and China. California: RAND Corporation. 10. Corporation IF (2008) The Business of Health in Africa: Partnering with the Private Sector to Improve People’s Lives. Washington DC: International Finance Corporation. 21. Bennett S, Hanson K, Kadama P, Montagu D (2005) Working with the non state sector to achieve public health goals. Making Health Systems Work Working Paper No 2. PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e27194 12
https://openalex.org/W4294754982	https://www.cambridge.org/engage/api-gateway/coe/assets/orp/resource/item/6316df700429d6007f943c87/original/rejection-of-the-use-of-thought-experiment-in-physics.pdf	English	null	Rejection of the Use of Thought Experiment in Physics	null	2,022	cc-by	1,991	Rejection of the Use of Thought Experiment in Physics Abolfazl Soltani * Department of Physics, University of Birjand, Birjand, Iran Email: soltani.a.physics@gmail.com In physics, whenever we cannot do an experiment in the real world for physical, technological, financial or ethical reasons, we use from "thought experiment" [1]. In this letter, we prove that sometimes a thought experiment may lead us to the wrong results; and to achieve the definitive and reliable results, all experiments must be done in the real world. Here we prove that thought analysis of an experiment before performing in the real world, namely thought experiment, is a wrong work. There are several thought experiments in physics. Schrodinger’s cat experiment [2], EPR paradox [3] and Einstein's light box [4],[5],[6] are some of the most famous of them. It is clear, all experiments are initially a thought experiment (As Ernest Mach said a thought experiment is any experiment that has not been realized yet [7]). Some of them remain a thought experiment due to the human inability to perform them in the real world (because of physical, technological, financial or ethical reasons), and some of them become real physical experiments. For example, due to the restrictions of today’s technology, we cannot perform the Einstein's light box experiment in the real world. Because, by today's tools, we cannot measure the change in mass of the box after emission of a photon. There is a very important point about thought experiments that seems to have been ignored so far: Sometimes a thought experiment may lead us to a wrong result. A thought experiment is an experiment which it is designed and performed in the brain of a scientist, by using the knowledge and science of that time, and since human knowledge has never been complete, a thought experiment can sometimes lead us to wrong conclusions. Let me illustrate this with an example. In Photoelectric effect, which was discovered by Philip Lenard in the 1990s, there is a threshold frequency that scientists of that time could not predict existence of it, based on the main theory of that time about light i.e. electromagnetic (EM) theory [8]. The existence of a threshold frequency for a given metal, a frequency below which no photoemission occurs, however great the light intensity, is completely inexplicable in electromagnetic terms [8]. Rejection of the Use of Thought Experiment in Physics Abolfazl Soltani * From the electromagnetic point of view the primary circumstance that determines whether or not photoemission will occur is the energy reaching the surface per unit time (or the intensity), but not the frequency [8]. If a person in the 1990s (namely in the years before the discovery of quantum aspect of light) wanted to predict the result of photoelectric experiment, could he predict the existence of threshold frequency? The existence of threshold frequency was discovered via perform the photoelectric experiment in the real world, not by thought investigation. The photoelectric experiment shows well that sometimes a thought experiment can lead us to a wrong result (which in this case is prediction of nonexistence of threshold frequency). Another example is the experiment of subatomic particles diffraction from double-slit. If we do this experiment for macroscopic particles (such as sand particles or salt grains), the pattern of diffraction will be random [9]. If we do this experiment with subatomic particles, like electrons, we expect to achieve the same result, but performing of the experiment in the real world leads us to an interference pattern, i.e. dark and light bands [9], (Figure 1), which is 1 contrary to prediction. Therefore, in this case, too, the prediction of thought experiment is untrue. contrary to prediction. Therefore, in this case, too, the prediction of thought experiment is untrue. Fig. 1. Diffraction pattern of electron from double-slit. Fig. 1. Diffraction pattern of electron from double-slit. Numerous other examples can be mentioned that show that the results of thought experiments are not reliable. As another example, most of us know the famous phenomenon of Chain Fountain (Figure 2). In this experiment, we expected the chain to fall from the edge of the glass, but the result of the experiment is different from our prediction, and the chain falls with a distance from the edge of the glass (Fig. 2). Fig. 2. Chain Fountain phenomenon Based on all of these discussions, the conclusion of this letter is as follows: A thought experiment is completely unreliable. All experiments must be performed in the real world so that we can accept their results; because, as we proved in this article, doing an experiment in the real world can yield results we never even imagined. Rejection of the Use of Thought Experiment in Physics Abolfazl Soltani * It means that, for example, in Einstein's light box experiment, before performing the experiment in the real world, we cannot talk about what is its definitive result and we cannot say that the conclusion of Niels Bohr was true, not Einstein (as you know, the winner of the debate over the result of this thought experiment was Bohr, not Einstein [4],[5],[6]). Because, with performing this experiment in the real world, some results may be observed that were not in Einstein's or Bohr's predictions. Fig. 2. Chain Fountain phenomenon Fig. 2. Chain Fountain phenomenon Fig. 2. Chain Fountain phenomenon Based on all of these discussions, the conclusion of this letter is as follows: A thought experiment is completely unreliable. All experiments must be performed in the real world so that we can accept their results; because, as we proved in this article, doing an experiment in the real world can yield results we never even imagined. It means that, for example, in Einstein's light box experiment, before performing the experiment in the real world, we cannot talk about what is its definitive result and we cannot say that the conclusion of Niels Bohr was true, not Einstein (as you know, the winner of the debate over the result of this thought experiment was Bohr, not Einstein [4],[5],[6]). Because, with performing this experiment in the real world, some results may be observed that were not in Einstein's or Bohr's predictions. Based on all of these discussions, the conclusion of this letter is as follows: A thought experiment is completely unreliable. All experiments must be performed in the real world so that we can accept their results; because, as we proved in this article, doing an experiment in the real world can yield results we never even imagined. It means that, for example, in Einstein's light box experiment, before performing the experiment in the real world, we cannot talk about what is its definitive result and we cannot say that the conclusion of Niels Bohr was true, not Einstein (as you know, the winner of the debate over the result of this thought experiment was Bohr, not Einstein [4],[5],[6]). Because, with performing this experiment in the real world, some results may be observed that were not in Einstein's or Bohr's predictions. Rejection of the Use of Thought Experiment in Physics Abolfazl Soltani * 2 Just like Photoelectric experiment, diffraction of subatomic particles from double-slit and Chain fountain experiment which their results, after performing in the real world, were unexpected; All experiments, after performing in the real world, may have unexpected results. Therefore, thought analysis of an experiment before performing in the real world, namely thought experiment, is a wrong and meaningless work. Because it is impossible for us to predict its unexpected results (if any). References: ( ) [3]. Einstein, A. Podolsky, B. Rosen, N. Can Quantum-Mechanical Description of Physical Reality Be Considered Complete? Phys. Rev. 47, 777 (1935) [4]. Mcevoy, J. Oscar, Z. Introducing Quantum Theory. (Icon Books Ltd, ed. 4, 2003) pp 146-149 [5] W lt I Ei t i Hi Lif d U i (Si & S h t 2008) 344 347 References: [1]. Brown, James Robert and Yiftach Fehige, "Thought Experiments", The Stanford Encyclopedia of Philosophy (Spring 2022 Edition), Edward N. Zalta (ed.) URL= https://plato.stanford.edu/archives/spr2022/entries/thought-experiment/ . [2]. Schrodinger, E. Die gegenwärtige Situation in der Quantenmechanik. Naturwissenschaften. volume 23, 823– 828 (1935) [3]. Einstein, A. Podolsky, B. Rosen, N. Can Quantum-Mechanical Description of Physical Reality Be Considered Complete? Phys. Rev. 47, 777 (1935) [4]. Mcevoy, J. Oscar, Z. Introducing Quantum Theory. (Icon Books Ltd, ed. 4, 2003) pp 146-149 [5]. Walter, I. Einstein: His Life and Universe. (Simon & Schuster, 2008) pp 344-347 [6]. Abraham, P. Subtle is the Lord: The Science and Life of Albert Einstein. (Oxford University Press, 2005) pp 446-448 [7]. Cucic, Dragoljub A. "A short insight about Thought experiment in Modern Physics." arXiv preprint arXiv:0812.2539 (2008). Page 2 [8]. Weidner, R. Sells, R. Elementary Modern Physics (Allyn and Bacon, ed. 2, 1973), pp 106 - 115 [9]. La Physique Autrement (2012) Wave Particle Duality. Aug 20. Available at: https://www.youtube.com/watch?v=qCmtegdqOOA&t=2s). [1]. Brown, James Robert and Yiftach Fehige, "Thought Experiments", The Stanford Encyclopedia of Philosophy (Spring 2022 Edition), Edward N. Zalta (ed.) [1]. Brown, James Robert and Yiftach Fehige, "Thought Experiments", The Stanford Encyclopedia of Philosophy (Spring 2022 Edition), Edward N. Zalta (ed.) URL= https://plato.stanford.edu/archives/spr2022/entries/thought-experiment/ . [2]. Schrodinger, E. Die gegenwärtige Situation in der Quantenmechanik. Naturwissenschaften. volume 23, 823– 828 (1935) [3]. Einstein, A. Podolsky, B. Rosen, N. Can Quantum-Mechanical Description of Physical Reality Be Considered Complete? Phys. Rev. 47, 777 (1935) [4]. Mcevoy, J. Oscar, Z. Introducing Quantum Theory. (Icon Books Ltd, ed. 4, 2003) pp 146-149 [5]. Walter, I. Einstein: His Life and Universe. (Simon & Schuster, 2008) pp 344-347 [6]. Abraham, P. Subtle is the Lord: The Science and Life of Albert Einstein. (Oxford University Press, 2005) pp 446-448 [7]. Cucic, Dragoljub A. "A short insight about Thought experiment in Modern Physics." arXiv preprint arXiv:0812.2539 (2008). Page 2 [8]. Weidner, R. Sells, R. Elementary Modern Physics (Allyn and Bacon, ed. 2, 1973), pp 106 - 115 [9]. La Physique Autrement (2012) Wave Particle Duality. Aug 20. Available at: https://www.youtube.com/watch?v=qCmtegdqOOA&t=2s). URL= https://plato.stanford.edu/archives/spr2022/entries/thought-experiment/ . [2]. Schrodinger, E. Die gegenwärtige Situation in der Quantenmechanik. Naturwissenschaften. volume 23, 823– 828 (1935) [3]. Einstein, A. Podolsky, B. Rosen, N. Can Quantum-Mechanical Description of Physical Reality Be Considered Complete? Phys. Rev. 47, 777 (1935) [4]. Mcevoy, J. Oscar, Z. Introducing Quantum Theory. (Icon Books Ltd, ed. 4, 2003) pp 146-149 [5]. Walter, I. [2]. Schrodinger, E. Die gegenwärtige Situation in der Quantenmechanik. Naturwissenschaften. volume 23, 823– 828 (1935) References: [1]. Brown, James Robert and Yiftach Fehige, "Thought Experiments", The Stanford Encyclopedia of Philosophy (Spring 2022 Edition), Edward N. Zalta (ed.) URL= https://plato.stanford.edu/archives/spr2022/entries/thought-experiment/ . [2]. Schrodinger, E. Die gegenwärtige Situation in der Quantenmechanik. Naturwissenschaften. volume 23, 823– 828 (1935) [3]. Einstein, A. Podolsky, B. Rosen, N. Can Quantum-Mechanical Description of Physical Reality Be Considered Complete? Phys. Rev. 47, 777 (1935) [4]. Mcevoy, J. Oscar, Z. Introducing Quantum Theory. (Icon Books Ltd, ed. 4, 2003) pp 146-149 [5]. Walter, I. Einstein: His Life and Universe. (Simon & Schuster, 2008) pp 344-347 [6]. Abraham, P. Subtle is the Lord: The Science and Life of Albert Einstein. (Oxford University Press, 2005) pp 446-448 [7]. Cucic, Dragoljub A. "A short insight about Thought experiment in Modern Physics." arXiv preprint arXiv:0812.2539 (2008). Page 2 [8]. Weidner, R. Sells, R. Elementary Modern Physics (Allyn and Bacon, ed. 2, 1973), pp 106 - 115 [9]. La Physique Autrement (2012) Wave Particle Duality. Aug 20. Available at: https://www.youtube.com/watch?v=qCmtegdqOOA&t=2s). [3]. Einstein, A. Podolsky, B. Rosen, N. Can Quantum-Mechanical Description of Physical Reality Be Considered Complete? Phys. Rev. 47, 777 (1935) [1]. Brown, James Robert and Yiftach Fehige, "Thought Experiments", The Stanford Encyclopedia of Philosophy (Spring 2022 Edition), Edward N. Zalta (ed.) References: Einstein: His Life and Universe. (Simon & Schuster, 2008) pp 344-347 [6]. Abraham, P. Subtle is the Lord: The Science and Life of Albert Einstein. (Oxford University Press, 2 446-448 [7]. Cucic, Dragoljub A. "A short insight about Thought experiment in Modern Physics." arXiv arXiv:0812.2539 (2008). Page 2 [8]. Weidner, R. Sells, R. Elementary Modern Physics (Allyn and Bacon, ed. 2, 1973), pp 106 - 115 3 3
https://openalex.org/W4231490925	https://f1000research.com/articles/5-2340/pdf	English	null	GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes	F1000Research	2,016	cc-by	5,124	SOFTWARE TOOL ARTICLE Evert van den Broek1,5, Stef van Lieshout1, Christian Rausch1,5, Bauke Ylstra1, Mark A. van de Wiel2,3, Gerrit A. Meijer1,5, Remond J.A. Fijneman1,5, Sanne Abeln 4 1Department of Pathology, VU University Medical Center, Amsterdam, 1081 HZ, The Netherlands 2Department of Pathology, Netherlands Cancer Institute, Amsterdam, 1066CX, The Netherlands 3Department of Mathematics, VU University Medical Center, Amsterdam, Amsterdam, 1081 HV, The Netherlands 4Department of Epidemiology & Biostatistics, VU University Medical Center, Amsterdam, 1081 HZ, The Netherlands 5Department of Computer Science, VU University Medical Center, Amsterdam, 1081 HV, The Netherlands Open Peer Review Approval Status 1 2 version 2 (revision) 06 Jul 2017 view version 1 19 Sep 2016 view view Tobias Marschall, Max-Planck Institute for Infomatics, Saarbrücken, Germany 1. Angel Rubio, University of Navarra, San Sebastian, Spain 2. Any reports and responses or comments on the article can be found at the end of the article. Open Peer Review Approval Status 1 2 version 2 (revision) 06 Jul 2017 view version 1 19 Sep 2016 view view Tobias Marschall, Max-Planck Institute for Infomatics, Saarbrücken, Germany 1. Angel Rubio, University of Navarra, San Sebastian, Spain 2. Any reports and responses or comments on the article can be found at the end of the article. First published: 19 Sep 2016, 5:2340 https://doi.org/10.12688/f1000research.9259.1 Latest published: 06 Jul 2017, 5:2340 https://doi.org/10.12688/f1000research.9259.2 v2 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 Abstract Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs) of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large) series of tumor samples. ‘GeneBreak’ is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS). First, ‘GeneBreak’ collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, ‘GeneBreak’, is implemented in R (www.cran.r- project.org) and is available from Bioconductor ( www.bioconductor.org/packages/release/bioc/html/GeneBreak.html). Tobias Marschall, Max-Planck Institute for Infomatics, Saarbrücken, Germany 1. Angel Rubio, University of Navarra, San Sebastian, Spain 2. Any reports and responses or comments on the article can be found at the end of the article. Page 1 of 10 Keywords structural chromosomal aberrations, recurrent breakpoint genes, molecular characterization, cancer genome, copy number aberration profile, computational method This article is included in the RPackage gateway. Breakpoint detection and filter optionsi pi p Breakpoints are defined by the chromosomal locations that separate the contiguous DNA copy number segments pinpointed by a segmentation algorithm. ‘GeneBreak’ identifies chromo- somal breakpoint locations for each individual DNA copy number profile. Instead of taking all detected breakpoints, users may want to define more precisely what breakpoints to take into account, based on the two flanking DNA copy number segment character- istics. One of the following three selection options can be applied. A) Copy number-deviation: this selects breakpoints where the shift in log2-ratio between two consecutive DNA copy number segments exceeds the user-defined threshold; B) CNA-associated breakpoints: this selects all breakpoints between consecutive DNA copy number segments, except for breakpoints flanked by two copy number neutral segments; C) CNA-breakpoints: this selects only those breakpoints flanked by segments with dissimilar discrete DNA copy number states. Tumor development is driven by irreversible somatic genomic aberra- tions such as single nucleotide variants (SNVs) and chromosomal aberrations including numerical as well as structural changes1,2. Genome-wide somatic DNA copy number aberrations (CNA) profiling is a widely established approach to characterize chromo- somal aberrations in cancer genomes. At present, application of computational methods has mainly been focused on the analysis of numerical aberrations of chromosomal segments. Evidence is emerging that genes affected by structural chromosomal aberra- tions, i.e. genes affected by chromosomal breaks, represent a bio- logically and clinically relevant class of mutations in many cancer types including solid tumors3–6. Importantly, the actual locations of chromosomal CNA-associated breakpoints, which are the points of copy number level shift in somatic CNA profiles, indicate underly- ing chromosomal breaks and thereby genomic locations affected by somatic structural aberrations5–12. Hence, the wide availability of large series of high-resolution DNA copy number data by for instance array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS) approaches enables to systematically search for regions and genes that are affected by CNA-associated structural chromosomal changes. Computational methods determining numerical CNAs, consequently, also yield CNA-associated breakpoint locations. However, it is not trivial to identify genes that are recurrently affected by CNA-associated chro- mosomal breakpoints across (large) series of cancer samples since this methodology also requires dedicated computational methods including comprehensive statistical evaluation. Breakpoints are defined by the genomic start position of the copy number segments. DNA copy number profiling data is typically granular due to the distance between microarray probes or bin size of WGS copy number data. F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 ‘GeneBreak’ takes DNA copy number data that are pre-processed by the R-package ‘CGHcall’13 or ‘QDNAseq’14, both based on the Circular Binary Segmentation algorithm15, as input. Alternatively, segmented values (log2-ratios) from a different copy number detection algorithm can be used. In addition, it is recommended to provide discrete DNA copy number states (e.g. loss, neutral, gain) that can be used for breakpoint selection. Bioconductor vignette and manual describe commands and workflows in detail (See Supplementary material). Amendments from Version 1 REVISED In this version we provide a much more extensive description of the underlying statistics for the detection of recurrent breakpoint events on genomic location- and gene-level. In addition, we rephrased a few sentences. See referee reports Breakpoint detection and filter optionsi This means that the genomic location of a breakpoint is not detected at nucleotide resolution but represents a chromosomal interval with a size that is determined by microarray probe density or WGS bin size. Cohort-based breakpoint statistics: breakpoint and gene level Identification of statistically recurrent breakpoint events across all samples can be performed on both chromosomal location- and gene-level. As features, i.e. microarray probes or bins of WGS copy number data, are (nearly) equally distributed over the genome, we assume that the null- probability for breakpoint occurrence is equal for all individual candidate breakpoints (features). It differs per sample, though, and equals ps = Ns/N, where N is the number of probes, and Ns the total number of breakpoint for samples. The test statistic is Tp is the total number of breakpoints for probe p across all samples. Then, under the null-hypothesis, Tp is simply a sum of independent Bernoulli (ps) random variables, the null-distribution of which is the same for all probes. It is quickly computed by using probability generating functions, giving also the p-values for any observed value of Tp. Breakpoint gene identificationi For identification of genes affected by chromosomal breakpoints the built-in gene annotations can be used. Alternatively, a user- defined gene annotation file can be provided (see Bioconductor vignette and manual for further details). The implemented mapping approach identifies genes that are associated with one or multiple chromosomal breakpoint intervals. We here provide a computational method, ‘GeneBreak’, that identifies chromosomal breakpoint locations using DNA copy number profiles. A tailored annotation approach maps breakpoint locations to genes for each individual profile. Moreover, dedicated comprehensive cohort-based statistical analysis including correc- tion for covariates that influence the probability to be a breakpoint gene and multiple testing pinpoints genes that are non-randomly and recurrently affected by chromosomal breaks across multiple tumor samples5. ‘GeneBreak’ is implemented in R (www.cran. r-project.org) and is available from Bioconductor (www.biocon- ductor.org/packages/release/bioc/html/GeneBreak.html). The Bioconductor vignette describes a detailed example workflow of CNA data obtained by analysis of 200 array-CGH samples. A schematic overview of computational methods is depicted in Figure 1. Corresponding authors: Remond J.A. Fijneman (r.fijneman@nki.nl), Sanne Abeln (s.abeln@vu.nl) Corresponding authors: Remond J.A. Fijneman (r.fijneman@nki.nl), Sanne Abeln (s.abeln@vu.nl) Author roles: van den Broek E: Conceptualization, Data Curation, Formal Analysis, Methodology, Software, Validation, Writing – Original Draft Preparation, Writing – Review & Editing; van Lieshout S: Methodology, Software, Writing – Review & Editing; Rausch C: Resources, Software, Validation, Writing – Review & Editing; Ylstra B: Resources, Writing – Review & Editing; van de Wiel MA: Conceptualization, Formal Analysis, Methodology, Software, Validation, Writing – Review & Editing; Meijer GA: Conceptualization, Funding Acquisition, Resources, Supervision, Writing – Review & Editing; Fijneman RJA: Conceptualization, Funding Acquisition, Methodology, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing; Abeln S: Conceptualization, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing How to cite this article: van den Broek E, van Lieshout S, Rausch C et al. GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes [version 2; peer review: 2 approved] F1000Research 2017, 5:2340 https://doi.org/10.12688/f1000research.9259.2 Page 2 of 10 DNA copy number profiles DNA copy number profiles The breakpoint detection method we provide is amenable for data from any DNA copy number discovery platform, e.g. array-CGH and (low-pass) WGS. For optimal results, Page 3 of 10 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 Figure 1. Schematic overview of computational methods. GeneBreak’ requires already segmented DNA copy number data from array-CGH or WGS approaches. The first step involves detection of breakpoint locations. Next, breakpoint locations will be mapped to gene annotations in order to identify genes affected by DNA breakpoints. The final step performs comprehensive cohort-based statistical analyses including correction for multiple testing to reveal both recurrent breakpoint locations and breakpoint genes. The breakpoint frequencies can be visualized with a built-in plot function. This example visualizes the breakpoint locations (vertical black bars) and breakpoint genes (horizontal red bars) on the p-arm of chromosome 20 identified in a cohort of 352 advanced colorectal cancers. The genes labeled with a name are statistically significant recurrent breakpoint genes (FDR<0.1). Figure 1. Schematic overview of computational methods. GeneBreak’ requires already segmented DNA copy number data from array-CGH or WGS approaches. The first step involves detection of breakpoint locations. Next, breakpoint locations will be mapped to gene annotations in order to identify genes affected by DNA breakpoints. The final step performs comprehensive cohort-based statistical analyses including correction for multiple testing to reveal both recurrent breakpoint locations and breakpoint genes. The breakpoint frequencies can be visualized with a built-in plot function. This example visualizes the breakpoint locations (vertical black bars) and breakpoint genes (horizontal red bars) on the p-arm of chromosome 20 identified in a cohort of 352 advanced colorectal cancers. The genes labeled with a name are statistically significant recurrent breakpoint genes (FDR<0.1). Figure 1. Schematic overview of computational methods. GeneBreak’ requires already segmented DNA copy number data from array-CGH or WGS approaches. The first step involves detection of breakpoint locations. Next, breakpoint locations will be mapped to gene annotations in order to identify genes affected by DNA breakpoints. The final step performs comprehensive cohort-based statistical analyses including correction for multiple testing to reveal both recurrent breakpoint locations and breakpoint genes. The breakpoint frequencies can be visualized with a built-in plot function. This example visualizes the breakpoint locations (vertical black bars) and breakpoint genes (horizontal red bars) on the p-arm of chromosome 20 identified in a cohort of 352 advanced colorectal cancers. DNA copy number profiles The genes labeled with a name are statistically significant recurrent breakpoint genes (FDR<0.1). Use casei The probe-based statistical analysis uses Benjamini-Hochberg false discovery rate (FDR) correction for multiple testing. For the intended use at gene level, a more advanced statistical null-model is required. For the gene level, the null-probability for a breakpoint to occur within an individual gene, depends on 1) the length of the gene, 2) the number of gene-associated features and 3) the number of breakpoints in the entire tumor profile for the specific sample. Therefore, at gene-level, we apply a linear regression-based cor- rection for covariates. These regression-estimates are then used as gene- and sample-specific breakpoint null-probabilities (pg,s). The test statistic remains the same, and so does the null-distribution computation, although it has to be repeated for each gene now. Finally, the Gilbert FDR correction that accounts for discreteness in the null-distribution16 is applied in this analysis to determine sig- nificance of recurrent breakpoint genes. Commands and example workflow can be found in Bioconductor vignette and manual. Identification of recurrent breakpoint genes in advanced colorectal cancers Identification of recurrent breakpoint genes in advanced colorectal cancers We applied our method to 352 high-resolution array-CGH sam- ples from a series of advanced colorectal cancers17 following CNA detection using ‘CGHcall’13. Array-CGH data are available in the Gene Expression Omnibus database under accession number GSE63216 (www.ncbi.nlm.nih.gov/projects/geo/). We selected for the CNA-associated breakpoints (setting: ‘CNA-associated’), used gene annotations from ensembl (human genome NCBI build36/hg18, release 54) and applied the dedicated Benjamini- Hochberg-type FDR correction (setting: ‘Gilbert’), for recur- rent breakpoint gene identification. A total of 748 genes appeared to be recurrently affected by chromosomal breaks (FDR<0.1)5. Breakpoint frequencies of chromosome 20p are visualized with the built-in plot function (Figure 1; see Bioconductor vignette and Page 4 of 10 Page 4 of 10 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 Latest source code: https://github.com/F1000Research/GeneBreak/ releases/tag/v1.0 manual for further details about this function). Interestingly, patient stratification based on recurrent gene breakpoints and well-known point mutations by propagation to the predefined STRING human protein interaction network revealed one CRC subtype with very poor prognosis, which supported clinical relevance of this class of somatic aberrations in advanced colorectal cancers5. Archived source code as at the time of publication: F1000Research/ Genebreak, doi: 10.5281/zenodo.15393718 License: GPL 2 Author contributions EvdB, GM, RF and SA conceived the study. EvdB, SvL, MvdW, GM, RF and SA designed the workflow and EvdB, SvL and MvdW developed and tested the code. MvdW provided expertise in biostatistics. CR and BY provided expertise in analysis of CNA data obtained by array-CGH and WGS. EvdB, RF and SA prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content. Conclusion Genome instability including numerical and structural somatic chromosomal aberrations is a hallmark of cancer. Several tools are available that focus on detection of numerical aberrations of large chromosome segments. The R-package ‘GeneBreak’ extracts additional information from CNA data. ‘GeneBreak’ provides an easy-to-use algorithm, which handles identification of genomic breakpoint locations, mapping of breakpoints to genes and includes a comprehensive statistical approach to reveal recurrent breakpoint genes from series of tumor samples. Therefore, ‘GeneBreak’ can be applied to detect CNA-associated chro- mosomal breaks in individual tumor samples and facilitates detection of recurrent breakpoint genes across multiple tumor samples. Competing interests No competing interests were disclosed. Supplementary material GeneBreak vignette. Click here to access the data. GeneBreak Manual. Click here to access the data. 1. Stratton MR, Campbell PJ, Futreal PA: The cancer genome. Nature. 2009; 458(7239): 719–724. PubMed Abstract \| Publisher Full Text \| Free Full Text 2. Forbes SA, Bindal N, Bamford S, et al.: COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011; 39(Database issue): D945–D950. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Mitelman F, Johansson B, Mertens F: The impact of translocations and gene fusions on cancer causation. Nat Rev Cancer. 2007; 7(4): 233–245. PubMed Abstract \| Publisher Full Text 4. Inaki K, Liu ET: Structural mutations in cancer: mechanistic and functional Data and software availability This work was supported by the VUmc-Cancer Center Amsterdam [to E.vd.B.]; performed within the framework of the Center for Translational Molecular Medicine, DeCoDe project [03O-101]; and CTMM-TraIT [05T-401 to EvdB, SvL, BY, GM, RF and SA]. Publicly available copy number data used for the use case is deposited at Gene Expression Omnibus database under acces- sion number GSE63216 (https://protect-eu.mimecast.com/ s/6LQhBmNGvCG). Software available from: C www.bioconductor.org/packages/ release/bioc/html/GeneBreak.html and https://protect-eu.mimecast. com/s/aLGhBqmpgF2 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplementary material insights. Trends Genet. 2012; 28(11): 550–559. PubMed Abstract \| Publisher Full Text 5. van den Broek E, Dijkstra MJ, Krijgsman O, et al.: High Prevalence and Clinical Relevance of Genes Affected by Chromosomal Breaks in Colorectal Cancer. PLoS One. 2015; 10(9): e0138141. PubMed Abstract \| Publisher Full Text \| Free Full Text 6. Malhotra A, Lindberg M, Faust GG, et al.: Breakpoint profiling of 64 cancer genomes reveals numerous complex rearrangements spawned by homology- independent mechanisms. Genome Res. 2013; 23(5): 762–776. PubMed Abstract \| Publisher Full Text \| Free Full Text 7. Edwards PA: Fusion genes and chromosome translocations in the common 458(7239): 719 724. PubMed Abstract \| Publisher Full Text \| Free Full Text References insights. Trends Genet. 2012; 28(11): 550–559. PubMed Abstract \| Publisher Full Text 5. van den Broek E, Dijkstra MJ, Krijgsman O, et al.: High Prevalence and Clinical Relevance of Genes Affected by Chromosomal Breaks in Colorectal Cancer. PLoS One. 2015; 10(9): e0138141. PubMed Abstract \| Publisher Full Text \| Free Full Text 6. Malhotra A, Lindberg M, Faust GG, et al.: Breakpoint profiling of 64 cancer genomes reveals numerous complex rearrangements spawned by homology- independent mechanisms. Genome Res. 2013; 23(5): 762–776. PubMed Abstract \| Publisher Full Text \| Free Full Text 7. Edwards PA: Fusion genes and chromosome translocations in the common Page 5 of 10 Page 5 of 10 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 epithelial cancers. J Pathol. 2010; 220(2): 244–254. PubMed Abstract \| Publisher Full Text 8. Hermsen M, Snijders A, Guervós MA, et al.: Centromeric chromosomal translocations show tissue-specific differences between squamous cell carcinomas and adenocarcinomas. Oncogene. 2005; 24(9): 1571–1579. PubMed Abstract \| Publisher Full Text 9. Muggeo VM, Adelfio G: Efficient change point detection for genomic sequences of continuous measurements. Bioinformatics. 2011; 27(2): 161–166. PubMed Abstract \| Publisher Full Text 10. Ritz A, Paris PL, Ittmann MM, et al.: Detection of recurrent rearrangement breakpoints from copy number data. BMC Bioinformatics. 2011; 12: 114. PubMed Abstract \| Publisher Full Text \| Free Full Text 11. Toloşi L, Theißen J, Halachev K, et al.: A method for finding consensus breakpoints in the cancer genome from copy number data. Bioinformatics. 2013; 29(14): 1793–1800. PubMed Abstract \| Publisher Full Text 12. Liu H, Zilberstein A, Pannier P, et al.: Evaluating translocation gene fusions by SNP array data. Cancer Inform. 2012; 11: 15–27. PubMed Abstract \| Publisher Full Text \| Free Full Text 13. van de Wiel MA, Kim KI, Vosse SJ, et al.: CGHcall: calling aberrations for array CGH tumor profiles. Bioinformatics. 2007; 23(7): 892–894. PubMed Abstract \| Publisher Full Text 14. Scheinin I, Sie D, Bengtsson H, et al.: DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly. Genome Res. 2014; 24(12): 2022–2032. PubMed Abstract \| Publisher Full Text \| Free Full Text 15. Olshen AB, Venkatraman ES, Lucito R, et al.: Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics. 2004; 5(4): 557–572. References Broek E: F1000Research/GeneBreak. Zenodo. 2016. Data Source References PubMed Abstract \| Publisher Full Text 16. Gilbert PB: A modified false discovery rate multiple-comparisons procedure for discrete data, applied to human immunodeficiency virus genetics. Appl Statist. 2005; 54(1): 143–158. Publisher Full Text 17. Haan JC, Labots M, Rausch C, et al.: Genomic landscape of metastatic colorectal cancer. Nat Commun. 2014; 5: 5457. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Broek E: F1000Research/GeneBreak. Zenodo. 2016. Data Source epithelial cancers. J Pathol. 2010; 220(2): 244–254. PubMed Abstract \| Publisher Full Text Hermsen M, Snijders A, Guervós MA, et al.: Centromeric chromosomal translocations show tissue-specific differences between squamous cell carcinomas and adenocarcinomas. Oncogene. 2005; 24(9): 1571–1579. PubMed Abstract \| Publisher Full Text Muggeo VM, Adelfio G: Efficient change point detection for genomic sequences of continuous measurements. Bioinformatics. 2011; 27(2): 161–166. PubMed Abstract \| Publisher Full Text Ritz A, Paris PL, Ittmann MM, et al.: Detection of recurrent rearrangement breakpoints from copy number data. BMC Bioinformatics. 2011; 12: 114. PubMed Abstract \| Publisher Full Text \| Free Full Text Toloşi L, Theißen J, Halachev K, et al.: A method for finding consensus breakpoints in the cancer genome from copy number data. Bioinformatics. 2013; 29(14): 1793–1800. PubMed Abstract \| Publisher Full Text Liu H, Zilberstein A, Pannier P, et al.: Evaluating translocation gene fusions by SNP array data. Cancer Inform. 2012; 11: 15–27. PubMed Abstract \| Publisher Full Text \| Free Full Text van de Wiel MA, Kim KI, Vosse SJ, et al.: CGHcall: calling aberrations for array CGH tumor profiles. Bioinformatics. 2007; 23(7): 892–894. PubMed Abstract \| Publisher Full Text 14. Scheinin I, Sie D, Bengtsson H, et al.: DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly. Genome Res. 2014; 24(12): 2022–2032. PubMed Abstract \| Publisher Full Text \| Free Full Text 15. Olshen AB, Venkatraman ES, Lucito R, et al.: Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics. 2004; 5(4): 557–572. PubMed Abstract \| Publisher Full Text 16. Gilbert PB: A modified false discovery rate multiple-comparisons procedure for discrete data, applied to human immunodeficiency virus genetics. Appl Statist. 2005; 54(1): 143–158. Publisher Full Text 17. Haan JC, Labots M, Rausch C, et al.: Genomic landscape of metastatic colorectal cancer. Nat Commun. 2014; 5: 5457. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Version 2 © 2017 Rubio A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.5256/f1000research.9967.r18598 © 2017 Rubio A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PubMed Abstract \| Publisher Full Text 16. Gilbert PB: A modified false discovery rate multiple-comparisons procedure for discrete data, applied to human immunodeficiency virus genetics. Appl Statist. 2005; 54(1): 143–158. Publisher Full Text 17. Haan JC, Labots M, Rausch C, et al.: Genomic landscape of metastatic colorectal cancer. Nat Commun. 2014; 5: 5457. PubMed Abstract \| Publisher Full Text \| Free Full Text 12. Liu H, Zilberstein A, Pannier P, et al.: Evaluating translocation gene fusions by SNP array data. Cancer Inform. 2012; 11: 15–27. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Broek E: F1000Research/GeneBreak. Zenodo. 2016. Data Source Page 6 of 10 Open Peer Review Current Peer Review Status: F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 Open Peer Review Angel Rubio Group of Bioinformatics, TECNUN, University of Navarra, San Sebastian, Spain The authors provided an brief explanation of the statistics involved in the detection of recurrent copy number changes. I would have liked a slightly deeper description but, for most users the explanation is sufficient and give an idea of the methodology. I think that this paper is ready. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Reviewer Report 13 February 2017 https://doi.org/10.5256/f1000research.9967.r16416 © 2017 Marschall T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Angel Rubio Angel Rubio 1 Group of Bioinformatics, TECNUN, University of Navarra, San Sebastian, Spain 2 Group of Bioinformatics, TECNUN, University of Navarra, San Sebastian, Spain The paper shows an inspiring vision of the copy number changes in the genome focusing on the g 1 Group of Bioinformatics, TECNUN, University of Navarra, San Sebastian, Spain 2 Group of Bioinformatics, TECNUN, University of Navarra, San Sebastian, Spain 1 Group of Bioinformatics, TECNUN, University of Navarra, San Sebastian, Spain 2 Group of Bioinformatics, TECNUN, University of Navarra, San Sebastian, Spain The paper shows an inspiring vision of the copy number changes in the genome focusing on the Page 7 of 10 Page 7 of 10 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 "changes" more than on the levels of change. The underlying reasoning is that a copy number change, if occurs within the loci occupied by a gene, implies an alteration in the coding sequence of the gene. In addition, it is shown that these changes occur recurrently, i.e. the loci where the copy number changes tend to be similar in different samples with the same type of cancer. The methodology has been uploaded to Bioconductor. The stringent quality checks of Bioconductor guarantees the availability for different platforms and, in fact, the vignette is easy to follow and use. My main concern with this paper is the (lack of) description of the statistical method to state the recurrence of the copy number changes. Within the methods section is only stated that there are two methods (genome location and gene-level) but the differences between them or the underlying statistical model is missing. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer Report 09 January 2017 https://doi.org/10.5256/f1000research.9967.r16416 Tobias Marschall 1 Center for Bioinformatics, Max-Planck Institute for Infomatics, Saarbrücken, Germa 1 Center for Bioinformatics, Max-Planck Institute for Infomatics, Saarbrücken, Germany 2 Center for Bioinformatics, Max-Planck Institute for Infomatics, Saarbrücken, Germany Center for Bioinformatics, Max-Planck Institute for Infomatics, Saarbrücken, Germany GeneBreak is an R package to help identifying recurrent breakpoints of copy number variants (CNVs). While the offered analyses are straightforward from a methodological point of view, this package can be valuable in practice, providing an easy and reproducible way to conduct such analyses. I appreciate that it is available from bioconductor (and hence easily installable), openly developed on github, and archived on zenodo. I merely have some minor suggestions for improvements: I merely have some minor suggestions for improvements: y gg p When trying to use the package, I couldn't open the example data (got a "data set ‘copynumber.data.chr20’ not found" error). Could you verify that it's available? ○ First sentence: SNV commonly means "single nucleotide variation" (not "small"). ○ When trying to use the package, I couldn't open the example data (got a "data set ‘copynumber.data.chr20’ not found" error). Could you verify that it's available? ○ First sentence: SNV commonly means "single nucleotide variation" (not "small"). P1, L9: "Recently, ..." Of course what you consider "recent" is a matter of taste, but here you ○ Page 8 of 10 Page 8 of 10 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 P1, Methods, L3: "... and copy number detection algorithm" Either explain what exactly you mean here, or remove. ○ P1, paragraph "Due to the typical granularity [...], in fact represent a chromosomal interval." I can guess what you mean here, but writing this more clearly would be good. ○ P1, "This method assumes the same permutation null- distribution for all candidate breakpoint events for the analysis of breakpoints at the level of genomic location." Could you describe in more detail how the null distribution is obtained? ○ P1, "In addition, a more comprehensive and powerful dedicated Benjamini-Hochberg FDR correction that accounts for discreteness in the null-distribution is supplied." The Benjamini- Hochberg procedure is a well defined statistical method. I would rephrase the respective sentence(s) to explicitly say that you are talking about Gilbert's method. ○ P1, "In addition, a more comprehensive and powerful dedicated Benjamini-Hochberg FDR correction that accounts for discreteness in the null-distribution is supplied." The Benjamini- Hochberg procedure is a well defined statistical method. I would rephrase the respective sentence(s) to explicitly say that you are talking about Gilbert's method. ○ Competing Interests: No competing interests were disclosed. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Reader Comment 29 May 2017 Evert van den Broek We thank the reviewer for careful evaluation of our work and providing helpful recommendations. As suggested by the reviewer, we rephrased some sentences and provided a more detailed description of the used statistics. With respect to the error by loading the example data of GeneBreak, we verified availability of the example data on different computers with different operating systems (MacOS and Linux) on which we installed the GeneBreak package from Bioconductor and CGHcall with all dependencies. The data (‘copynumber.data.chr20.rda’) was also available in the ‘data’ directory that was retrieved from Bioconductor. The exact code we used is provided by https://www bioconductor org/packages/release/bioc/vignettes/GeneBreak/inst/doc/GeneBrea p y https://www.bioconductor.org/packages/release/bioc/vignettes/GeneBreak/inst/do Competing Interests: No competing interests were disclosed. Page 9 of 10 F1000Research 2017, 5:2340 Last updated: 19 FEB 2024 The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com The benefits of publishing with F1000Research: The benefits of publishing with F1000Research: Page 10 of 10
https://openalex.org/W2966645254	https://www.iiste.org/Journals/index.php/JEP/article/download/48825/50444	English	null	Democratic Education of Students in Elementary School	null	2,019	cc-by	3,065	Abstract d t iti h ld h f l thi k f l iti l thi ki kill t b f th i i ht The results of this study will be comparing the differences between the groups involved in the research. Students citizens should choose freely think freely critical thinking skills to be aware of their rig y p g g p Students, citizens should choose freely, think freely, critical thinking skills, to be aware of their rights and responsibilities. In this paper there will be information about the students democratic education, which is a major factor of democracy. Keywords: democratic education;students; critical thinking; mass media; tolerance. DOI: 10 7176/JEP/10-20-04 Keywords: democratic education;students; critical thinking; mass media; tolerance. DOI: 10.7176/JEP/10-20-04 Publication date:July 31st 2019 INTRODUCTION Democratization of society requires democratization of the school, and this undoubtedly emphasizes the model towards respect for the individual and personality. Only with such a pattern of education can the principles of humanism, individualism, differentiation of education and assessment of personal interests of students be applied, the democratization of education is increasingly recognized as one of the high-value values for success in reform efforts in the field of education. Raising tolerant person can be perceived as a contribution to human culture and social good manners. T l i i f d h i i i h b i f d i d l li i i i i Raising tolerant person can be perceived as a contribution to human culture and social good manners. Tolerance is a virtue of moderate human society, it is the basis of a democratic and pluralistic society, it is an attitude that should be held regularly accompanied by understanding and compromise for respecting different ideas, the approach and the identity of others. pp y Schools in this research have been randomly selected. Democratic Education of Students in Elementary School Rabije Murati1 Agnesa Besimi2 1.Associate Professor, University of Tetova, Faculty of Pedagogy, Ilinden Street bb, 1200 Tetovo, Republic of North Macedonia 2.Master's student, University of Tetova, Faculty of Pedagogy, Ilinden Street bb, 1200 Tetovo, Republic of North Macedonia www.iiste.org www.iiste.org Journal of Education and Practice Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) DOI: 10.7176/JEP Vol.10, No.20, 2019 Abstract s paper we will try to outline the role of civic and democratic education of students in elementary schools ubject of this survey is the civic education, aims to prepare students as an active citizen of the future. In this paper we will try to outline the role of civic and democratic education of students in elementary schools. The subject of this survey is the civic education, aims to prepare students as an active citizen of the future. This study is quantitative research where the students are selected in a random way. j y , p p his study is quantitative research where the students are selected in a random way. y q y The population of the survey is consisted of the students of primary school. The sample survey includes students of primary schools in the city of Tetova and village in the surroundings of Tetova. p y y g g We took two schools similar by number of students, but different by location. The urban school is elementary school “Istikball” in Tetovo and the rural school is elementary school “Sami Frasheri” in village of Pirok. As a method and technique of research are applied questionnaires for students in primary school, who were part of the research. The research methods are: method of data collection, theoretical analysis method, comparative method. Research Techniques and Instruments are: analysis of pedagogical documentation, survey, survey charts. Independent variables are: City, Nationality, Type of the educational institution, pupil`s. Dependent variables: - The opinion of pupil`s from rural and urban areas about democratic, civic education; - The level of development of critical thinking and opinion of tolerance. g p All the data obtained by the research are also statistically processed through: numerical data, scales, etc. Th l f hi d ill b i h diff b h i l d i h h y y p g , , The results of this study will be comparing the differences between the groups involved in the research. Students, citizens should choose freely, think freely, critical thinking skills, to be aware of their rights and responsibilities. he results of this study will be comparing the differences between the groups involved in the research. Methodology of survey It is assumed that mass media have a significant impact on the development of democracy; 4. It is assumed that the development of critical thinking at school is at a satisfactory level. 1.4. Research Methods 1.4. Research Methods In this quantitative study, the research methods are: In this quantitative study, the research methods are: - Method of data collection; - Theoretical analysis method, - Comparative method, 1.5. Research Techniques and Instruments - Analysis of pedagogical documentation - Survey with the students - survey sheet for students 1.6. Sample survey 1.6. Sample survey p y In this research will be included two elementary schools with 40 (forty) participants ninth grade pupil`s, by 20 (twenty) 10 (ten) boys and 10 (ten) girls in each school, one of the municipality of Tetovo and one of the municipality of Bogovinje. And they are as follows “Istikball” in Tetovo, municipality of Tetovo and the other is “Sami Frasheri” in village of Pirok, municipality of Bogovinje. This means that the total number of involved pupil`s in primary schools would be 40 pupil`s and all of them are of albanian ethnicity. The time for realization the research is the school year 2017-2018. In the research survey are applied questionnaires intended for pupil``s in primary schools. The questionnaires are composed of questions of closed type and evaluation scales in order to obtain more objective responses and to come to a more relevant knowledge about the issue that we research. 1.7. Variables Independent variables: - City – Nationality - Type of the educational institu 1.7. Variables Independent variables: Independent variables: - City – Nationality - Type of the educational institution - Students gender Dependent variables: p - The opinion of pupil`s from rural and urban areas about democratic, civic education - The level of development of critical thinking and opinion of tolerance. - The opinion of pupil`s from rural and urban areas about democratic, civic education h l l f d l f i i l hi ki d i i f l - The opinion of pupil s from rural and urban areas about democratic, civic education - The level of development of critical thinking and opinion of tolerance. - The level of development of critical thinking and opinion of tolerance. Methodology of survey 1.1. Subject of the survey Contemporary education, therefore, contemporary teaching should enable the individual to communicate effectively, not only with other people but also with technology and science in general. In 21th century education must emphasize being democratic and tolerant person and be inclusive, to prepare individuals for a better future. From this, the subject of the study of this paper is the influence of democratic education in the preparation of students for democratic life. 1.2. Purpose and Research Tasks p The main purpose of the research is to examine the importance of democratic education in the society. The research tasks are: The main purpose of the research is to examine the importance of democratic education in the societ The research tasks are: The research tasks are: - To explore the impact of civic education on raising democratic awareness among students; - To review how students understand the notion of tolerance; - To review how students understand the notion of tolerance; To explore the impact of mass media on the development of democracy - To explore the impact of mass media on the development of democracy xplore the impact of mass media on the development 26 Journal of Education and Practice www.iiste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) DOI: 10.7176/JEP Vol.10, No.20, 2019 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) DOI: 10.7176/JEP Vol.10, No.20, 2019 - To explore the development of critical thinking in school. 1.3. Hypothesis 1.3. Hypothesis The general hypothesis is: It is considered that democratic education prepares students for democratic life. The specific hypotheses are: 1. It is assumed that the subject of civic education affects the raising of democratic awareness among students; 2. It is assumed that students understand and know the notion of tolerance; 3. It is assumed that mass media have a significant impact on the development of democracy; 4. It is assumed that the development of critical thinking at school is at a satisfactory level. The general hypothesis is: The specific hypotheses are: 1. It is assumed that the subject of civic education affects the raising of democratic awareness among students; 2. It is assumed that students understand and know the notion of tolerance; 3. It is assumed that mass media have a significant impact on the development of democracy; 4. It is assumed that the development of critical thinking at school is at a satisfactory level. 3. 1.8. Statistical processing of data The answers given in the interview list is as follows: 0 1 2 3 4 5 6 7 8 9 elementary school-Istikball - Tetovo elementary school-Sami Frasheri-Pirok Which implies that the majority or the greater number of pupil`s understand the notion of tolerance as respect for the free opinion of others. - The third hypothesis was that mass media have a significant impact on the development of democracy. The answers given in the interview list is as follows: 0 2 4 6 8 10 12 Elementary school School Istikball- Tetovo Elementary school School Sami Frasheri-Pirok substantial intermediate none From the table we can conclude that most students think that the subject of civic education has an impact on raising the democratic awareness of the pupil`s. - The second hypothesis was that pupil`s understand and know the notion of tolerance. The answers given in the interview list is as follows: the table we can conclude that most students think that the subject of civic education has an impact on g the democratic awareness of the pupil`s. g p p - The second hypothesis was that pupil`s understand and know the notion of tolerance. The answers given in the interview list is as follows: 0 1 2 3 4 5 6 7 8 9 elementary school-Istikball - Tetovo elementary school-Sami Frasheri-Pirok Which implies that the majority or the greater number of pupil`s understand the notion of tolerance as respect for the free opinion of others. - The third hypothesis was that mass media have a significant impact on the development of democracy. The answers given in the interview list is as follows: - The second hypothesis was that pupil`s understand and know the notion of tolerance. The answers given in the interview list is as follows: 0 1 2 3 4 5 6 7 8 9 elementary school-Istikball - Tetovo elementary school-Sami Frasheri-Pirok Which implies that the majority or the greater number of pupil`s understand the notion of tolerance as respect for the free opinion of others. - The third hypothesis was that mass media have a significant impact on the development of democracy. The answers given in the interview list is as follows: Which implies that the majority or the greater number of pupil`s understand the notion of tolerance as respect for the free opinion of others. 1.8. Statistical processing of data The data obtained by the research are statistically processed through: numerical data, percentages, scales, etc. The purpose of statistical data processing was to establish the rate to which the democratic education of elementary school students are affected. 1.9.Analysis of the results obtained by the research The survey included school students in urban and rural areas, 20 pupil`s in each school (out of which 10 girls and 10 boys), that is, a total of 40 pupil`s; 20 girls and 20 boys. Below is present in a tabular way the gender presentation of students: y y The survey included school students in urban and rural areas, 20 pupil`s in each school (out of which 10 girls and 10 boys), that is, a total of 40 pupil`s; 20 girls and 20 boys. Below is present in a tabular way the gender presentation of students: Girls Boys Total Elementary school - Istikball Tetovo 10 10 20 Elementary school - Sami Frashëri Pirok 10 10 20 Total 20 20 40 27 iiste.org Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) DOI: 10.7176/JEP Vol.10, No.20, 2019 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) DOI: 10.7176/JEP Vol.10, No.20, 2019 The first hypothesis is to investigate and clarify how much is the impact of civic education on raising Survey results are as follows: The first hypothesis is to investigate and clarify how much is the impact of civic education on raising Survey results are as follows: From the table we can conclude that most students think that the subject of civic education has an impact on raising the democratic awareness of the pupil`s. - The second hypothesis was that pupil`s understand and know the notion of tolerance. The answers given in the interview list is as follows: 0 2 4 6 8 10 12 14 16 Elementary school School Istikball- Tetovo Elementary school School Sami Frasheri-Pirok substantial intermediate none From the table we can conclude that most students think that the subject of civic education has an impact on raising the democratic awareness of the pupil`s. - The second hypothesis was that pupil`s understand and know the notion of tolerance. 1.8. Statistical processing of data - The third hypothesis was that mass media have a significant impact on the development of democracy. The answers given in the interview list is as follows: 28 www.iiste.org Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) DOI: 10.7176/JEP Vol.10, No.20, 2019 From the responses received from the students of both primary schools, mass media have a major impact on the development of democracy in the country. As to the question of which kind of media has more impact on raising and informing you as a student, answers are as follows: The impact of the media in the developing of democracy substantial intermediate none The impact of the media in the developing of democracy substantial intermediate none The impact of the media in the developing of democracy From the responses received from the students of both primary schools, mass media have a major impact on the development of democracy in the country. As to the question of which kind of media has more impact on raising and informing you as a student, answers are as follows: 0 2 4 6 8 10 12 14 TV Radio Internet - facebook, instagram, twitter. elementary school-Istikball- Tetovo elementary school-Sami Frasheri-Pirok - The fourth hypothesis that the growth of critical thinking in the school is in satisfactory level the result is as follows: The impact of the media in the developing of democracy substantial intermediate none From the responses received from the students of both primary schools, mass media have a major impact on the development of democracy in the country. As to the question of which kind of media has more impact on raising and informing you as a student, answers are as follows: 0 2 4 6 8 10 12 14 TV Radio Internet - facebook, instagram, twitter. elementary school-Istikball- Tetovo elementary school-Sami Frasheri-Pirok - The fourth hypothesis that the growth of critical thinking in the school is in satisfactory level the result is as follows: 0 2 4 6 8 10 12 14 TV Radio Internet - facebook, instagram, twitter. Conclusion and recommendation Conclusion Democratic education of students is an important factor for social cohesion, mutual understanding, intercultural and religious dialogue, and solidarity. Education in democratic society must be independent. In the sense of respecting education independence is considered as an important segment, which greatly influences the development of society as a whole through the development and formation of the individual as an important resource in the development of the state. It is considered that the most educated people are inclined or tend to more liberal and democratic values. Recommendation Democratic education of students in schools should be realized through their active participation in school and teaching. Students get familiar with their rights, but also be aware of their responsibilities in a democratic society. The student must be respected in class and at school. p Students involved in establishing school policies and changes that must take place in the school. The teacher must respect the individuality of students. 1.8. Statistical processing of data elementary school-Istikball- Tetovo elementary school-Sami Frasheri-Pirok elementary school-Istikball- Tetovo - The fourth hypothesis that the growth of critical thinking in the school is in satisfactory level the result is as follows: 29 www.iiste.org Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) DOI: 10.7176/JEP Vol.10, No.20, 2019 0 2 4 6 8 10 12 Elementary school Istikball- Tetovo Elementary school Sami Frasheri-Pirok substantial intermediate none The result shows that the largest number of pupil`s, especially those in the urban school, thinks that the their school and class develops critical thinking at a satisfactory level. The result shows that the largest number of pupil`s, especially those in the urban school, thinks that the their school and class develops critical thinking at a satisfactory level. References 1. Shosh, Edita. 1987, Demokratizacija obrazovanja, Zagreb 1. Shosh, Edita. 1987, Demokratizacija obrazovanja, Zagreb 2. Murati,Rabije, 2016, Demokratizimi i sistemit edukativ-arsimor në Republikën e MaqedonisëLuma grafik,, Tetovë 3. Murati,Rabije, 2016, Demokratizimi i institucioneve edukative-arsimor në Republikën e Maqedonisë, Luma grafik, Tetovë 4. Murati, Rabije, 2019, Demokracia në shkollë, Luma grafik, Tetovë 1. Shosh, Edita. 1987, Demokratizacija obrazovanja, Zagreb 2. Murati,Rabije, 2016, Demokratizimi i sistemit edukativ-arsimor në Republikën e MaqedonisëLuma grafik,, Tetovë 3. Murati,Rabije, 2016, Demokratizimi i institucioneve edukative-arsimor në Republikën e Maqedonisë, Luma grafik, Tetovë 4 M i R bij 2019 D k i ë hk llë L fik T ë 2. Murati,Rabije, 2016, Demokratizimi i sistemit edukativ-arsimor në Republikën e MaqedonisëLuma Tetovë 2. Murati,Rabije, 2016, Demokratizimi i sistemit edukativ-arsimor në Republikën e MaqedonisëLuma grafik,, Tetovë 3. Murati,Rabije, 2016, Demokratizimi i institucioneve edukative-arsimor në Republikën e Maqedonisë, Luma 3. Murati,Rabije, 2016, Demokratizimi i institucioneve edukative-arsimor në Republikën e Maqedonisë, Luma grafik, Tetovë 4. Murati, Rabije, 2019, Demokracia në shkollë, Luma grafik, Tetovë 30
https://openalex.org/W2352604051	https://www.frontiersin.org/articles/10.3389/fgene.2016.00087/pdf	English	null	Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress	Frontiers in genetics	2,016	cc-by	27,368	REVIEW REVIEW published: 13 May 2016 doi: 10.3389/fgene.2016.00087 published: 13 May 2016 doi: 10.3389/fgene.2016.00087 Edited by: Kristijan Ramadan, University of Oxford, UK Reviewed by: Dana Branzei, IFOM, The FIRC Institute of Molecular Oncology, Italy Aga Gambus, University of Birmingham, UK Correspondence: Helle D. Ulrich h.ulrich@imb-mainz.de †These authors have contributed equally to this work. Edited by: Kristijan Ramadan, University of Oxford, UK Reviewed by: Dana Branzei, IFOM, The FIRC Institute of Molecular Oncology, Italy Aga Gambus, University of Birmingham, UK Edited by: Kristijan Ramadan, University of Oxford, UK Reviewed by: Dana Branzei, IFOM, The FIRC Institute of Molecular Oncology, Italy Aga Gambus, University of Birmingham, UK Correspondence: Helle D. Ulrich h.ulrich@imb-mainz.de Keywords: ubiquitin, SUMO, DNA replication, DNA replication stress, DNA damage, DNA repair, genome stability †These authors have contributed equally to this work. Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress Néstor García-Rodríguez†, Ronald P. Wong† and Helle D. Ulrich* Institute of Molecular Biology, Mainz, Germany Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and ﬂexible responses, for which posttranslational protein modiﬁcations that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance speciﬁcally as they relate to DNA replication. We will consider cases where the modiﬁers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. Keywords: ubiquitin, SUMO, DNA replication, DNA replication stress, DNA damage, DNA repair, genome stability INTRODUCTION Specialty section: This article was submitted to Cancer Genetics, a section of the journal Frontiers in Genetics Received: 21 March 2016 Accepted: 27 April 2016 Published: 13 May 2016 Citation: García-Rodríguez N, Wong RP and Ulrich HD (2016) Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress. Front. Genet. 7:87. d i 10 3389/f 2016 00087 Specialty section: This article was submitted to Cancer Genetics, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Cancer Genetics, a section of the journal Frontiers in Genetics Received: 21 March 2016 Accepted: 27 April 2016 Published: 13 May 2016 DNA replication in eukaryotes is a multi-step process that is tightly coupled to both cell cycle progression and the DNA damage response (Leman and Noguchi, 2013; Siddiqui et al., 2013; Berti and Vindigni, 2016). After completion of mitosis during the G1 stage of the cell cycle, replication origins are prepared for activation in a process called origin licensing (Siddiqui et al., 2013). This reaction results in the formation of pre-replicative complexes (pre-RCs) at replication origins, which include key components of the replicative helicase, albeit in an inactive form. Licensing restricts origin ﬁring to once per cell cycle, thus preventing genome instability induced by re-replication. At the entry into S phase, DNA replication is initiated by the action of cell cycle-regulated kinases, resulting in the activation of the replicative helicase and the separation of strands to form the ﬁrst replication forks. This is helicase and several DNA polymerases, but also a large number of accessory factors responsible accompanied by the assembly of replisomes, multi-protein complexes that comprise not only the for monitoring replication fork Received: 21 March 2016 Accepted: 27 April 2016 Published: 13 May 2016 Contributions of Ubiquitin and SUMO to Origin Licensing and Replication Initiation At the entry into S phase, ubiquitin functions predominantly as an inducer of proteasomal degradation, owing to its prominent role in cell cycle regulation (Teixeira and Reed, 2013). Preparation for DNA replication requires loading of the hexameric ring-shaped Mcm2-7 complex onto origins of replication, mediated by the origin recognition complex (ORC) and two auxiliary factors, Cdt1 and Cdc6 (Siddiqui et al., 2013). Establishment of the pre-RC can only proceed late in mitosis and during G1 phase, when cyclin-dependent kinase (CDK) levels are low. This is achieved by a large, multi-subunit ubiquitin ligase, the anaphase promoting complex (APC/C), which induces degradation of mitotic cyclins and of the CDK- activating phosphatase Cdc25 (King et al., 1995; Donzelli et al., 2002; Teixeira and Reed, 2013). In vertebrates, the APC/C also targets the Cdt1 inhibitor geminin for degradation (McGarry and Kirschner, 1998). p g g p Accurate control over all stages of DNA replication is of vital importance for the maintenance of genome integrity in proliferating cells. Both incomplete replication and over- replication interfere with proper chromosome segregation, and defects in replication ﬁdelity pose a serious threat to genome stability due to an increased mutation load. Hence, the mechanisms ensuring complete and accurate replication need to be considered as part of a cell’s repertoire to defend itself against insults to its genome. By reversibly altering the properties of their target proteins, various diﬀerent posttranslational protein modiﬁcations contribute signiﬁcantly to these processes. Over the past decade, we have witnessed the emergence of ubiquitin and SUMO as key regulators of genome maintenance pathways (Ulrich and Walden, 2010; Jackson and Durocher, 2013). Although best known for mediating protein degradation, ubiquitin can convey a variety of non-proteolytic signals. This can partly be ascribed to the eﬀects of mono-ubiquitylation, but also to ubiquitin’s ability to form polymeric chains of diﬀerent geometries, recognized by highly chain-selective eﬀector proteins (Komander and Rape, 2012). More recently, it has been realized that SUMO can also trigger degradation of its targets by forming polymeric SUMO chains interacting with a class of enzymes known as SUMO-targeted ubiquitin ligases (STUbLs; Prudden et al., 2007; Sriramachandran and Dohmen, 2014). Hence, both proteolytic and non-proteolytic contributions need to be considered in discussing the eﬀects of ubiquitin and SUMO on DNA replication. REPLICATION OF INTACT DNA The function of ubiquitylation in unperturbed DNA replication has been the subject of an excellent recent review (Moreno and Gambus, 2015), to which the reader is referred for details, particularly with respect to proteolytic functions of ubiquitylation. Here we complement this with information on the roles of protein SUMOylation, and we discuss the recurring problem of distinguishing modiﬁcations that are inherently part of the replication process from those occurring in response to spontaneous problems based on diﬃcult-to-replicate sequences or chromatin regions. Citation: García-Rodríguez N, Wong RP and Ulrich HD (2016) Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress. Front. Genet. 7:87. doi: 10.3389/fgene.2016.00087 May 2016 \| Volume 7 \| Article 87 1 Frontiers in Genetics \| www.frontiersin.org Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. progression, generating checkpoint and damage signals, and coordination of DNA synthesis with chromatin assembly (Leman and Noguchi, 2013). In eukaryotes, origin ﬁring follows a temporally regulated program throughout S phase, giving rise to distinct early- and late-replicating regions of the genome (Renard-Guillet et al., 2014). The pattern of origin ﬁring is ﬂexible and reacts to situations such as the stalling of individual forks or the perception of a global damage signal by the cell. DNA synthesis proceeds bi-directionally, initiated by the deposition of short RNA primers that are subsequently extended by DNA polymerase α. Leading and lagging strand replication by the main replicative DNA polymerases ε and δ, respectively, is closely coordinated with the unwinding of the template DNA. As a consequence, accumulation of extended regions of single- stranded (ss)DNA is perceived as a sign of fork stalling and triggers a checkpoint response that suppresses the ﬁring of late replication origins and prevents entry into mitosis (Leman and Noguchi, 2013). The nicks in the emerging lagging strand, arising from its discontinuous synthesis, are successively sealed by DNA ligase. As replication units (replicons) from neighboring origins meet, replication forks merge and replication is terminated by the disassembly of the replisomes. Since DNA replication takes place in the context of chromatin, removal of nucleosomes in front of the helicase and their renewed deposition after passage of the replication fork need to be synchronized with DNA synthesis (Groth, 2009). This coordination, actively mediated by components of the replisome, also protects against the loss of epigenetic marks during replication. replication elongation and termination, and with regard to chromatin assembly and nuclear structure. Another important aspect will be the response to replication stress. As much of the recent progress in the ﬁeld can be ascribed to large-scale siRNA screens and proteomic approaches, mechanistic information is often lagging behind the identiﬁcation of novel modiﬁcation targets and conjugation factors. We will therefore refrain from giving a comprehensive account of all the enzymes and substrates involved in DNA replication and rather focus on representative examples where a relevant functional context is available. Frontiers in Genetics \| www.frontiersin.org Contributions of Ubiquitin and SUMO to Origin Licensing and Replication Initiation In order to initiate S phase, two helicase coactivators – Cdc45 and the GINS complex – are recruited to pre-RCs, assembling the active replicative helicase, the CMG complex (Cdc45-Mcm2-7- GINS). Once the initial unwinding occurs, DNA polymerases and the sliding clamp, PCNA, are recruited to assemble the replisome and establish the replication fork (Leman and Noguchi, 2013). Origin ﬁring requires a rise in CDK activity. Accordingly, APC/C activity is downregulated, mainly by inhibition of its regulatory subunit Cdh1 (Eldridge et al., 2006; Fukushima et al., 2013; Lau et al., 2013), but also by autoubiquitylation and degradation of its cognate ubiquitin conjugating enzyme (E2), UbcH10, a process induced in the absence of APC/C substrates (Rape and Kirschner, 2004). This allows an accumulation of G1-speciﬁc This review will cover the functions of ubiquitin and SUMO during unperturbed replication, i.e., during origin licensing, May 2016 \| Volume 7 \| Article 87 2 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. cyclins. Additionally, CDK inhibitors, such as p27 and p21, are degraded (Starostina and Kipreos, 2012). Three diﬀerent E3s, KPC, Pirh2 and the Skp1-Cullin-F-box complex SCFSkp2, are known to act on p27 in a temporally and spatially ordered fashion during G1 and early S phase. The p21 protein is also a substrate of SCFSkp2, but in addition, this factor is targeted by an intriguing mechanism that directly couples ubiquitylation to S phase entry (Abbas et al., 2008; Kim et al., 2008). The cognate E3, Cullin-RING ligase CRL4Cdt2, recognizes its substrate only in conjunction with the replication clamp, PCNA, and only when PCNA is encircling DNA. The relevant degradation signal, which includes a PCNA-interacting peptide (PIP), is also found in other factors whose removal is associated with S phase, such as the ﬁssion yeast inhibitor of ribonucleotide reductase, Spd1, and the G1-speciﬁc transcription factor E2F1 from Drosophila melanogaster. (Havens and Walter, 2011; Ulrich, 2014). Thus, by coupling substrate recognition to binding of loaded PCNA, CRL4Cdt2 is able to read the state of the replication machinery as an activating signal. (de Albuquerque et al., 2016). Moreover, it is important to note that other components of pre-RCs have also been identiﬁed as SUMOylation targets, among them the subunits of ORC (Golebiowski et al., 2009). Contributions of Ubiquitin and SUMO to Origin Licensing and Replication Initiation Hence, it remains to be established whether the negative eﬀect of SUMO on origin ﬁring observed in this study is due to the modiﬁcation of an individual Mcm subunit, the Mcm2-7 complex in its entirety, or a general accumulation of SUMO around the pre-RC. Proteomic Analyses of Replicating Chromatin A wealth of information has emerged from the isolation of chromatin-associated proteins from proliferating cells, followed by mass spectrometry. Proteome-wide analyses identiﬁed numerous replication factors as ubiquitylation targets in human cells, including integral components of the replisome such as GINS and the Mcm2-7 helicase complex, the Replication Factor C (RFC) clamp loader complex, as well as all the replicative DNA polymerases and many associated factors (Wagner et al., 2011). Comparison of substrate spectra in the absence and presence of the proteasome inhibitor MG132 revealed both proteolytic and non-proteolytic roles of ubiquitylation. Similarly, a systematic screen in the budding yeast Saccharomyces cerevisiae identiﬁed a signiﬁcant number of replisome components targeted by SUMO, including components of the Mcm2-7 complex, subunits of DNA polymerases and the RFC complex, the Rad27 ﬂap endonuclease and topoisomerases Top1 and Top2 (Cremona et al., 2012). A recent study, using a procedure to isolate proteins on nascent DNA (iPOND) followed by mass spectrometry, characterized proteins enriched in the proximity of replisomes in an unprecedented spatial resolution. Interestingly, SUMOylation was predominant on factors near the replisome, while ubiquitylated proteins prevailed on mature chromatin (Lopez-Contreras et al., 2013). Although the implications of this distribution are not well understood, an appropriate balance appears to be important for replication and genome stability, as the ubiquitin isopeptidase USP7 was found to be responsible for maintaining SUMOylated proteins at replication forks by means of protecting them from ubiquitylation (Lecona et al., 2016). USP7 activity was found essential for origin ﬁring as well as replisome progression, and intriguingly, one of its functions appears to be the deubiquitylation of SUMO itself. Firing of origins needs to be strictly limited to once per cell cycle to avoid problems of re-replication. This is achieved through a process known as origin licensing that restricts pre- RC assembly to G1 (Moreno and Gambus, 2015). In order to render the process irreversible, essential loading factors, such as Cdt1 and Cdc6, are eliminated when cells enter S phase. In many organisms, this is again mediated by ubiquitin-mediated proteolysis. Human Cdt1 is ubiquitylated by at least two diﬀerent E3s of the CRL family, SCFSkp2 and – as described above – CRL4Cdt2 (Li et al., 2003; Zhong et al., 2003). Cdc6 is deactivated either by export from the nucleus or by degradation following its ubiquitylation by CRL4Cdt2 (Saha et al., 1998; Clijsters and Wolthuis, 2014). Proteomic Analyses of Replicating Chromatin In budding yeast, SCFCdc4 mediates ubiquitylation of Cdc6 (Drury et al., 1997). q y ( y , ) In contrast to the pervasive inﬂuence of ubiquitin, SUMO appears to exert more subtle regulatory eﬀects on replication initiation. In a cell-free system based on Xenopus laevis egg extracts, inhibition of SUMOylation was found to increase replication rates by allowing a larger number of origins to ﬁre (Bonne-Andrea et al., 2013). The negative eﬀect of SUMO on origin ﬁring was attributable to the modiﬁcation of cyclin E following recruitment of the cyclin E-CDK complex to pre-RCs. The notion that most cells only use a sub-set of their potential origins in each S phase suggests that SUMO may in this context contribute to limiting excessive origin ﬁring. In the budding yeast, Wei and Zhao (2016) recently reported an apparently unrelated phenomenon that likewise suggests a negative impact of SUMO on origin ﬁring. They observed a cell-cycle regulated SUMOylation of Mcm2-7, peaking at the pre-RC stage when the complex is loaded onto origins, but declining upon origin ﬁring at the G1-to-S transition. Artiﬁcial enhancement of local SUMOylation inhibited CMG assembly and origin ﬁring, most likely by means of recruiting a phosphatase that reversed essential phosphorylation events required for CMG activation. Intriguingly, both SUMOylation and deSUMOylation of Mcm proteins are accomplished by multiple E3s and isopeptidases in a subunit-speciﬁc manner, and signiﬁcant diﬀerences were noted in the cell cycle regulation of individual Mcm subunits Despite these observations, the functions of most replisome- associated modiﬁcations remain to be explored, and the notion that many of the SUMOylation events were found to be enriched after exposure of the cells to DNA damage (Cremona et al., 2012) raises the question of whether these modiﬁcations are inherent in the replication process or represent a response to spontaneous replication problems or low-level DNA damage. Frontiers in Genetics \| www.frontiersin.org PCNA Modiﬁcations during Unperturbed DNA Replication FIGURE 1 \| Modiﬁcations of PCNA by ubiquitin and SUMO during replication of intact and damaged DNA. (A) During unperturbed replication, budding yeast PCNA is SUMOylated at K164 and to a minor extent at K127. Modiﬁcation at K164 is mediated by SUMO E2-E3 Ubc9-Siz1. PCNASUMO recruits anti-recombinogenic helicase Srs2 to counteract Rad51 ﬁlament formation. RFC-like complex RLC-Elg1 interacts with PCNASUMO and unloads PCNA from DNA. (B) Upon replication stalling and exposure of ssDNA, E2-E3 complex Rad6-Rad18 is recruited by interaction with the RPA complex and (in yeast) SUMO and monoubiquitylates PCNA at K164. This modiﬁcation is removed by Ubp10 (yeast) or USP1 (humans). Monoubiquitylated PCNA recruits damage-tolerant DNA polymerases for translesion synthesis (TLS), while polyubiquitylated PCNA initiates template switching (TS) by a poorly deﬁned mechanism. Auxiliary factors DVC1 and SNM1A modulate TLS. DVC1 cooperates with the AAA ATPase p97 to extract polymerase η from chromatin. Mgs1/WRNIP1 and ZRANB3 bind to polyubiquitylated PCNA and might contribute to TS. Ubiquitylation of TLS polymerases prevents association with PCNAUb and may induce their degradation. FIGURE 1 \| Modiﬁcations of PCNA by ubiquitin and SUMO during replication of intact and damaged DNA. (A) During unperturbed replication, budding yeast PCNA is SUMOylated at K164 and to a minor extent at K127. Modiﬁcation at K164 is mediated by SUMO E2-E3 Ubc9-Siz1. PCNASUMO recruits anti-recombinogenic helicase Srs2 to counteract Rad51 ﬁlament formation. RFC-like complex RLC-Elg1 interacts with PCNASUMO and unloads PCNA from DNA. (B) Upon replication stalling and exposure of ssDNA, E2-E3 complex Rad6-Rad18 is recruited by interaction with the RPA complex and (in yeast) SUMO and monoubiquitylates PCNA at K164. This modiﬁcation is removed by Ubp10 (yeast) or USP1 (humans). Monoubiquitylated PCNA recruits damage-tolerant DNA polymerases for translesion synthesis (TLS), while polyubiquitylated PCNA initiates template switching (TS) by a poorly deﬁned mechanism. Auxiliary factors DVC1 and SNM1A modulate TLS. DVC1 cooperates with the AAA ATPase p97 to extract polymerase η from chromatin. Mgs1/WRNIP1 and ZRANB3 bind to polyubiquitylated PCNA and might contribute to TS. Ubiquitylation of TLS polymerases prevents association with PCNAUb and may induce their degradation. cell cycle regulation (Moldovan et al., 2007; Ulrich and Takahashi, 2013). ring-shaped complex that encircles DNA and functions as a processivity factor for DNA polymerases. PCNA Modiﬁcations during Unperturbed DNA Replication Posttranslational modiﬁcations heavily modulate the function of the eukaryotic sliding clamp. PCNA is a homotrimeric, May 2016 \| Volume 7 \| Article 87 3 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. FIGURE 1 \| Modiﬁcations of PCNA by ubiquitin and SUMO during replication of intact and damaged DNA. (A) During unperturbed replication, budding yeast PCNA is SUMOylated at K164 and to a minor extent at K127. Modiﬁcation at K164 is mediated by SUMO E2-E3 Ubc9-Siz1. PCNASUMO recruits anti-recombinogenic helicase Srs2 to counteract Rad51 ﬁlament formation. RFC-like complex RLC-Elg1 interacts with PCNASUMO and unloads PCNA from DNA. (B) Upon replication stalling and exposure of ssDNA, E2-E3 complex Rad6-Rad18 is recruited by interaction with the RPA complex and (in yeast) SUMO and monoubiquitylates PCNA at K164. This modiﬁcation is removed by Ubp10 (yeast) or USP1 (humans). Monoubiquitylated PCNA recruits damage-tolerant DNA polymerases for translesion synthesis (TLS), while polyubiquitylated PCNA initiates template switching (TS) by a poorly deﬁned mechanism. Auxiliary factors DVC1 and SNM1A modulate TLS. DVC1 cooperates with the AAA ATPase p97 to extract polymerase η from chromatin. Mgs1/WRNIP1 and ZRANB3 bind to polyubiquitylated PCNA and might contribute to TS. Ubiquitylation of TLS polymerases prevents association with PCNAUb and may induce their degradation. h d l h l d f ll l l ( ld l l h d k h h FIGURE 1 \| Modiﬁcations of PCNA by ubiquitin and SUMO during replication of intact and damaged DNA. (A) During unperturbed replication, budding yeast PCNA is SUMOylated at K164 and to a minor extent at K127. Modiﬁcation at K164 is mediated by SUMO E2-E3 Ubc9-Siz1. PCNASUMO recruits anti-recombinogenic helicase Srs2 to counteract Rad51 ﬁlament formation. RFC-like complex RLC-Elg1 interacts with PCNASUMO and unloads PCNA from DNA. (B) Upon replication stalling and exposure of ssDNA, E2-E3 complex Rad6-Rad18 is recruited by interaction with the RPA complex and (in yeast) SUMO and monoubiquitylates PCNA at K164. This modiﬁcation is removed by Ubp10 (yeast) or USP1 (humans). Monoubiquitylated PCNA recruits damage-tolerant DNA polymerases for translesion synthesis (TLS), while polyubiquitylated PCNA initiates template switching (TS) by a poorly deﬁned mechanism. Auxiliary factors DVC1 and SNM1A modulate TLS. DVC1 cooperates with the AAA ATPase p97 to extract polymerase η from chromatin. Mgs1/WRNIP1 and ZRANB3 bind to polyubiquitylated PCNA and might contribute to TS. Ubiquitylation of TLS polymerases prevents association with PCNAUb and may induce their degradation. PCNA Modiﬁcations during Unperturbed DNA Replication In addition, PCNA serves as an interaction platform for numerous factors involved in DNA replication, repair, chromatin dynamics, cohesion and During unperturbed replication, budding yeast PCNA is modiﬁed by SUMO at a highly conserved lysine, K164, and May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 4 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. to a minor extent at K127 (Hoege et al., 2002; Figure 1A). Modiﬁcation at K164 is mediated by the SUMO E2 Ubc9 in combination with the SUMO E3 Siz1 and is triggered by loading of the clamp onto DNA (Parker et al., 2008). SUMOylation at K127 in vivo requires Siz2 (Parker et al., 2008). The modiﬁcation enhances interaction with an antirecombinogenic helicase, Srs2, at replication forks. Srs2 interacts with PCNASUMO via its carboxy-terminal tail containing a PIP-like PCNA interaction motif adjacent to a canonical SUMO interacting motif (SIM; Pfander et al., 2005; Armstrong et al., 2012). Recruitment of Srs2 prevents unwanted homologous recombination (HR) by disrupting Rad51 ﬁlaments (Krejci et al., 2003; Veaute et al., 2003; Papouli et al., 2005; Pfander et al., 2005). In addition, the presence of SUMO on PCNA boosts the damage-induced activity of the ubiquitin ligase Rad18 toward PCNA, again through a SIM in the E3 sequence (Parker and Ulrich, 2012). As a consequence, upon encounter of replication-stalling DNA lesions, damage processing is channeled into a bypass pathway that depends on PCNA ubiquitylation (Figure 1B, and see below). Hence, PCNA SUMOylation appears to function as a pre-emptive defense measure to inﬂuence pathway choice in response to replication stress. The modiﬁcation also appears to enhance interaction with an alternative clamp loader complex, RLC-Elg1, which has been proposed to mediate PCNA unloading during replication (Parnas et al., 2010; Kubota et al., 2013). However, SUMOylation is not essential for Elg1 action on PCNA. SUMOylation at K164 has been observed not only in budding yeast, but also in X. laevis egg extracts, chicken DT40 cells and, more recently, in mammalian cells (Leach and Michael, 2005; Arakawa et al., 2006; Gali et al., 2012; Moldovan et al., 2012). In human cells, expression of a PCNA-SUMO fusion Replication Termination y All replicative DNA polymerases have been identiﬁed as ubi- quitin and/or SUMO targets in budding yeast and mammalian cells (Wagner et al., 2011; Cremona et al., 2012). Mammalian DNA polymerase δ, responsible mainly for lagging strand synthesis, consists of four subunits (Hubscher et al., 2002), two of which, p12 and p66, are ubiquitylated during a normal S phase without leading to proteasomal degradation (Liu and Warbrick, 2006). Additionally, p66 is modiﬁed by SUMO at two diﬀerent residues, K258 and K433 (Liu and Warbrick, 2006). Although the biological signiﬁcance of these modiﬁcations remains unclear, it has been proposed that they might regulate protein–protein interactions within the polymerase complex or with other replication factors (Liu and Warbrick, 2006). A study in Schizosaccharomyces pombe showed that the catalytic subunit of the leading strand polymerase ε, Pol2, is polyubiquitylated and undergoes signiﬁcant proteasome-dependent degradation during unperturbed S phase, involving the ubiquitin ligase SCFPof3 (the homolog of budding yeast SCFDia2; Roseaulin et al., 2013). In contrast, Pol3, the catalytic subunit of polymerase δ, remained stable despite being ubiquitylated. The authors propose that the high rate of Pol2 turnover might ensure a continuous supply of “fresh” polymerase at the leading strand, while the discontinuous nature of lagging strand synthesis would not require an active exchange mechanism (Roseaulin et al., 2013). It will be interesting to address whether polymerase ε degradation serves a regulatory or a quality control purpose, and whether the phenomenon is conserved in other organisms. Convergence of two replication forks leads to replication termination via disassembly of the replicative helicase. This process must be tightly controlled, as the CMG complex cannot be reloaded after initiation and must remain associated with the replication fork until completion of the replication unit, the replicon. However, in contrast to replication initiation, the mechanism of replisome disassembly is not well understood. Two recent reports have helped to shed light on this reaction in budding yeast and X. laevis egg extracts, uncovering a key role for the ubiquitin system (Figure 2; Maric et al., 2014; Moreno et al., 2014). Helicase disassembly is triggered by K48- polyubiquitylation of the helicase subunit Mcm7 by a member of the SCF family of ubiquitin ligases. In budding yeast, the relevant F-box protein is Dia2 (Maric et al., 2014). FIGURE 2 \| Replication termination via ubiquitin-mediated CMG helicase extraction. A model, derived from observations in budding yeast and Xenopus laevis egg extracts, proposes ubiquitylation of Mcm7 by the replisome-associated E3 SCFDia2 at the sites of replication termination where two forks converge. The CMG helicase (Mcm2-7, Cdc45, and GINS) is subsequently extracted from the chromatin by Cdc48/p97 in a ubiquitin-dependent manner. FIGURE 2 \| Replication termination via ubiquitin-mediated CMG helicase extraction. A model, derived from observations in budding yeast and Xenopus laevis egg extracts, proposes ubiquitylation of Mcm7 by the replisome-associated E3 SCFDia2 at the sites of replication termination where two forks converge. The CMG helicase (Mcm2-7, Cdc45, and GINS) is subsequently extracted from the chromatin by Cdc48/p97 in a ubiquitin-dependent manner. FIGURE 2 \| Replication termination via ubiquitin-mediated CMG helicase extraction. A model, derived from observations in budding yeast and Xenopus laevis egg extracts, proposes ubiquitylation of Mcm7 by the replisome-associated E3 SCFDia2 at the sites of replication termination where two forks converge. The CMG helicase (Mcm2-7, Cdc45, and GINS) is subsequently extracted from the chromatin by Cdc48/p97 in a ubiquitin-dependent manner. FIGURE 2 \| Replication termination via ubiquitin-mediated CMG helicase extraction. A model, derived from observations in budding yeast and Xenopus laevis egg extracts, proposes ubiquitylation of Mcm7 by the replisome-associated E3 SCFDia2 at the sites of replication termination where two forks converge. The CMG helicase (Mcm2-7, Cdc45, and GINS) is subsequently extracted from the chromatin by Cdc48/p97 in a ubiquitin-dependent manner. May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. Modiﬁcation of Mcm10 Similarly, PCNA monoubiquitylation has been detected during replication of undamaged DNA in X. laevis egg extracts and was found to be required for eﬃcient chromosomal replication (Leach and Michael, 2005). It is currently unclear, however, whether PCNA ubiquitylation contributes to the normal replication process itself or rather reﬂects higher levels of endogenous damage or fork problems in these systems. Modiﬁcation of Mcm10 protein inhibits spontaneous as well as damage-induced HR (Gali et al., 2012). Furthermore, a novel PCNA-interacting factor, the helicase PARI, has been suggested to function analogously to Srs2 in humans: it contains PIP and SIM motifs for interaction with PCNASUMO and suppresses HR by removing Rad51 from DNA (Moldovan et al., 2012). However, SUMOylated PCNA is present at very low levels in mammalian when compared to yeast cells, and its detection requires overexpression of epitope- tagged SUMO alleles (Gali et al., 2012). Whether this reﬂects the need for a tighter regulation of the process in the yeast system with its naturally higher rate of recombination remains to be explored. The essential, conserved minichromosome maintenance protein 10 (Mcm10) facilitates initiation of DNA replication. The protein is loaded onto replication origins at the G1/S transition, where it promotes strand separation either by activating the helicase or by stabilizing the formation of ssDNA, but it is dispensable for assembly of the helicase itself (Kanke et al., 2012; van Deursen et al., 2012; Thu and Bielinsky, 2013). A contribution of Mcm10 to the elongation step of DNA replication remains controversial (Thu and Bielinsky, 2013). Mcm10 has been shown to interact with the catalytic subunit of DNA polymerase α (Pol1) and regulate its stability, suggesting a role of Mcm10 in lagging strand synthesis (Ricke and Bielinsky, 2004). Ricke and Bielinsky (2004) reported that a small fraction of Mcm10 is monoubiquitylated at two distinct lysine residues during G1 and S phase of the cell cycle (Das-Bradoo et al., 2006). The modiﬁcation promotes interaction with PCNA, but inhibits binding of Mcm10 to polymerase α. Moreover, mutations within Mcm10’s PIP box render cells inviable, suggesting that the interaction between Mcm10 and PCNA is essential (Das-Bradoo et al., 2006). Based on these ﬁndings, it was speculated that ubiquitylation of Mcm10 might induce a conformational change to expose its PIP box, thus allowing interaction with PCNA and release of polymerase α after the priming event. This might in turn facilitate the recruitment of polymerase δ and thereby Okazaki fragment extension (Das- Bradoo et al., 2006; Thu and Bielinsky, 2013). p In response to DNA damage, PCNA is mono- and polyubiquitylated at K164 (Hoege et al., 2002), which facilitates the bypass of replication-blocking lesions (see below). In ﬁssion yeast, however, these modiﬁcations are observed during S phase even in the absence of exogenous DNA-damaging agents (Frampton et al., 2006). UBIQUITIN AND SUMO IN DNA REPLICATION STRESS g p Homologous recombination serves as a means to repair DNA DSBs, to promote exchange of genetic material and proper chromosome segregation during meiotic cell divisions, and to rescue stalled or collapsed replication forks (Krejci et al., 2012). The process is initiated by strand breaks or – in particular at stalled replication forks – regions of ssDNA, tightly bound by the Replication Protein A (RPA) complex. RPA is exchanged for the recombination factor RAD51. In yeast, this step is promoted by the RAD52 protein. In human cells, the exchange is mainly mediated by BRCA2 (Jensen et al., 2010; Liu J. et al., 2010; Thorslund et al., 2010). The RAD51- ssDNA ﬁlament invades dsDNA, forming a so-called D-loop, and exchange of genetic material proceeds via a combination of DNA synthesis, branch migration and resolution or dissolution of recombination intermediates by the action of nucleases. Numerous auxiliary factors, among them DNA helicases and DNA-dependent ATPases, modulate HR activity either positively or negatively at every step (Krejci et al., 2012), and many of them are modulated in their activities by ubiquitin and/or SUMO. Replication stress is broadly deﬁned as a condition that interferes with replication fork progression (Zeman and Cimprich, 2014). It is caused by a range of intrinsic or exogenous factors, including polymerase inhibition or nucleotide depletion, imbalances in the levels of replication proteins, interference from ongoing transcription, incorporation of ribonucleotides, or physical barriers to the DNA polymerases, such as sequences inherently prone to form secondary structures, tightly bound proteins, or DNA lesions arising from chemical alterations or strand breaks. Conditions that impair the replicative DNA polymerases without impeding strand unwinding by the helicase result in an accumulation of ssDNA. This in turn initiates a replication- speciﬁc checkpoint response via the protein kinase ATR in order to stabilize stalled replication intermediates, suppress the ﬁring of late origins and prevent entry into mitosis (Jossen and Bermejo, 2013). Depending on the nature of the blockage, ATR signaling promotes replication fork rescue or restart in one of several ways, for example by means of re-priming downstream of the problematic region, fork reversal, translesion synthesis or strand exchange between the sister chromatids (Jossen and Bermejo, 2013; Leman and Noguchi, 2013). Prolonged replication fork stalling or lack of an appropriate checkpoint response can cause replication fork collapse. Proteomic Analyses of the DNA Replication Stress Response of the helicase is currently unknown. One possible scenario is that ubiquitylation is triggered by a DNA-mediated signal: while during replication the CMG helicase encircles ssDNA, it must enclose dsDNA upon termination (Bell, 2014). The mechanism of CMG helicase disassembly is likely to be conserved in higher eukaryotes. Homologs of Dia2 have yet to be identiﬁed, but other ubiquitin ligases might be involved in the process. Notably, disassembly of the CMG helicase requires the ubiquitin- dependent segregase p97, also called VCP (in yeast: Cdc48; Maric et al., 2014; Moreno et al., 2014), an AAA ATPase that remodels and thus extracts ubiquitylated proteins from protein complexes, membranes or chromatin, in many cases presenting them for proteasomal degradation (Vaz et al., 2013; Franz et al., 2016). Inactivation of p97 led to the accumulation of ubiquitylated forms of CMG on the chromatin, while inhibition of the proteasome did not block CMG disassembly (Maric et al., 2014; Moreno et al., 2014). Thus, whether Mcm7Ub is degraded after extraction remains to be seen, although the K48-linkage of the polyubiquitin chain on Mcm7 would imply proteasomal action. of the helicase is currently unknown. One possible scenario is that ubiquitylation is triggered by a DNA-mediated signal: while during replication the CMG helicase encircles ssDNA, it must enclose dsDNA upon termination (Bell, 2014). The mechanism of CMG helicase disassembly is likely to be conserved in higher eukaryotes. Homologs of Dia2 have yet to be identiﬁed, but other ubiquitin ligases might be involved in the process. Notably, disassembly of the CMG helicase requires the ubiquitin- dependent segregase p97, also called VCP (in yeast: Cdc48; Maric et al., 2014; Moreno et al., 2014), an AAA ATPase that remodels and thus extracts ubiquitylated proteins from protein complexes, membranes or chromatin, in many cases presenting them for proteasomal degradation (Vaz et al., 2013; Franz et al., 2016). Inactivation of p97 led to the accumulation of ubiquitylated forms of CMG on the chromatin, while inhibition of the proteasome did not block CMG disassembly (Maric et al., 2014; Moreno et al., 2014). Thus, whether Mcm7Ub is degraded after extraction remains to be seen, although the K48-linkage of the polyubiquitin chain on Mcm7 would imply proteasomal action. Proteomic Analyses of the DNA Replication Stress Response p p A number of large-scale proteomic studies and systematic analyses of chromatin-associated factors have illustrated the dynamics of ubiquitylation and SUMOylation speciﬁcally in response to replication stress (Povlsen et al., 2012; Bursomanno et al., 2015; Xiao et al., 2015). According to those studies, when replication forks encounter DNA lesions, a plethora of SUMO and ubiquitin modiﬁcations on multiple factors is upregulated to either protect replication forks or initiate DNA repair mechanisms. In many cases, their consequences are mechanistically and functionally not well characterized, and it is clear today that modiﬁcation of entire protein groups is sometimes more important than ubiquitylation or SUMOylation of individual factors (Psakhye and Jentsch, 2012). Moreover, a clear distinction between replication stress triggered by fork stalling and a full-blown damage response that might result from subsequent fork collapse has not always been attempted. Replication Termination Interestingly, SCFDia2 had previously been identiﬁed as a component of the replication progression complex (RPC), tethered to the Ctf4 and Mrc1 subunits via a TPR domain within Dia2 (Mimura et al., 2009; Morohashi et al., 2009). SCFDia2 was also proposed to mediate degradation of Ctf4 and Mrc1 (Mimura et al., 2009); however, a recent study has challenged this model and reported instead that SCFDia2 tethering to the RPC is important for the eﬃcient ubiquitylation of Mcm7 (Maculins et al., 2015). Inhibition of replication fork progression prevents Mcm7 ubiquitylation, suggesting that Mcm7 ubiquitylation is restricted to terminating replisomes (Maric et al., 2014; Moreno et al., 2014). How cells distinguish these from elongating complexes to avoid premature ubiquitylation and disassembly May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 6 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. UBIQUITIN AND SUMO IN DNA REPLICATION STRESS This poorly deﬁned event may include a dissociation of the replisome and/or the formation of strand breaks, caused either passively or by the action of nucleases. Importantly, fork collapse triggers the transition to a genuine DNA damage response, mediated by the checkpoint kinase ATM, as is generally observed in response to DSBs. Over the past decade it has become clear that ubiquitin and SUMO are key regulators of both the replication- and the damage-associated branches of the checkpoint response (Ulrich, 2012; Jackson and Durocher, 2013). Frontiers in Genetics \| www.frontiersin.org Replication Protein A (RPA) However, whether RPA1SUMO is indeed the in vivo target of this SIM relevant for recruitment of RAD51 to damage sites needs to be demonstrated, considering that SUMOylation of other proteins might act synergistically or redundantly in the assembly of repair complexes (Psakhye and Jentsch, 2012). The yeast homolog of RPA1, Rfa1, is also modiﬁed by SUMO upon treatment with the alkylating agent methyl- methanesulfonate (MMS; Burgess et al., 2007; Cremona et al., 2012), although the functional signiﬁcance of this modiﬁcation remains unclear. might promote the exchange of RPA1 for RAD51 on ssDNA, stimulating HR (Galanty et al., 2012). This mechanism and the recruitment of RAD51 through RPASUMO are not mutually exclusive, as both could cooperate in promoting RAD51 ﬁlament formation (Figure 3A). However, the direct ubiquitylation of SUMO-modiﬁed RPA1 by RNF4 has yet to be demonstrated. On the other hand, RPA is also ubiquitylated under conditions complementary to those that trigger its SUMOylation (Figure 3B). Ubiquitylation on multiple sites of all three RPA subunits was observed in response to replication fork stalling upon UV irradiation or treatment with other fork-stalling agents such as 4-nitroquinoline oxide or HU, but not after exposure to ionizing radiation (Elia et al., 2015). Thus, apparently cells respond to diﬀerent types of damage in distinct ways, either ubiquitylating or SUMOylating RPA. Ubiquitylation of RPA, mediated by the E3 RFWD3, does not lead to proteasomal degradation. Inhibition of the modiﬁcation by RFWD3 depletion or by means of a ubiquitylation-deﬁcient RPA mutant caused defects in fork restart and persistence of γ-H2AX foci after release from prolonged HU treatment, as well as a reduction in HR in response to both fork stalling and direct induction of DSBs. These ﬁndings imply that RFWD3-dependent ubiquitylation of RPA promotes fork stability and HR-mediated restart of collapsed forks upon exposure to replication stress (Elia et al., 2015). The exact mechanism by which RPAUb stimulates these eﬀects remains elusive, even though it has been suggested that the modiﬁcation may promote release of the RPA complex from DNA and/or facilitate the recruitment of HR factors, similar to RPASUMO (Elia et al., 2015). Whether ubiquitin and SUMO can coexist on the same RPA complex remains to be explored. An independent study identiﬁed another ubiquitin ligase, PRP19, as the E3 responsible for RPA ubiquitylation (Marechal et al., 2014). More recently, Galanty et al. Replication Protein A (RPA) Replication protein A is a ssDNA-binding protein complex with a central role as a scaﬀold in virtually all DNA transactions. In eukaryotes, RPA consists of three subunits: RPA1, RPA2, and RPA3 (Zou et al., 2006). In mammals, the largest subunit, RPA1, is stably associated with the Sentrin/SUMO- speciﬁc protease SENP6 during S phase, which keeps RPA1 in a hypoSUMOylated state (Dou et al., 2010; Figure 3A). In response to replication-mediated or radiation-induced DSBs, SENP6 dissociates, resulting in modiﬁcation of RPA1 with SUMO through the action of unknown SUMO ligases. Two lysine residues were identiﬁed as SUMO acceptor sites: K449 was modiﬁed by a poly-SUMO chain, whereas K577 was mono- SUMOylated. Importantly, treatment with hydroxyurea (HU) or May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 7 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. UV irradiation, which stalls replication forks without causing DSBs, did not alter the association between SENP6 and RPA1. SUMOylation of RPA enhanced its interaction with RAD51 in vitro and promoted HR in vivo (Figure 3A). Taken together, RPA1SUMO seems to facilitate recruitment of RAD51 to collapsed forks and DSBs, thereby initiating HR (Dou et al., 2010). Interestingly, RAD51 contains a SIM motif that is necessary for its accumulation at damage sites (Shima et al., 2013). However, whether RPA1SUMO is indeed the in vivo target of this SIM relevant for recruitment of RAD51 to damage sites needs to be demonstrated, considering that SUMOylation of other proteins might act synergistically or redundantly in the assembly of repair complexes (Psakhye and Jentsch, 2012). The yeast homolog of RPA1, Rfa1, is also modiﬁed by SUMO upon treatment with the alkylating agent methyl- methanesulfonate (MMS; Burgess et al., 2007; Cremona et al., 2012), although the functional signiﬁcance of this modiﬁcation remains unclear. UV irradiation, which stalls replication forks without causing DSBs, did not alter the association between SENP6 and RPA1. SUMOylation of RPA enhanced its interaction with RAD51 in vitro and promoted HR in vivo (Figure 3A). Taken together, RPA1SUMO seems to facilitate recruitment of RAD51 to collapsed forks and DSBs, thereby initiating HR (Dou et al., 2010). Interestingly, RAD51 contains a SIM motif that is necessary for its accumulation at damage sites (Shima et al., 2013). BLM The RecQ DNA helicase BLM plays an important role in genome maintenance by facilitating HR-mediated DNA repair in various ways (Bohm and Bernstein, 2014). BLM protein levels are regulated during the cell cycle, being lowest in G1 and peaking in late S phase (Dutertre et al., 2000). BLM normally resides in promyelocytic leukaemia (PML) nuclear bodies but re-localizes to stalled replication forks in response to DNA damage (Sengupta et al., 2003). At replication forks, BLM can exert both pro- and anti-recombinogenic functions (Figure 4): it protects replication forks by suppressing the formation of aberrant recombination events or, upon fork collapse, it promotes repair by HR. Posttranslational modiﬁcations of BLM by ubiquitin and/or SUMO make key contributions to the regulation of these processes (Bohm and Bernstein, 2014). Monoubiquitylation of BLM in the absence of DNA damage appears to be important for its normal localization in PML nuclear bodies (Figure 4A). Following HU treatment, BLM is further polyubiquitylated with K63-linked chains at K105, K225, and K259 by the E3s RNF8 and RNF168. Polyubiquitylation of BLM was found to be required for its recruitment to stalled replication forks, mediated via interaction with the ubiquitin- interacting motifs of the adaptor protein RAP80 (Tikoo et al., 2013). Once at stalled replication forks, BLM suppresses excessive HR (Tikoo et al., 2013) by dismantling RAD51-ssDNA ﬁlaments and disrupting D-loops (Bugreev et al., 2007). Polyubiquitylation of BLM might also potentiate the protein’s anti-recombinogenic eﬀect. However, constitutive association of BLM with chromatin, achieved by fusion with histone H2AX or the FHA domain of MDC1, was suﬃcient to suppress the elevated levels of HR caused by depletion of either RNF8 or RNF168, indicating that polyubiquitylation of BLM might function more as a means to recruit rather than to activate the protein (Tikoo et al., 2013). Sgs1 modiﬁcations also appear to impinge on the protein’s subcellular localization (Bohm et al., 2015): During S phase, Sgs1 forms nuclear foci that likely indicate spontaneous recombination events, as they increase with ionizing radiation treatment. Upon replication fork stalling by nucleotide depletion, the number of these foci is strongly reduced in a manner depending on the STUbL Slx5/8 – suggesting that STUbL- mediated ubiquitylation contributes to removing Sgs1 from stalled forks, thus possibly preventing unwanted recombination. However, as overall Sgs1 levels do not decrease, the process does not appear to involve degradation of the helicase, but rather its re- localization. Replication Protein A (RPA) (2012) posited a plausible mechanism for the transition from RPA to RAD51on ssDNA, relying on the SUMO-targeted ubiquitin ligase (STUbL) RNF4: in RNF4-depleted cells RAD51 fails to accumulate and RPA persists at lesions. A SUMOylation-defective RPA1 mutant exhibited a similar behavior. Based on these ﬁndings, RNF4 was proposed to target RPA1SUMO for proteasomal degradation (Figure 3A). Consistent with this model, RNF4 and RPA1 coimmunoprecipitated in a manner dependent on the SIM region of RNF4, suggesting that RNF4 directly recognizes RPA1SUMO as a ubiquitylation target. As a consequence, RPA1 accumulates in RNF4-depleted cells after exposure to DNA damage, and several proteasome subunits become detectable at damage sites in an RNF4-dependent manner. Thus, RNF4-mediated RPA1 turnover FIGURE 3 \| Ubiquitylation and SUMOylation of RPA under conditions of replicative stress. (A) SUMOylation of RPA1 is counteracted by the protease SENP6 during unperturbed S phase. In response to replication-mediated DSBs, SENP6 dissociates, allowing SUMOylation of RPA1 in order to facilitate HR via recruitment of RAD51. In addition, RPASUMO is recognized by the STUbL RNF4, which mediates proteasomal turnover of RPA1, thereby promoting exchange of RPA1 for RAD51. (B) Upon replication fork stalling, the ubiquitin E3 RFWD3 ubiquitylates RPA at multiple sites, thereby promoting replication fork restart and HR repair. FIGURE 3 \| Ubiquitylation and SUMOylation of RPA under conditions of replicative stress. (A) SUMOylation of RPA1 is counteracted by the protease SENP6 during unperturbed S phase. In response to replication-mediated DSBs, SENP6 dissociates, allowing SUMOylation of RPA1 in order to facilitate HR via recruitment of RAD51. In addition, RPASUMO is recognized by the STUbL RNF4, which mediates proteasomal turnover of RPA1, thereby promoting exchange of RPA1 for RAD51. (B) Upon replication fork stalling, the ubiquitin E3 RFWD3 ubiquitylates RPA at multiple sites, thereby promoting replication fork restart and HR repair. May 2016 \| Volume 7 \| Article 87 8 Frontiers in Genetics \| www.frontiersin.org Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. In this study, depletion of PRP19 was found to reduce damage- induced RPA ubiquitylation and compromise the accumulation of the ATR-ATRIP checkpoint complex at sites of damage. In addition, ATRIP was reported to exhibit an aﬃnity for K63-linked ubiquitin chains, suggesting that this modiﬁcation on RPA might contribute to the recruitment of ATR-ATRIP (Marechal et al., 2014). Frontiers in Genetics \| www.frontiersin.org Replication Protein A (RPA) However, these ﬁndings have been called into question by the observation of an unintentional side eﬀect of the siRNAs used for the depletion of PRP19, on exogenously expressed ubiquitin (Elia et al., 2015). Hence, an involvement of PRP19 in RPA ubiquitylation needs to be reconﬁrmed. et al., 2009). However, in contrast to its ubiquitylation, its SUMOylation was not required for the traﬃcking of BLM itself to stalled forks (Ouyang et al., 2009). Thus, BLM SUMOylation might function as a molecular switch to regulate its activity: unSUMOylated, polyubiquitylated BLM is recruited to stalled replication forks, protecting them from deleterious HR, while BLMSUMO facilitates HR by promoting RAD51 recruitment to collapsed forks (Figure 3; Ouyang et al., 2009). Future studies will certainly provide insight into the molecular mechanism by which these modiﬁcations regulate BLM function. Little is known about posttranslational modiﬁcations of the BLM ortholog in budding yeast, Sgs1. In the absence of Sgs1, cells accumulate Rad51-dependent cruciform structures at damaged replication forks (Liberi et al., 2005). The same is observed in mutants of the SUMO-conjugating enzyme, ubc9 (Branzei et al., 2006), and interestingly, Sgs1 is indeed a target of SUMOylation, suggesting the possibility that the modiﬁcation might be important to prevent the accumulation of aberrant recombinogenic structures during replication of damaged templates (Branzei et al., 2006). However, in contrast to BLM modiﬁcation, SUMOylation of Sgs1 does not seem to inﬂuence recombination frequencies (Lu et al., 2010). BLM The mechanism is likely conserved, since BLMSUMO is also targeted by the mammalian STUbL RNF4 (Galanty et al., 2012). Thus, SUMOylation of BLM/Sgs1 seems essential for the ﬁne-tuning of the protein’s function: it facilitates HR repair at collapsed forks, but it also induces removal of the protein from stalled forks, adding an additional level of regulation. SRS2 A recent study by Urulangodi et al. (2015) has uncovered a new mechanism promoting local recombination at sites of compromised replication in budding yeast. As described above, PCNASUMO recruits the helicase Srs2 to prevent unwanted recombination during unperturbed S phase. Hence, removal of Srs2 should be critical in order to engage HR after fork stalling. Urulangodi et al. (2015) identiﬁed Esc2, a protein containing two SUMO-like domains (SLDs), as a new factor associated with stalled replication forks and controlling Srs2 levels. Via its SLDs, Esc2 interacts with the SIM of Srs2, thereby promoting interaction of Srs2 with the STUbL complex Slx5/8 and subsequent degradation by the proteasome. Consistent with these ﬁndings, Srs2 SUMOylation is induced by DNA damage (Saponaro et al., 2010). Thus, local down- regulation of Srs2 appears to enable recruitment of Rad51 and thereby HR-mediated rescue of stalled forks (Urulangodi In addition to being ubiquitylated, BLM is modiﬁed by SUMO at multiple sites, preferentially at K317 and K331 (Eladad et al., 2005). Expression of a SUMOylation-defective BLM mutant induces an excess of γ-H2AX foci, DSBs and cell death under conditions of replication stress, such as prolonged HU treatment, uncovering a role of BLM SUMOylation in protecting and/or restarting replication forks (Figure 4B). Interestingly, cells unable to SUMOylate BLM also fail to recruit RAD51 and to induce HR at stalled replication forks (Ouyang et al., 2009). In fact, as described for RPA (Dou et al., 2010), SUMOylation of the helicase enhances binding to RAD51 in vitro (Ouyang May 2016 \| Volume 7 \| Article 87 9 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. FIGURE 4 \| Regulation of BLM activity under conditions of replicative stress. (A) Upon replication fork stalling, BLM is polyubiquitylated by the ubiquitn E3s RNF8 and RNF168. Polyubiquitylated BLM is recognized by RAP80, which mediates relocation of BLM from PML nuclear bodies and recruitment to stalled forks. At the fork BLM suppresses unwanted HR events. (B) Upon collapse of a stalled fork, BLM is SUMOylated, thereby facilitating the recruitment of RAD51 and repair by HR. FIGURE 4 \| Regulation of BLM activity under conditions of replicative stress. (A) Upon replication fork stalling, BLM is polyubiquitylated by the ubiquitn E3s RNF8 and RNF168. Polyubiquitylated BLM is recognized by RAP80, which mediates relocation of BLM from PML nuclear bodies and recruitment to stalled forks. At the fork BLM suppresses unwanted HR events. SLX4 The binding of a multitasking protein to either SUMO or ubiquitin can modulate its function by conveying diﬀerent contextual speciﬁcities. An example is provided by the scaﬀold protein SLX4, which coordinates multiple DNA repair pathways through its ability to bind several nucleases. Human SLX4 contains two ubiquitin-binding zinc ﬁnger (UBZ) domains that are essential for its role in the FA pathway, facilitating repair of DNA interstrand crosslinks (ICLs; see below and Coleman and Huang, 2016). In addition, SLX4 contains a cluster of SIMs, which recognizes SUMO chains. How much this cluster contributes to the repair of ICLs is not entirely clear, as expression of a SIM-defective mutant of SLX4 was able to eﬀectively rescue the mitomycin C (MMC) sensitivity of SLX4-deﬁcient human cells (Guervilly et al., 2015; Ouyang et al., 2015), whereas rescue was only partial in mouse cells (Gonzalez-Prieto et al., 2015). However, the SIM cluster is exclusively required for SRS2 (B) Upon collapse of a stalled fork, BLM is SUMOylated, thereby facilitating the recruitment of RAD51 and repair by HR. et al., 2015). In addition, it has been shown in vitro that Srs2 SUMOylation and interaction with PCNASUMO are mutually inhibitory (Kolesar et al., 2012), suggesting that Esc2 might help to dismantle the association between PCNASUMO and Srs2. Upon dissociation from PCNASUMO, Srs2 would be free to undergo SUMOylation, which would disfavor re-association with PCNASUMO. Alternatively, Esc2 might bypass the need for Srs2 SUMOylation by acting as a platform to recruit Slx5/8 to its substrate via physical interaction of Esc2 with Slx5 (Urulangodi et al., 2015). eﬃcient recruitment and retention of SLX4 to laser-induced DNA damage sites, where it might enhance the association of SLX4 with multiple SUMOylated targets, including RPA and MRN (Gonzalez-Prieto et al., 2015; Ouyang et al., 2015). Once at stalled forks, SLX4 might, for instance, promote replication fork restart when associated with the endonuclease MUS81 (Hanada et al., 2007). Interestingly, the SIMs perform yet another, unexpected function: by interacting with SUMO-charged Ubc9, they promote the SUMOylation of SLX4 itself and its binding partner, XPF. This presumed SUMO ligase activity appears to be toxic under some conditions, as mild overexpression of SLX4, but not mutation of the SIM or BTB domain, sensitizes cells to replication fork stalling upon HU treatment and promotes DSBs. In contrast, E3 activity was found to be required to prevent mitotic catastrophe at chromosome fragile sites, suggesting that promotion of DSB formation might be beneﬁcial in diﬃcult-to-replicate regions of the genome (Guervilly et al., 2015). Additional work will be needed to understand how the SUMO ligase activity of SLX4 contributes to genome stability and whether it can target other substrates besides SLX4 and XPF. Nse2/Mms21, a SUMO ligase associated with the Smc5/6 complex Nse2/Mms21, a SUMO ligase associated with the Smc5/6 complex Nse2 associates with the coiled-coil domain of Smc5 via an essential N-terminal domain. In contrast, its C-terminal SUMO E3 domain is dispensable for survival, but important for resistance to DNA damage (McDonald et al., 2003; Andrews et al., 2005; Potts and Yu, 2005; Zhao and Blobel, 2005; Duan et al., 2009). Cells lacking the Nse2 SUMO E3 activity accumulate recombination intermediates following DNA replication stress, similar to smc5/6 hypomorphic mutants (Branzei et al., 2006; Chavez et al., 2010). This suggests that the Smc5/6 complex responds to DNA damage primarily through its associated SUMOylation activity. g ( g ) In most organisms, all subunits of the Smc5/6 complex are essential for cell survival. Hypomorphic mutants of Smc5/6 show mark sensitivity to perturbation of replication such as reduced dNTP levels and DNA damage (Murray and Carr, 2008; Stephan et al., 2011). Moreover, Smc5/6 localizes to natural replication pausing sites such as rDNA, centromeres and telomeres, and to collapsed forks (Ampatzidou et al., 2006; Lindroos et al., 2006; Menolﬁet al., 2015). It has been shown that HR intermediates such as X-shaped molecules accumulate in Smc5/6 mutants in the course of replication in yeast. This leads to lethality in mitosis due to failure of these mutants to properly segregate their chromosomes (Ampatzidou et al., 2006; Branzei et al., 2006; Chen et al., 2009; Irmisch et al., 2009; Bermudez-Lopez et al., 2010; Choi et al., 2010). Interestingly, restricting Smc5/6- activity to G2, i.e., after completion of genome replication, is compatible with survival (Bermudez-Lopez et al., 2010; Menolﬁ et al., 2015). These data suggest that Smc5/6 is important for resolving recombination structures formed during DNA replication. A recent study has provided new insight into the activation mechanism of Nse2’s SUMO E3 activity toward its mostly chromatin-bound targets. Bermudez-Lopez et al. (2015) reported that ATP binding by the globular head of Smc5 induces a conformational change in the coiled-coil region, which was found to enhance E3 activity of Nse2. This mechanism appears to couple the loading of Smc5/6 onto chromatin to the activation of its enzymatic activity and suggests that the Smc5/6 complex as a whole behaves like a giant SUMO E3. In contrast to the FIGURE 5 \| Ubiquitylation and SUMOylation targets of Smc5/6 and their associated processes. The Smc5/6 complex harbors SUMO ligase (Nse2) and ubiquitin ligase (Nse1-Nse3) subunits. Structural Maintenance of Chromosomes 5 and 6 (Smc5/6) The structural maintenance of chromosomes 5 and 6 (Smc5/6) complex (Figure 5) belongs to a family of multisubunit ATPases that also includes cohesion and condensin. The complex consists of eight subunits, Smc5, Smc6 and six non-Smc element (Nse) May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 10 García-Rodríguez et al. Ubiquitin and SUMO in DNA Replication subunits, Nse1– 6 (Murray and Carr, 2008). Smc5 and Smc6 adopt extended coiled-coil structures with globular heads at the C- and N-termini that form an ATPase domain. It is believed that Smc5/6, like the related cohesin and condensin complexes, is able to embrace DNA double-strands and thereby inﬂuence higher chromatin organization. Consistent with this idea, Smc5/6 has been shown to sequester sister chromatid intertwinings and assist replication fork rotation to relieve super-helical tension generated as DNA unwinds ahead of the fork (Kegel et al., 2011). One of the Smc5/6 subunits, Nse2, also known as Mms21, is known to be a SUMO ligase (Andrews et al., 2005; Potts and Yu, 2005; Zhao and Blobel, 2005), whereas Nse1 was proposed and subsequently shown to harbor ubiquitin ligase activity (Pebernard et al., 2008; Doyle et al., 2010). Nse2/Mms21, a SUMO ligase associated with the Smc5/6 complex DNA binding stimulates the ATPase domains at the globular heads of Smc5/6, inducing a conformational change in the coiled-coil region of Smc5 that activates the SUMO E3 activity of Nse2. Smc5/6 likely selects its ubiquitylation and/or SUMOylation targets at relevant loci where the complex is recruited. FIGURE 5 \| Ubiquitylation and SUMOylation targets of Smc5/6 and their associated processes. The Smc5/6 complex harbors SUMO ligase (Nse2) and ubiquitin ligase (Nse1-Nse3) subunits. DNA binding stimulates the ATPase domains at the globular heads of Smc5/6, inducing a conformational change in the coiled-coil region of Smc5 that activates the SUMO E3 activity of Nse2. Smc5/6 likely selects its ubiquitylation and/or SUMOylation targets at relevant loci where the complex is recruited. May 2016 \| Volume 7 \| Article 87 11 Frontiers in Genetics \| www.frontiersin.org Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. SUMO ligases of the PIAS family, Nse2 lacks a DNA-binding domain (Jackson, 2001; Ulrich, 2014). Therefore, loading of the entire Smc5/6 complex is likely required for selecting chromatin- associated substrates. In fact, many of Nse2’s targets have been found to co-localize with Smc5/6 or with its associated repair sites. Not surprisingly, Nse2 SUMOylates several subunits within the Smc5/6 complex, such as Smc5, Smc6, Nse3, and Nse2 itself (Andrews et al., 2005; Potts and Yu, 2005; Zhao and Blobel, 2005). Interestingly, SUMOylation by the Smc5/6 complex impinges on the structurally related cohesin complex: in response to DNA damage Nse2 SUMOylates all cohesin subunits, Smc1, Smc3, and Scc1. The modiﬁcation is required for proper loading of the cohesin complex under these conditions. Abolishing SUMOylation of cohesin by point mutations or by tethering a SUMO-speciﬁc isopeptidase to the complex caused defects in the establishment of sister chromatid cohesion and impaired cellular survival (Almedawar et al., 2012; Wu et al., 2012). Other substrates include DNA repair factors such as Ku70 and TRAX (Potts and Yu, 2005; Zhao and Blobel, 2005). In human cells, the complex modiﬁes telomere-binding proteins like RAP1, TRF1, and TRF2 (Potts and Yu, 2007). In budding yeast, rDNA- associated proteins such as RNA polymerase I, Fob1, and Tof2, and the replication factors Pol2 and Mcm6 have been identiﬁed as substrates (Albuquerque et al., 2013; Hang et al., 2015). The functional consequences of these SUMOylation events are yet to be clariﬁed. Nse1, an Smc5/6 subunit with ubiquitin ligase activity q g y The Nse1 subunit of the Smc5/6 complex, a RING ﬁnger protein, exhibits weak ubiquitin ligase activity on its own (Pebernard et al., 2008). This activity is signiﬁcantly enhanced in the presence of its direct interaction partner, Nse3. In collaboration with the E2 Ubc13/Mms2, Nse1/3 is capable of assembling K63-linked ubiquitin chains (Doyle et al., 2010). In S. pombe, the RING- like motif of Nse1 is not essential, but inactivation of the domain leads to hypersensitivity toward genotoxic stress (Pebernard et al., 2008). Recently, Nse3 was found to harbor DNA binding activity, and mutations in the relevant domain caused damage sensitivity and chromosome aberrations (Zabrady et al., 2015). These data indicate that Nse1/3 contribute to the activity of the Smc5/6 complex in chromosome maintenance upon genotoxic stress. However, the targets of such ubiquitin ligase activity have not been identiﬁed. Mono- and Polyubiquitylation of PCNA Mono and Polyubiquitylation of PCNA The profound impact of damage bypass on replication eﬃciency and ﬁdelity is reﬂected by an intricate regulation of the pathway in cells (Ulrich, 2009; McIntyre and Woodgate, 2015). Central to its activation is the ubiquitylation of PCNA on a conserved lysine residue, K164 (Figure 1B). Whereas monoubiquitylation by the E2-E3 pair Rad6-Rad18 promotes TLS, extension of the modiﬁcation to a K63-linked polyubiquitin chain by the heterodimeric E2 Ubc13-Mms2 triggers error-free TS (Hoege et al., 2002; Stelter and Ulrich, 2003). The cognate E3 in budding yeast is the RING ﬁnger protein Rad5; its human homologs are HLTF and SHPRH (Motegi et al., 2008). Rad18, which is rate-limiting for both TLS and TS, is recruited by RPA-covered ssDNA through physical interactions with the RPA complex (Davies et al., 2008; Niimi et al., 2008). In budding yeast, damage-independent SUMOylation of PCNA (see above) provides a second signal that strongly stimulates Rad18’s activity toward PCNA (Parker and Ulrich, 2012). Additional E3s have been reported to operate on mammalian PCNA, such as RNF8 and CRL4Cdt2 (Simpson et al., 2006; Zhang et al., 2008; Terai et al., 2010; Krijger et al., 2011). Moreover, large- scale mass spectrometry studies have identiﬁed multiple other ubiquitylation sites (McIntyre and Woodgate, 2015). However, the relevance of these conjugation factors and modiﬁcations for damage bypass is still a matter of debate, and links to proteasomal degradation may not be excluded (Yu et al., 2009; Cazzalini et al., 2014). Nse2/Mms21, a SUMO ligase associated with the Smc5/6 complex distinguished, which diﬀer signiﬁcantly in their overall accuracy: on the one hand, specialized damage-tolerant DNA polymerases can copy damaged DNA in a process named translesion synthesis (TLS). Due to the low ﬁdelity of the enzymes involved, this pathway is a major cause of damage-induced mutagenesis. On the other hand, error-free damage bypass can be accomplished by means of a so-called template switching (TS) pathway, which altogether avoids the use of the damaged DNA as a replication template and instead relies on the (undamaged) sister chromatid to provide accurate sequence information. This process involves recombination factors and joint molecules as intermediates (Giannattasio et al., 2014), but appears to be distinct from the classical HR mechanism used for DSB repair. Both branches of damage bypass can act in a postreplicative manner; thus, they are not necessarily coupled to replication fork progression (Daigaku et al., 2010; Karras and Jentsch, 2010). DNA Damage Bypass In addition, the ubiquitin-like modiﬁer ISG15 was recently found to contribute to the termination of TLS by modiﬁcation of PCNA, which in turn mediated the recruitment of USP10 for PCNA deubiquitylation and dissociation of polymerase η (Park et al., 2014). Intriguingly, a viral isopeptidase, BPLF1, was also shown to deubiquitylate human PCNA during replication of the Epstein–Barr genome, thus inhibiting polymerase η recruitment during the lytic phase of infection (Whitehurst et al., 2012). How an inhibition of TLS may promote viral replication is not yet understood. In yeast, PCNA deubiquitylation is mediated by Ubp10; however, despite an accumulation of PCNAUb, inactivation of the enzyme does not cause a noticeable increase in mutation rates, indicating that reversal of the modiﬁcation may be less critical for damage bypass in this organism (Gallego- Sanchez et al., 2012). convincing yeast homolog has not been identiﬁed, which argues for an auxiliary function of ZRANB3 rather than a key role in activating TS. g Analysis of the TS pathway is further complicated by the multi-functionality of Rad5 and its two human homologs, whose catalytic RING domains are embedded in SWI/SNF- like domains with helicase and DNA-dependent ATPase activity. Although this helicase function can be genetically separated from Rad5’s role in ubiquitin-dependent TS, it does contribute to survival of replication stress (Choi et al., 2015). Interestingly, Rad5 and its homologs have been implicated not only in TS, but also in TLS in budding and ﬁssion yeast as well as humans (Minesinger and Jinks-Robertson, 2005; Gangavarapu et al., 2006; Pages et al., 2008; Coulon et al., 2010; Lin et al., 2011; Kuang et al., 2013; Xu et al., 2016). Mechanistically, this activity remains controversial, as some studies have invoked PCNA polyubiquitylation in the process, whereas others have reported a RING- and ATPase- independent function or a dependence on a physical interaction with the TLS polymerase Rev1. Moreover, although both HLTF and SHPRH are capable of polyubiquitylating PCNA in vitro, they have been postulated to fulﬁll non-redundant functions in cooperation with TLS polymerases η and κ, respectively, depending on the nature of the damaging agent (Lin et al., 2011). Based on these observations, Lin et al. (2011) have put forth a model where a damage-tolerant polymerase harboring multiple UBDs, such as polymerase κ, might preferentially recognize polyubiquitylated PCNA, while monoubiquitylation might stimulate those polymerases with only one UBD, such as polymerase η. DNA Damage Bypass Along similar lines, Fuchs and coworkers proposed that polyubiquitylated PCNA might serve to simultaneously attract several diﬀerent TLS polymerases for cooperation in damage bypass (Coulon et al., 2010). In contrast, observations by Yang et al. (2014) in an in vitro set-up have led to the opposite conclusion: rather than promoting TLS, polyubiquitylation of PCNA was found to inhibit the activity of polymerase η in the bypass of an abasic site, suggesting that the K63-chains trap the polymerase in a non-productive mode. In budding yeast, genetic analysis supports a positive eﬀect of Rad5 on TLS in some situations. At the same time, however, PCNA polyubiquitylation promotes damage resistance even in the absence of any damage-tolerant polymerase, thus clearly implying a TLS-independent function in TS (Zhao and Ulrich, 2010). In summary, the consequences of PCNA polyubiquitylation remain to be elucidated in molecular terms, and future studies will be needed in order to gain insight into how the balance between mutagenic TLS and error-free TS is controlled in vivo. How polyubiquitylation of PCNA triggers TS is still an unresolved question. From experiments using linear head-to- tail fusions of ubiquitin moieties as mimics of polyubiquitin chains it was inferred that the K63-linkage itself is important for TS activity (Zhao and Ulrich, 2010). Although putative eﬀectors that preferentially interact with polyubiquitylated PCNA have been identiﬁed, they are unlikely to be directly responsible for activating TS: the human ATPase WRNIP1 and its yeast homolog Mgs1 accumulate at stalled replication intermediates in a manner that depends on their UBZ domain as well as PCNA ubiquitylation (Crosetto et al., 2008; Saugar et al., 2012). However, even though a subset of the phenotypes of mgs1 mutants is consistent with a function downstream of PCNAUb (Hishida et al., 2006; Saugar et al., 2012), no obvious TS defects are observed in such mutants. In human cells, WRNIP1 appears to contribute to checkpoint activation as a bridging factor that promotes interaction of PCNAUb with the ATM-associated ATMIN protein (Kanu et al., 2015). Yet, this function is unlikely to be related to polyubiquitylation, as a single ubiquitin moiety is suﬃcient to stimulate the interaction between WRNIP1/Mgs1 with PCNA (Saugar et al., 2012). DNA Damage Bypass Activation of TLS by monoubiquitylated PCNA can largely be explained by the presence of ubiquitin-binding domains within the major family of damage-tolerant polymerases, which convey an enhanced aﬃnity for the modiﬁed form of PCNA (Watanabe et al., 2004; Bienko et al., 2005; Bi et al., 2006; Plosky et al., 2006). In mammals, direct interactions with Rad18 also contribute to the recruitment of TLS polymerases (Watanabe et al., 2004). Whereas in yeast TLS-mediated damage-induced mutagenesis nearly completely depends on PCNA ubiquitylation, the process appears to be less dependent on this modiﬁcation in vertebrate cells (Stelter and Ulrich, 2003; Edmunds et al., 2008; Hendel et al., 2011). In addition to the damage-tolerant polymerases, a number of auxiliary factors have been proposed DNA damage bypass, also called DNA damage tolerance, is important in situations where fork stalling has been triggered by lesions in the replication template that cannot be copied by the replicative DNA polymerases (Saugar et al., 2014). Such lesions mostly represent damage that is subject to base or nucleotide excision repair, i.e., small or bulky adducts, oxidative lesions, abasic sites and UV-induced pyrimidine dimers. In order to prevent a permanent replication arrest, damage bypass ensures complete duplication of the aﬀected region without actually removing the lesion, and excision-based repair can act subsequently when the DNA has regained its double- stranded form. Two major pathways of damage bypass can be May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 12 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. to modulate TLS via recognition of monoubiquitylated PCNA in mammals. These include the UBZ domain-containing proteins SNM1A, a nuclease that might provide a link between TLS and the repair of ICLs, and DVC1 (also called Spartan), an adaptor for the ubiquitin-dependent chaperone p97 (Yang et al., 2010; Centore et al., 2012). The selectivity of DVC1’s UBZ domain for PCNAUb has been contested, however, and it has been proposed that the protein binds to other ubiquitylated proteins at sites of replication stalling, where it would mediate extraction of polymerase η in order to limit TLS activity (Davis et al., 2012; Mosbech et al., 2012). Downregulation of TLS appears to be important for preventing excessive mutagenesis during replication. In human cells, this is accomplished mainly by PCNA deubiquitylation via the isopeptidase USP1 (Huang et al., 2006). Frontiers in Genetics \| www.frontiersin.org The Fanconi Anemia Pathway The Fanconi Anemia Pathway DNA interstrand cross-links (ICLs) are strongly replication fork-stalling lesions that are not only refractory to copying by replicative DNA polymerases, but also prevent strand separation and passage of the helicase. Accordingly, their processing in replicating cells requires an intricate operation involving components of several repair pathways, namely TLS polymerases, HR proteins and structure-speciﬁc nucleases. In vertebrate cells, cooperation between these factors is mediated by the FA pathway, named after a rare hereditary disease associated with bone-marrow failure, congenital abnormalities, cancer predisposition and a marked sensitivity to ICL-causing agents (Kottemann and Smogorzewska, 2013; Walden and Deans, 2014). Nineteen genes have been assigned to this pathway by means of epistasis analysis, and eight of these encode subunits of a multimeric ubiquitin ligase, the FA core complex (Coleman and Huang, 2016). This E3 is recruited to chromatin upon stalling of the replisome upstream of an ICL, where its catalytic subunit, FANCL, monoubiquitylates a heterodimer of two other FA proteins, FANCD2 and FANCI (Alpi et al., 2008; Longerich et al., 2009; Sato et al., 2012). The ubiquitylated form of this “ID complex” initiates ICL processing, which involves the generation of a collapsed fork as a step toward the unhooking of the cross-link by dual incisions on either side of the lesion. It is followed by TLS-mediated repair synthesis and HR-mediated reactivation of the replication fork. How these downstream events are accomplished has only recently been elucidated. Central to the unhooking step is the scaﬀold protein, SLX4/FANCP, which recognizes the ubiquitylated ID complex by means of two ubiquitin-binding UBZ domains and interacts with a number of structure-speciﬁc nucleases that mediate the actual incisions (Zhang and Walter, 2014). Ubiquitin binding by SLX4 is required for cellular resistance speciﬁcally toward DNA cross-linking agents (Kim et al., 2013), and mutations in SLX4 confer a FA phenotype in humans, highlighting the importance of SLX4 for ICL repair (Kim et al., 2011; Stoepker et al., 2011). p Another recurring theme in the regulation of damage bypass is the protection of critical factors from proteolysis. This may be achieved by the SUMOylation of a protein, as is observed in the nematode Caenorhabditis elegans, where SUMOylation of polymerase η and potentially κ prevents their ubiquitin- mediated degradation during early embryonic development (Kim and Michael, 2008; Roerink et al., 2012). Alternatively, deubiquitylation can eﬀectively stabilize a chromatin-associated protein by preventing its extraction or proteasomal degradation. DNA Damage Bypass A second ATPase, ZRANB3, has also been implicated in PCNA-dependent damage bypass, based on its localization to laser-induced DNA damage, its preferential interaction with polyubiquitylated PCNA, and a general sensitivity to replication stress upon depletion of the protein (Ciccia et al., 2012; Weston et al., 2012). However, a function in the TS pathway has yet to be properly established by means of genetic analysis. Moreover, a Ubiquitylation of Other Damage Bypass Factors Besides PCNA, numerous other factors involved in DNA damage bypass have been identiﬁed as ubiquitylation and/or SUMOylation targets (McIntyre and Woodgate, 2015). As many of the modiﬁcations were detected in the context of large- scale proteomics screens, their relevance for damage bypass has not always been conﬁrmed. Nevertheless, some common patterns indicate potential regulatory impacts. For example, all May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 13 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. human TLS polymerases of the Y-family are ubiquitylated, and in many cases this depends on their own ubiquitin-binding domains. Although this is reminiscent of the E3-independent phenomenon of coupled ubiquitylation (Hoeller et al., 2007), relevant ubiquitin ligases have actually been identiﬁed, such as Pirh2, Mdm2, and TRIP in the case of polymerase η (Jung et al., 2010, 2011, 2012; Wallace et al., 2014). While Pirh2 attaches monoubiquitin, which apparently inhibits TLS by preventing interaction of the polymerase with PCNAUb (Bienko et al., 2010), Mdm2 achieves the same eﬀect by targeting polymerase η for polyubiquitylation and proteasomal degradation. In contrast, polyubiquitylation by the TRAF-interacting protein TRIP was reported to promote polymerase η localization to nuclear foci. The D. melanogaster homolog of TRIP, NOPO, is known to assemble K63-linked chains, possibly indicating a regulatory function of TRIP-mediated polymerase η modiﬁcation as well, and interactions of both TRIP and NOPO with several Y-family polymerases suggest a conservation of the process (Wallace et al., 2014). In budding yeast, polymerase η has been found to be ubiquitylated as well; however, the eﬀects of this modiﬁcation on protein stability remain controversial (Parker et al., 2007; Skoneczna et al., 2007; Pabla et al., 2008; Plachta et al., 2015). Ubiquitin-mediated proteolysis also controls the levels of budding yeast TLS polymerase Rev1 along the cell cycle, thus limiting the bulk of its mutagenic activity to the G2/M phase (Waters and Walker, 2006). Finally, McIntyre et al. Frontiers in Genetics \| www.frontiersin.org DNA Damage Bypass (2013) showed that ubiquitylation of human Y-family polymerases appears to promote mutual interactions via their ubiquitin- binding domains and – as a consequence – facilitate their cooperation in TLS. replication and was therefore proposed to prevent damage- induced mutagenesis during cell cycle-independent processes such as other DNA repair events. In summary, USP7 appears to be an important modulator of replication eﬃciency and ﬁdelity not only during unperturbed replication, but also during DNA damage bypass. The Fanconi Anemia Pathway In human cells, the isopeptidase USP7 appears to play a major role in this manner. As described above, its activity is important even during unperturbed replication (Lecona et al., 2016). In response to replication stress, USP7 deubiquitylates a number of proteins, among them polymerase η, Mdm2, Rad18, and HLTF. By acting on polymerase η and Mdm2, USP7 directly and indirectly stabilizes the polymerase and thereby facilitates TLS (Qian et al., 2015). Deubiquitylation of Rad18 and HLTF was also observed to stabilize the E3s and thus contribute positively to PCNA mono- and polyubiquitylation, respectively (Qing et al., 2011; Zlatanou et al., 2016). In addition, Zeman et al. (2014) reported that a failure to deubiquitylate Rad18 prevented its eﬃcient recruitment and interaction with SHPRH, thus promoting mutagenic TLS at the expense of error-free TS. Surprisingly, USP7 also deubiquitylates PCNA (Kashiwaba et al., 2015). However, unlike the S phase-associated activity of USP1, USP7 activity toward PCNA was not found to be coupled to Another structure-speciﬁc endonuclease, FANCD2/FANCI- associated nuclease 1 (FAN1), was identiﬁed to act downstream of the ID complex (Kratz et al., 2010; Liu T. et al., 2010; MacKay et al., 2010; Smogorzewska et al., 2010). Like SLX4, FAN1 carries a UBZ domain that was reported to mediate the recruitment to damage sites via binding to monoubiquitylated FANCD2. However, FAN1 was found to be dispensable for ICL incision in a cell-free system (Klein Douwel et al., 2014). Moreover, patients carrying a FAN1 homozygous microdeletion do not suﬀer from typical FA conditions (Trujillo et al., 2012), thus arguing against a contribution of the nuclease to the FA pathway. Insight into this conundrum has very recently come from the observation that FAN1 instead prevents May 2016 \| Volume 7 \| Article 87 14 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. Ubiquitylation of Histones H2A and H2B Histone H2A was the ﬁrst protein discovered to be modiﬁed by ubiquitin (Goldknopf et al., 1975). In fact, H2A and H2B are two of the most abundant ubiquitylation targets in the nucleus (Cao and Yan, 2012). Both H2A and H2B are predominantly modiﬁed by monoubiquitin. H2B was found to be monoubiquitylated at K123 in S. cerevisiae or K123 and 120 in human cells, which plays an important role in transcriptional regulation (Henry et al., 2003; Wood et al., 2003; Kao et al., 2004; Nakanishi et al., 2009; Song and Ahn, 2010). The Fanconi Anemia Pathway In yeast, H2B monoubiquitylation is mediated by the E2 Rad6 and the E3 Bre1 (Robzyk et al., 2000; Wood et al., 2003). The mammalian homologs of Bre1, RNF20, and RNF40 (Kim et al., 2005), cooperate with the E2s hRad6 and UbcH6 (Koken et al., 1991). H2BUb promotes di- and tri-methylation of H3 at K4, which controls various aspects of transcription (Dover et al., 2002; Sun and Allis, 2002; Krogan et al., 2003), among them a stabilization of the histone chaperone complex FACT (Fleming et al., 2008). genomic instability induced by replication fork stalling events unrelated to ICLs (Lachaud et al., 2016). Thus, ubiquitylation of the ID complex by the FA core complex appears to serve a twofold purpose in response to replication stress, depending on the downstream eﬀectors: a highly specialized ICL repair pathway triggered by SLX4 recruitment, and an independent, more general fork protection mechanism by means of FAN1. Interestingly, the FA pathway appears to be intimately connected with another system for replication fork protection, the Rad18- and PCNA-dependent damage bypass mechanism described above. Not only does ICL processing require the activity of TLS polymerases, but the central initiating event of the FA pathway, the activation of the ID complex, was actually found to depend on Rad18, the E3 responsible for PCNA monoubiquitylation. The exact relationship between the two pathways is still a matter of controversy, as one study observed an interaction between FANCL and PCNAUb that was required for eﬃcient recruitment of FANCL to chromatin (Geng et al., 2010), whereas another report postulated a direct role of Rad18 in binding and recruitment of FANCD2 in a manner independent of PCNA modiﬁcation (Williams et al., 2011). Another piece of evidence for a tight coordination between the two pathways is the notion that the isopeptidase USP1 mediates deubiquitylation of both PCNA and the ID complex (Nijman et al., 2005; Huang et al., 2006). Controlling the ubiquitylation of these two key players appears to be essential for proper replication fork maintenance, as loss of USP1 causes high levels of genome instability and mutagenesis (Huang et al., 2006). ) Beyond its role in transcriptional regulation, H2BUb has been implicated in DNA replication (Figure 6A). This connection was established by the observation that Bre1 is enriched around replication origins, where it contributes to maintaining H2BUb levels on newly replicated DNA (Trujillo and Osley, 2012). The Fanconi Anemia Pathway Whereas a ubiquitylation-deﬁcient mutant of H2B, K123R, is highly sensitive to replication fork-stalling agents (Trujillo and Osley, 2012; Lin et al., 2014), H3 mutants that abolish methylation are signiﬁcantly less sensitive (Trujillo and Osley, 2012). This argues that the contribution of H2BUb to replication is independent of its regulatory role in transcription, mediated through histone methylation. In cells lacking H2BUb, despite eﬃcient formation of the pre-RC, association of replisome components such as polymerases ε and α and RPA with origins is impaired (Trujillo and Osley, 2012), as is replication progression after HU treatment (Lin et al., 2014). Also, PCNA associates normally at origins, but its levels are reduced at more distal sites, suggesting that the H2B-K123R mutant does not aﬀect origin ﬁring, but rather fork progression under conditions of replication stress (Trujillo and Osley, 2012). In addition, lack of H2BUb leads to a defect in the binding of the FACT complex and reduced nucleosome occupancy in newly replicated DNA under the same stress condition (Trujillo and Osley, 2012; Lin et al., 2014). H2BUb’s eﬀect on FACT in this context is reminiscent of its role during transcription. Since FACT is known to stimulate the activity of the Mcm helicase (Tan et al., 2006), it was speculated that H2BUb could play a role in facilitating the unwinding of DNA ahead of the fork to promote replication progression. However, this view was challenged by a recent report from Lin et al. (2014), who postulated that H2BUb may instead function to limit uncontrolled fork progression. They observed signiﬁcant elongation of replication tracts in the absence of H2BUb after HU treatment, together with increased levels of H2A phosphorylation, a sign of fork damage (Lin et al., 2014). In support of this model, fork progression under conditions of replication stress is also strongly enhanced in rad61 cells (Yu et al., 2014). A recent study discovered a regulatory circuit of polyubiquitin and SUMO that also appears to contribute to controlling FA pathway activity at sites of replication problems (Gibbs-Seymour et al., 2015): upon treatment with replication fork-stalling agents, FANCD2 and FANCI are SUMOylated by two SUMO E3 ligases, PIAS1 and PIAS4, in a manner dependent on prior activation of the ID complex by monoubiquitylation. The modiﬁcation targets the proteins for RNF4-mediated polyubiquitylation and subsequent extraction from the chromatin by the p97 segregase in complex with DVC1. The Fanconi Anemia Pathway Hence, this mechanism may limit ID complex dosage at the sites of replication stress in order to terminate the response or avoid excessive activity of the FA pathway. Frontiers in Genetics \| www.frontiersin.org REPLICATION OF CHROMATIN Genome replication occurs in the context of chromatin. Hence, for eﬃcient copying of genomic DNA, nucleosomes must be disrupted ahead of an advancing replication fork. Upon passage of the fork, chromatin structure must rapidly be restored, and loss of epigenetic information in the process needs to be avoided. It is therefore not surprising that many chromatin components are targets of the ubiquitin and/or SUMO system for regulatory purposes, and these modiﬁcations are known to be important for the replication process itself. May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 15 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. Functions of histone ubiquitylation in DNA replication. (A) Budding yeast E2–E3 complex Rad6-Bre1 is recruited to sites of replication stress for lation at K123. H2BUb regulates fork speed and nucleosome assembly behind the fork. Via H3K9 methylation, it independently contributes to al regulation. (B) The Polycomb Repressive Complex (PRC) is recruited to sites of replication stress or at problematic sequences to ubiquitylate H2A at b recruits BAP1, which maintains fork stability by protecting chromatin-remodeling INO80 complex from proteasomal degradation. (C) The E3 SCFRtt101 H3 at K121, 122, and 125. The reaction is stimulated by acetylation of H3 at K56 by histone acetyltransferase Rtt109. This facilitates transfer of the mer to CAF-1 for nucleosome deposition behind the fork. Rtt101 also ubiquitylates the chromatin-reorganizing FACT complex. FIGURE 6 \| Functions of histone ubiquitylation in DNA replication. (A) Budding yeast E2–E3 complex Rad6-Bre1 is recruited to sites of replication stress for H2B ubiquitylation at K123. H2BUb regulates fork speed and nucleosome assembly behind the fork. Via H3K9 methylation, it independently contributes to transcriptional regulation. (B) The Polycomb Repressive Complex (PRC) is recruited to sites of replication stress or at problematic sequences to ubiquitylate H2A at K119. H2AUb recruits BAP1, which maintains fork stability by protecting chromatin-remodeling INO80 complex from proteasomal degradation. (C) The E3 SCFRtt101 ubiquitylates H3 at K121, 122, and 125. The reaction is stimulated by acetylation of H3 at K56 by histone acetyltransferase Rtt109. This facilitates transfer of the H3-H4 tetramer to CAF-1 for nucleosome deposition behind the fork. Rtt101 also ubiquitylates the chromatin-reorganizing FACT complex. FIGURE 6 \| Functions of histone ubiquitylation in DNA replication. (A) Budding yeast E2–E3 complex Rad6-Bre1 is recruited to sites of replication stress for H2B ubiquitylation at K123. H2BUb regulates fork speed and nucleosome assembly behind the fork. Ubiquitylation of Histone H3 in Replication-Coupled Nucleosome Assembly H2A is well known to be ubiquitylated at the conserved residue K119 by the polycomb repressive complex 1 (PRC1), which comprises the RING-E3 subunits RING1A or RING1B and BMI1 together with the E2 UbcH5c (Gao et al., 2012; McGinty et al., 2014). The mark is essential for establishing repressive chromatin during development (Lanzuolo and Orlando, 2012; Di Croce and Helin, 2013). However, H2AUb may also play a role in the replication of intact and damaged DNA (Figure 6B). RING1B localizes to sites of replication (Lee et al., 2014; Piunti et al., 2014), and several Polycomb proteins were also found to be recruited to sites of DNA damage (Bergink et al., 2006; Chou et al., 2010; Ginjala et al., 2011), suggesting that H2AUb may contribute to damage signaling at replication forks. In fact, loss of PRC function causes an increase in asymmetric forks, indicating perturbed replication dynamics (Piunti et al., 2014; Bravo et al., 2015). Conversely, enhancement of H2A ubiquitylation by depletion of the ubiquitin-speciﬁc protease USP3 in mammalian cells causes delays in S phase progression and increased formation of ssDNA and DNA breaks (Nicassio et al., 2007). These observations are consistent with H2AUb acting as a damage signal that – when present in excess – leads to a hyperactivation of the damage response that would generate abnormal replication or repair structures causing genomic instability. In order to ensure proper restoration of chromatin structure upon genome replication, the nucleosome assembly machinery is tightly coupled to replication fork progression. In budding yeast, this is achieved by means of a pathway involving acetylation of histone H3, a marker of newly synthesized histones (Figure 6C). In front of a replication fork, nucleosomes are disassembled by the action of the Mcm2-7 complex and histone chaperone Asf1 (Groth et al., 2007; Huang et al., 2015). Behind the fork, both parental and newly synthesized histones contribute to the restoration of chromatin structure. In S. cerevisiae, preferential binding of Asf1 to the H3-H4 dimer stimulates acetylation of newly synthesized H3 at K56 by the histone acetyltransferase Rtt109 (Masumoto et al., 2005; Driscoll et al., 2007; Han et al., 2007a). H3K56ac enhances binding of H3-H4 to histone chaperones Rtt106 and CAF-1 (Li et al., 2008). CAF-1 in turn interacts with PCNA and assists in histone deposition behind the fork (Shibahara and Stillman, 1999; Moggs et al., 2000; Zhang et al., 2000). Ubiquitylation of Histone H3 in Replication-Coupled Nucleosome Assembly H3 acetylation peaks in S phase and is removed upon completion of genome replication (Masumoto et al., 2005). H3K56ac is also detectable in mammalian cells, although in much lower abundance compared to yeast (Garcia et al., 2007; Das et al., 2009; Tjeertes et al., 2009; Yuan et al., 2009; Jasencakova et al., 2010), suggesting that either the modiﬁcation is much more transient, or other acetylation sites can substitute for H3K56. Defects in the Asf1-Rtt109-H3K56ac pathway result in various aspects of genome instability, including reduced replisome function under conditions of replication stress (Franco et al., 2005; Schulz and Tyler, 2006; Han et al., 2007b; Clemente- Ruiz et al., 2011), sensitivity to DNA-damaging agents (Driscoll et al., 2007; Li et al., 2008), loss of sister chromatid cohesion, excessive recombination and high rates of gross chromosomal rearrangements (Myung et al., 2003; Prado et al., 2004; Ramey et al., 2004; Thaminy et al., 2007; Kadyrova et al., 2013; Munoz- Galvan et al., 2013). A recent study suggests a special role of H2AUb in the replication of pericentromeric heterochromatic domains, which are duplicated late in S phase (Bravo et al., 2015). Cells deﬁcient in all RING1 activities were found to accumulate high levels of ssDNA in these regions, along with increased spontaneous levels of γH2AX and a delayed transition from middle to late S phase. Consistent with these ﬁndings, H2AUb colocalizes with PCNA in late S phase (Vassilev et al., 1995). Interestingly, selective restoration of H2AUb within the pericentromeric heterocromatic domains by means of a fusion construct of RING1B, BMI1 and methyl-CpG binding domain protein 1 (MBD1) rescued the defect in S phase progression in RING1- deﬁcient cells (Bravo et al., 2015). Given the enrichment of major satellite repeats in pericentric heterochromatin and their propensity to form secondary structures, the strong eﬀect of H2AUb in these regions may well reﬂect a general contribution of the modiﬁcation to the replication of problematic sequences. Intriguingly, a large-scale genetic screen in budding yeast identiﬁed the CRL ubiquitin ligase complex, Rtt101Mms1/Mms22, as a downstream eﬀector of the pathway (Collins et al., 2007b). Rtt101Mms1 is believed to be the budding yeast ortholog of human CRL4DDB1 and assembles with the substrate adaptor Mms22 in a DNA damage-induced manner (Zaidi et al., 2008; Han et al., 2010, 2013). REPLICATION OF CHROMATIN Via H3K9 methylation, it independently contributes to transcriptional regulation. (B) The Polycomb Repressive Complex (PRC) is recruited to sites of replication stress or at problematic sequences to ubiquitylate H2A at K119. H2AUb recruits BAP1, which maintains fork stability by protecting chromatin-remodeling INO80 complex from proteasomal degradation. (C) The E3 SCFRtt101 ubiquitylates H3 at K121, 122, and 125. The reaction is stimulated by acetylation of H3 at K56 by histone acetyltransferase Rtt109. This facilitates transfer of the H3-H4 tetramer to CAF-1 for nucleosome deposition behind the fork. Rtt101 also ubiquitylates the chromatin-reorganizing FACT complex. May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org Frontiers in Genetics \| www.frontiersin.org 16 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. (Papamichos-Chronakis and Peterson, 2008; Shimada et al., 2008; Vincent et al., 2008; Falbo et al., 2009; Vassileva et al., 2014). Taken together, ubiquitylation of H2B appears to coordinate nucleosome assembly with fork progression, an activity that becomes particularly important when the replisome is challenged by replication stress such as nucleotide depletion or DNA damage. However, the precise mechanism and the eﬀectors of the modiﬁcation are yet to be deﬁned. Frontiers in Genetics \| www.frontiersin.org Ubiquitylation of Histone H3 in Replication-Coupled Nucleosome Assembly Inactivation of the complex causes damage sensitivity and defects in fork progression through damaged DNA and natural replication pause sites such as ribosomal DNA loci, and these defects are epistatic with the lack of Rtt109 (Luke et al., 2006; Duro et al., 2008; Zaidi et al., 2008; Wurtele et al., 2012). Moreover, Rtt109 was indeed found to recruit Rtt101 to chromatin (Roberts et al., 2008). The mechanism by which H2AUb inﬂuences DNA replication is still unknown, but some insight comes from the observation that BRCA1-associated protein-1 (BAP1) recognizes H2AUb at replication forks and recruits the ATP-dependent chromatin remodeler Ino80. BAP1 deubiquitylates INO80 and thereby protects the protein from ubiquitin-mediated proteolysis (Lee et al., 2014). Hence, via BAP1 recruitment H2AUb might allow the INO80 complex to exert its well-known role in stabilizing stalled replication forks and assisting fork restart Despite the strong genetic link between the Asf1-Rtt109- H3K56ac nucleosome assembly pathway and Rtt101Mms1/Mms22 May 2016 \| Volume 7 \| Article 87 17 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. in replisome functions and genome maintenance, their cooperation is not well understood in mechanistic terms. A recent report might provide insight into the process. Han et al. (2013) found that Rtt101Mms1/Mms22 preferentially binds H3K56ac-H4 over unmodiﬁed H3-H4 and can directly ubiquitylate H3 at lysine residues 121, 122, and 125. This in turn weakens H3 interaction with Asf1 and instead facilitates association with CAF-1 for subsequent deposition behind the replication fork. This function appears to be conserved in human cells, as depletion of CRL4DDB1 results in enhanced interaction of H3-H4 with Asf1 and reduced deposition of new H3 (Han et al., 2013). Hence, Rtt101Mms1/Mms22-mediated ubiquitylation of H3 appears to assist in a hand-oﬀmechanism that ensures the transfer of H3–H4 from Asf1 ahead of an advancing fork to other chaperones such as CAF-1 and Rtt106 behind the fork. humans), is recruited to nascent DNA after replication (Lopez- Contreras et al., 2013). UHRF1 binds to hemi-methylated DNA through its SET and RING ﬁnger-associated (SRA) domain (Arita et al., 2008; Avvakumov et al., 2008). Recently, Nishiyama et al. (2013) found that UHRF1 ubiquitylates histone H3 at K23 in X. laevis egg extracts. Methylation is then restored by DNMT1, which recognizes H3K23Ub through its replication foci targeting sequence. A similar mechanism is observed in mammalian cells, where UHRF1 ubiquitylates H3 at K18. SPATIAL REGULATION OF UBIQUITYLATION AND SUMOYLATION DURING DNA REPLICATION Intriguingly, however, in this case a K63- linked ubiquitin chain was detected on FACT (Han et al., 2010). Hence, it is still an open question whether Rtt101Mms1/Mms22 plays any role in proteasomal degradation mediated via K48- chains. A number of ubiquitin- and SUMO-related enzymes are found at the nuclear pore. For instance, SUMO protease Ulp1 is anchored to the nucleoporin Nup60 through myosin-like proteins (MLPs) Mlp1 and Mlp2 (Zhao et al., 2004). Mutation of ULP1 or loss of MLPs shows synthetic eﬀects when combined with mutations in HR (Zhao et al., 2004; Collins et al., 2007a; Palancade et al., 2007), and deleting MLPs leads to mislocalization of Ulp1, DNA damage sensitivity and clonal lethality (Zhao et al., 2004). Moreover, cells with impaired Ulp1function accumulate ssDNA during replication (Soustelle et al., 2004). It is therefore conceivable that the presence of deSUMOylating activity at the nuclear pore either prevents the accumulation of toxic recombination intermediates during replication or is required to resolve these structures. Proper localization of the SUMO conjugation system thus impinges on the process of DNA replication itself. Ubiquitylation of Histone H3 in Replication-Coupled Nucleosome Assembly Here, DNMT1 binds to H3K18Ub via a ubiquitin-interacting UIM motif (Qin et al., 2015). This is an interesting example of how cells can use the ubiquitin system to establish other epigenetic marks following DNA replication. SPATIAL REGULATION OF UBIQUITYLATION AND SUMOYLATION DURING DNA REPLICATION How does nucleosome assembly inﬂuence replisome stability? There is growing evidence indicating that coupling of nucleosome assembly and replication progression is essential for maintenance of intact replisomes. This view is supported by the observation that deregulation of histone supply causes replication forks to collapse, followed by recombination-mediated rescue (Groth et al., 2007; Clemente-Ruiz and Prado, 2009; Takayama and Toda, 2010; Clemente-Ruiz et al., 2011; Mejlvang et al., 2014). This eﬀect is reminiscent of the situation where lack of the Asf1-Rtt109-H3K56ac or Rtt101Mms1/Mms22 pathway causes a decoupling of nucleosome assembly and replication progression. The ubiquitin and SUMO systems are organized within the cell in a spatially controlled manner. One important hub for the coordination of nuclear ubiquitylation and SUMOylation activities appears to be the nuclear pore complex (NPC). The NPC is responsible for the transport of macromolecules between the nucleus and the cytoplasm, but genetic data from budding yeast suggest that it has additional functions in coordinating DNA damage signaling and repair (Figure 7). For example, it has been observed that cells deﬁcient in components of the Nup84 nuclear pore subcomplex are hypersensitive to DNA-damaging agents (Bennett et al., 2001; Loeillet et al., 2005; Therizols et al., 2006) and accumulate spontaneous recombination foci in S and G2 phase (Loeillet et al., 2005; Palancade et al., 2007; Nagai et al., 2008). Mutations in both Nup84 and the HR pathway are synthetically lethal (Loeillet et al., 2005). These ﬁndings suggest that the NPC plays a role in replication during both unperturbed and stress conditions, and HR-based mechanisms to resolve fork problems become essential when NPC function is compromised. p g y p p g Nevertheless, many mechanistic questions remain. For example, the majority of Cullin-based ubiquitin ligases are known to produce K48-linked polyubiquitin chains that target their substrates for proteasomal degradation (Komander and Rape, 2012; Mattiroli and Sixma, 2014), and the E2 Cdc34 that associates with Rtt101 is also known to assemble K48-chains (Ye and Rape, 2009). Yet, H3 itself is unlikely to be a substrate of the proteasome. This has led to the idea that Rtt101Mms1/Mms22 may target other components at stalled forks for degradation in order to facilitate repair or restart. Indeed, one such substrate could be the FACT complex (Han et al., 2010), which requires Rtt101 speciﬁcally for localization to sites of replication, but not to transcription sites. Ubiquitylation of Histone H3 in Replication-Coupled Epigenetic Inheritance In order to maintain its identity and gene expression patterns, it is crucial for a cell to restore its epigenetic information after every round of replication. Due to the semiconservative nature of DNA replication, DNA is hemi-methylated after every replication cycle, and full DNA methylation has to be restored in order to reestablish gene silencing. It is known that a RING- type ubiquitin ligase, UHRF1 (ubiquitin-like with PHD and ring ﬁnger domains 1, also known as NP95 in mouse and ICBP90 in Intriguingly, the nuclear pore is also the site of accumulation of STUbLs in yeast (Nagai et al., 2011). Deletion of the STUbL complex Slx5/8 renders yeast hypersensitive to DNA-damaging May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 18 García-Rodríguez et al. Ubiquitin and SUMO in DNA Replication FIGURE 7 \| Spatial organization of ubiquitin and SUMO metabolism in the nucleus. Both ubiquitin and SUMO conjugating and deconjugating enzymes are enriched at the Nuclear Pore Complex (NPC). Collapsed forks relocalize to NPCs, which modulates local ubiquitylation and/or SUMOylation of relevant components in order to facilitate fork restart or prevent toxic recombination events. FIGURE 7 \| Spatial organization of ubiquitin and SUMO metabolism in the nucleus. Both ubiquitin and SUMO conjugating and deconjugating enzymes are enriched at the Nuclear Pore Complex (NPC). Collapsed forks relocalize to NPCs, which modulates local ubiquitylation and/or SUMOylation of relevant components in order to facilitate fork restart or prevent toxic recombination events. FIGURE 7 \| Spatial organization of ubiquitin and SUMO metabolism in the nucleus. Both ubiquitin and SUMO conjugating and deconjugating enzymes are enriched at the Nuclear Pore Complex (NPC). Collapsed forks relocalize to NPCs, which modulates local ubiquitylation and/or SUMOylation of relevant components in order to facilitate fork restart or prevent toxic recombination events. However, it is still not fully understood how these activities are coordinated at the pore, for instance whether a certain factor is deSUMOylated by Ulp1 or directed to proteasomal degradation through STUbL activity. How such events would impact on the outcome of repair and the consequences for genome integrity awaits further investigation. agents and replication stress, and the mutants show higher rates of spontaneous gross chromosomal rearrangements (Zhang et al., 2006; Prudden et al., 2007; Nagai et al., 2008). Consistent with these ﬁndings, collapsed replication forks – like DSBs – are redirected to the nuclear pore (Nagai et al., 2008; Horigome et al., 2014). Ubiquitylation of Histone H3 in Replication-Coupled Epigenetic Inheritance These observations prompted the hypothesis that relocalization to the nuclear pore facilitates HR-mediated fork restart by means of STUbL activity, possibly via degradation of SUMOylated proteins such as Srs2 (Urulangodi et al., 2015). In support of this idea, Su et al. (2015) recently observed that sites of replication blockage created by expanded CAG repeats are relocated to nuclear pores particularly in late S phase. The authors suggested that such relocalization may target Rad52SUMO for degradation, which would then alter the outcome of HR pathways in the context of replication restart. In humans, it has been proposed that the PML nuclear bodies functionally resemble the yeast nuclear pores as a site where the mammalian STUbL RNF4 accumulates (Nagai et al., 2011). However, it remains to be tested whether perturbed replication forks are redirected to PML bodies in human cells. 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CONCLUDING REMARKS The extensive range of mechanisms by which ubiquitin and SUMO impinge on eukaryotic DNA replication is a very good reﬂection of the diversity of these two posttranslational modiﬁcation systems in general. Several recurring concepts, including proteasomal targeting, either by ubiquitin alone or in a SUMO-dependent manner as mediated by the SUMO- targeted ubiquitin ligases, but also SUMO-mediated protection from ubiquitylation and proteolysis, can be observed to operate on replicating chromatin. Non-proteolytic functions, such as the enhancement of protein–protein interactions via SUMOylation, monoubiquitylation or linkage-speciﬁc polyubiquitylation, play an even more prominent role in the recruitment of various regulatory factors to active or stalled replisomes. Importantly, when compared to replication initiation, which is largely coupled to cell cycle regulatory events, replication fork progression appears to be an extremely delicate condition in which numerous Considering the large number of repair factors and replisome components that are SUMOylated during replication (Cremona et al., 2012; Psakhye and Jentsch, 2012), directing a collapsed fork to the nuclear pore may provide a window of opportunity for cells to ﬁne-tune repair events by altering the fate of various repair and replication factors via posttranslational modiﬁcation. May 2016 \| Volume 7 \| Article 87 19 Ubiquitin and SUMO in DNA Replication García-Rodríguez et al. all the modiﬁcation events that have by now been detected in system-wide screens, distinguishing relevant from bystander events, analyzing their regulation, and ﬁnally assigning a physiological role to them will occupy many laboratories for a long time to come. modulating modiﬁcations are needed to ﬁne-tune the activity of various components or stabilize weakly associated complexes in order to maintain fork integrity. No matter whether individual factors or entire groups of proteins are concerned, there is a large gray area between those modiﬁcations that regulate unperturbed replication and those that are initiated in response to replication problems and stress conditions. FUNDING Work in this lab is funded by an advanced investigator grant from the European Research Council to HU. ACKNOWLEDGMENT The authors would like to thank Román González-Prieto and Brian Luke for critical comments on the manuscript. AUTHOR CONTRIBUTIONS Perhaps not surprisingly, a few key replication factors emerge as nodes in a network of posttranslational modiﬁcation targets around the replication fork, such as PCNA, RPA and several central recombination proteins. Complementary to these prime targets, a few conjugation and deconjugation enzymes appear to dominate the replication-associated modiﬁcation landscape and might thus be critical for coordinating diﬀerent pathways involved in signaling or damage processing. These include ubiquitin ligases such as Rad18, RNF4 and the CRL4Cdt2 complex, but also prominent isopeptidases like USP1 and USP7. Undoubtedly, the range of identiﬁed targets and functions will continue to expand with the growing interest in these factors. 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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. Zhang, S., Chea, J., Meng, X., Zhou, Y., Lee, E. Y., and Lee, M. Y. (2008). PCNA is ubiquitinated by RNF8. Cell Cycle 7, 3399–3404. doi: 10.4161/cc.7. 21.6949 Zhang, Z., Shibahara, K., and Stillman, B. (2000). PCNA connects DNA replication to epigenetic inheritance in yeast. Nature 408, 221–225. Frontiers in Genetics \| www.frontiersin.org REFERENCES doi: 10.1038/35048530 May 2016 \| Volume 7 \| Article 87 Frontiers in Genetics \| www.frontiersin.org 28
https://openalex.org/W2899627107	https://revistas.javeriana.edu.co/index.php/scientarium/article/download/18526/21096	English	null	Complete mitochondrial genome of the nesting Colombian Caribbean loggerhead turtle: first approach of tRNAs and phylogenetic analysis	Universitas Scientiarum	2,018	cc-by	9,713	Bogotá Bogotá Univ. Sci. 23 (3): 355-381, 2018. doi: 10.11144/Javeriana.SC23-3.cccl Funding: Electronic supplementary material: Supp. 1. Electronic supplementary material: Supp. 1. Keywords: Mitogenome; Caretta caretta; Cheloniidae; coding genes; sea turtle phylogeny. Complete Colombian Caribbean loggerhead turtle mitochondrial genome: tRNA structure analysis and revisited marine turtle phylogeny Katherin Otálora 1, Javier Hernández-Fernández 1, * Abstract Juan Carlos Salcedo-Reyes (salcedo.juan@javeriana.edu.co) The loggerhead marine turtle, Caretta caretta, is a widely distributed and endangered species that is facing critical population decline, especially in Colombian Caribbean rookeries. Mitochondrial DNA sequence data are of great importance for the description, monitoring, and phylogenetic analyses of migratory turtle populations. In this study, the ﬁrst full mitochondrial genome of a loggerhead turtle nesting in the Colombian Caribbean was sequenced and analyzed. This mitochondrial genome consists of 16 362 bp with a nucleotide composition of T: 25.7 %, C: 27 %, A: 35 % and G: 12 %. Sequence annotation of the assembled molecule revealed an organization and number of coding and functional units as reported for other vertebrate mitogenomes. This Colombian loggerhead turtle (Cc-AO-C) showed a novel D-Loop haplotype consisting of thirteen new variable sites, sharing 99.2 % sequence identity with the previously reported Caribbean loggerhead CC-A1 D-Loop haplotype. All 13 protein-coding genes in the Cc-AO-C mitogenome were compared and aligned with those from four other loggerhead turtles from different locations (Florida, Greece, Peru, and Hawaii). Eleven of these genes presented moderate genetic diversity levels, and genes COII and ND5 showed the highest diversity, with average numbers of pair-wise differences of 16.6 and 25, respectively. In addition, the ﬁrst approach related to t-RNAs 2D and 3D structure analysis in this mitogenome was conducted, leading to observed unique features in two tRNAs (tRNATrp and tRNALeu). The marine turtle phylogeny was revisited with the newly generated data. The entire mitogenome provided phylogenetically informative data, as well as individual genes ND5, ND4, and 16S. In conclusion, this study highlights the importance of complete mitogenome data in revealing gene ﬂow processes in natural loggerhead turtle populations, as well as in understanding the evolutionary history of marine turtles. 1. Facultad de Ciencias Naturales e Ingeniería. Grupo de Investigación en Genética, Biología Molecular y Bioinformática – genbimol, Universidad Jorge Tadeo Lozano, Cra 4 No 22-61, Bogotá, Colombia, South America. Received: 02-10-2017 Accepted: 16-04-2018 Published on line: 31-10-2018 Received: 02-10-2017 Accepted: 16-04-2018 Published on line: 31-10-2018 Introduction Marine turtles (superfamily Chelonioidae) comprise seven existing species grouped into two families: Cheloniidae, including the ﬂatback (Natator depressus), olive ridley (Lepidochelys olivacea), Kemp’s ridley (Lepidochelys 356 Mitochondrial genome of Caretta caretta kempii), loggerhead (C. caretta), hawksbill (Eretmochelys imbricata), and green turtle (Chelonia mydas) species (Pritchard & Mortimer, 1999); and Dermochelyidae which currently comprises a single species, the leatherback sea turtle (Dermochelys coriacea). The loggerhead turtle, Caretta caretta (Cc) is distributed around the oceans of the world in tropical and subtropical latitudes (Amorocho, 2003). Its main nesting locations have been reported in the coasts of the peninsula of Florida (FWC 2015), in the western Brazilian Atlantic Ocean, in the Eastern Mediterranean Sea, in the Omani Arabian Sea, in Madagascar, and in Japan (Dodd 1988, Lancheros & Hernández 2013, Hernández et al. 2017). Despite its wide global distribution, it is considered as an endangered species (IUCN 2016). Loggerhead populations are directly threatened by several anthropic activities including: ﬁsheries bycatch, excessive ﬁshing/hunting, and illegal trade of eggs and meat. In addition, Loggerhead turtle populations are affected by habitat deterioration, coastal development, pollution, pathogens and climate change (Eckert et al. 2000, Lancheros & Hernández, 2013, Machado & Bermejo, 2012). Loggerhead turtles reach their sexual maturity at around 20-30 years of age (Machado & Bermejo, 2012), which does not offset the rampant overall population decline of the species. The threat to Loggerhead turtles has been well documented the Colombian Caribbean (Amorocho, 2003, Ceballos-Fonseca, 2004), where the world’s second highest number of catches per year (approximately 600 turtles) has been reported (Humber et al. 2014). This, despite existing national laws and international agreements to protect the species from anthropic threats (SWOT 2012, IUCN 2016). In all vertebrate taxa, the mitochondrial genome (mitogenome) is arranged as a single, circular, and haploid DNA molecule that features a uniquely high mutation rate, is non-recombining, maternally inherited, and has a specific organization and expression mode (Avise, 1994). Stretches from the mitogenome constitute the most commonly used molecular markers for genetic analysis of loggerhead turtle populations (Drosopoulou et al. 2012, Duchene et al. 2012). The loggerhead turtle mitogenome contains 37 coding units including two ribosomal RNAs (rRNAs) genes, 22 transfer RNAs (tRNAs) genes, 13 protein-coding genes, and one non-coding region of approximately 1 100 bp called the D-Loop or control region. This D-Loop contains the origin of the H replication strand and signals for mitochondrial replication and transcription (Drosopoulou et al. Introduction 2012, Duchene et al. 2012, Chiari et al. 2012). ^ ^ In sea turtles, as well as in other vertebrates, point mutations in tRNA genes are likely to alter the 3D structure and function of this machinery, hence compromising peptide synthesis and possibly leading to systemic lifespan-threatening conditions. Despite the key role of mitochondrial Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 357 Otálora & Hernández-Fernández tRNAs, their study has almost exclusively been undertaken in humans (MITOMAP, 2018). But, the availability of large databases containing thousands of tRNA sequences from hundreds of complete genomes has promoted the development of the new ﬁeld of “tRNAomics” (Marck & Grosjean, 2002). Furthermore, the understanding of sea turtle tRNA secondary and tertiary structures can beneﬁt greatly from such structural biology resources (Popenda et al. 2012). Mitochondrial D-Loop haplotypes have been useful in the identiﬁcation of sea turtle individuals, nesting colonies, and their relationship with foraging areas. Studies have been carried out with mitochondrial haplotypes addressing important aspects of the phylogeography, phylopathy (natal homing), genetic structure, and maternal lineages of loggerhead turtles (Bowen et al. 1995, Bowen & Karl, 2007). The most thorough analyses of loggerhead turtle nesting colonies, based on D-Loop sequence data, have been carried out in Brazil (Reis et al. 2010), Southeastern United States (Francisco et al. 1999), the Atlantic-Mediterranean (Encalada et al. 1998), and the Paciﬁc Ocean (FitzSimmons et al. 1996, Nobetsu et al. 2004 and Hatase et al. 2002). Several studies have employed data from single mitochondrial regions (e.g. the Cytochrome b gene (Cytb), ND4 gene, and the D-Loop) to best explain the phylogeny of different animal taxa (Dutton et al. 1996, Scotto 2006, Adebambo 2009, Duc ^ hene et al. 2011). However, there is an ongoing debate whether single mitochondrial markers are ideal to trace phyologentic histories (Scotto, 2006). The entire mitogenome is becoming the marker of choice for phylogenetic reconstructions, since it provides better phylogenetic resolution and precision than single traditional markers (Duc ^ hene et al. 2011, Novelleto et al. 2016, Miya et al. 2003, Kim et al. 2005, Jung et al. 2006, Parham et al. 2006, Drosopoulou et al. 2012). The reconstruction of the evolutionary history of the Cheloniidae has been performed via phylogenetic tree analyses based on genetic data from the entire mitogenome (Kim et al. 2005, Duc ^ hene et al. 2012, Drosopoulou et al. 2012). Sampling, DNA extraction and quantiﬁcation Sampling, DNA extraction and quantiﬁcation A single blood sample was collected from a loggerhead turtle. This turtle was found at Don Diego beach (11◦16’ N - 73◦45’ W) in the Colombian Caribbean. This turtle showed the morphological characteristics of the logger head species Suppl. 1, was in good health condition, and had no evident physical anomalies. The sample was obtained from the dorsal cervical sinus of the turtle according to Dutton’s method (1996). The blood sample was taken in a test tube with Tris-EDTA buffer to avoid coagulation and transported at 4 ◦C to the Molecular Biology Laboratory at the Universidad Jorge Tadeo Lozano, in Bogotá. Total genomic DNA was extracted by using the GF-1 Tissue DNA Extraction Kit (Vivantis, Subang Jaya, Malaysia) according to manufacturer’s instructions. The amount of DNA extracted was measured with a Nanodrop 1000 and analyzed with the ND-1000 V3.7.1 program (Thermo Scientiﬁc, Waltham, USA). Primer design The sequencing strategy for the entire mitogenome was based on PCR ampliﬁcation of overlapping fragments of 800 - 2 500 bp in length. The overlap among fragments was of 50 - 200 bp to facilitate full sequence assembly. A total of 22 primer pairs were employed to sequence the mitogenome of the Colombian Caribbean loggerhead turtle (Table 1). Seventeen primer pairs were designed using the Overlapping Primer Sets Program (Whitehead Institute, Cambridge, USA) based on the mitochondrial genome sequence of another loggerhead sea turtle (Cc-MS-G, GenBank accession number NC_016923.1). The remaining ﬁve primer pairs were used as previously described by Drosopoulou et al. (2012). Different analyses were performed to identify chimeras between the mitochondrial genome and nuclear paralog sequences. First, the mitochondrial DNA was assembled with the reference genome of the loggerhead turtle (GenBank accession number NC_016923.1). Then, the mitogenome was aligned with mitogenomes of other four loggerhead turtles, and a phylogenetic tree was built using the complete mitogenome sequences of all six sea turtle species. Complete mtDNA ampliﬁcation and sequencing Introduction In this study, the complete mitochondrial genome of a loggerhead turtle of the Colombian Atlantic Ocean (Cc-AO-C) was sequenced and analyzed with three purposes: (1) to compare the characteristics of mitochondrial genome with all previously reported mitogenomes of loggerhead individuals nesting in Hawaii, Paciﬁc Ocean (Cc-PO-H); Peru, Paciﬁc Ocean (Cc-PO-P); Greece, Mediterranean Sea (Cc-MS-G) and Florida, Atlantic Ocean (Cc-AO-F). (2) Assessing the mutations in the tRNAs genes and their possible implications in 2D and 3D structures, and (3) revisiting the phylogeny of the superfamily Chelonioidae using data from 13 protein-coding genes, the 16S rRNA gene, and the complete mitochondrial genome. Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 358 Mitochondrial genome of Caretta caretta Materials and methods Materials and methods http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Complete mtDNA ampliﬁcation and sequencing PCR reactions were carried out to a ﬁnal volume of 25 µl. Each PCR reaction included: 1X PCR buffer (160 mM (NH4)2SO4, 67 mM Tris-HCl [pH 8.8 to 25 ◦C], 0.1 % Tween-20), (Bioline Inc., Oxnard, USA), 1.5-3.0 mM de MgCl2, http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Universitas Scientiarum Vol. 23 (3): 355-381 359 Otálora & Hernández-Fernández Table 1. Sequence, position and ampliﬁed genes of the loggerhead turtle Cc-AO-C mitogenome with primer pairs used as described in the text. Oligo 5´-3´ Forward 3´-5´ Reverse Gene Position Reference CC1 GGCAGTAAAGTTCATTCGTTCCTC GCCGATTGGTTGTTAGTTTGGG D-Loop-, 12S 15925-358 Drosopoulou et al. 2012 CC2 GCCACCGCGGTTATACAAG CAGTTAGCTACACCTTGACC 12S 315-788 Drosopoulou et al. 2012 CC3 ATCTACCTCACCATCCCTTGCC GTCTTTCCACTCTTTTGCCACAG 12S, 16S 662-1441 Drosopoulou et al. 2012 CC4 CCTAAACAATTAATAAGTCA TTAAGTACTTTATGTTGTTT 12s-16s 750-1550 Present study CC5 TAGCTGGTTGCTCAATAAAA TCGCCCCAACCAAAAATATAG 16s 1500-2300 Drosopoulou et al. 2012 CC6 CTGACTAAGCCCTAAAAAGCAAAG CCCTGGGGTAGCTTGGTTCGTTGAT 16S 1269-2437 Present study CC7 GGACTCCCACCTATACCATAG TTGTAATTATTCATCCTAGATGGG ND2 2250-3050 Present study CC8 TACGAAAAATCATAGCATTC ATTGCAAATTTAAAGATATATCT ND2 3750-4550 Present study CC9 TAAAAAGCGGGAAAACCCAG GTTGTATTTAGATTTCGGTCTGT COI 4500-5300 Present study CC10 GTACTCGCCGCAGGCATTACCA ATAACTACTGCTACTATAGAGA COI 6000-6800 Present study CC11 AACTCTATTTCATCAATTGG TCAGCTGAGATTAGTATTC COI-COII 6750-7550 Present study CC12 ATCGCATAGTAATACCAATA TCATATTGGAATAGCTAGTC COII- ATP8-ATP6 7500-8300 Present study CC13 CTACTAGGCCTTCTACCCTA TGGGGTAATTCCTGTAGGTG ATP6- COIII 8250-9050 Present study CC14 CCCCTACCCCAGAACTAGGA AAGGTGAGAGTTGGGGATGG COIII-ND3 9000-9800 Present study CC15 CGCCTGATACTGACACTTCG CGTTTTCGTGAGGTTGGTTC ND3-ND4L 8930-9830 Present study CC16 AACCCATGGTTCAGACCAAC GTGTAATTGCGCCGGTGTAG ND4L-ND4 9730-10630 Present study CC17 CACTGAAAACGGTTCCTCATC GGGTGATTAGGGCTAAGAGG ND4-ND5 10530-11430 Present study CC18 ACACAATGAGGGGAAACACC CTAGGCAAAGGCAGGTTGAG ND5 11330-12230 Present study CC19 CAGGAAAATCAGCCCAATTC GATGTGTTGCGATGTTTTGG ND5-ND6 12230-13030 Present study CC20 GGAGTAATCCAGGTCGGTTTC GGAATAGAATGGTGGTTAGGG ND1 12930-13830 Present study CC21 GCCTCAAACTCCAAATACGC GTGGTAGGCCTGAGAAGGTG ND1 13730-14730 Present study CC22 TACCCCACGAGATAAACCACG TAAGTATTCTGCACGGCCAATCA ND6, CytB 13636-16135 Drosopoulou et al. 2012 Universitas Scientiarum Vol. 23 (3): 355-381 360 Mitochondrial genome of Caretta caretta 0.4-1.0 µM of each forward and reverse primer, 200 µM of each dNTP, 1U of Taq Polymerase and 60 ng of DNA (Bioline Inc., Oxnard, USA). The employed thermocycling program consisted of an initial denaturation step at 94 ◦C for 5 min, followed by 35 cycles at 94 ◦C for 1 min, 30 ◦C - 50 ◦C (depending on each primer pair) for 1 min, 72 ◦C for 1 min, and a ﬁnal extension step at 72 ◦C for 10 min. The PCR reaction was carried out in a block PTC-100TM Programmable Thermal Controller Thermocycler (MJ Research, Madison, USA). Complete standardization to this protocol was described by Beltran-Torres et al (2013). The PCR-ampliﬁed electrophoretic bands were puriﬁed using the GF-1 Gel DNA Recovery kit (Vivantis Malaysia HQ). Complete mtDNA ampliﬁcation and sequencing Puriﬁed material was used for double strand (5’-3’ and 3’-5’) sequencing reactions, using the automated tag DyeDeoxy Terminator Cycle-sequencing method in an ABI 3730XL sequencer (Applied Biosystems, Foster City, USA) at SSIGMOL, Universidad Nacional de Colombia, Bogotá, Colombia. Mitogenome assembly The 22 obtained sequences were aligned using ClustalW (Thompson et al. 1994) and assembled by means of the Geneious R6 program (Biomatters Ltd., Auckland, New Zealand) using the Cc-MS-G mitogenome as reference sequence (GenBank accession number NC_016923.1). To poceed with the assembly of the Cc-AO-C loggerhead sea turtle mtDNA reads to the reference sequence as FASTA ﬁles, the following options were used: File, import from ﬁle and ﬁnally Map to Reference. To determine the nucleotide composition of the assembled mitogenome, the “statistics” option was run. The Geneious Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990) was used to identify sequence similarities among the newly generated assembly and other loggerhead mitogenome assemblies previously mentioned. Functional annotation of the Cc-AO-C mitogenome assembly was made using BLASTX. Genetic variation analysis Genetic variation analysis Standard diversity indices, such as number of haplotypes (k), number of polymorphic sites (S), haplotype diversity (H), average number of differences between pairs of sequences (Π), and nucleotide diversity (π) according to Nei (1987) were estimated for each one of the thirteen mitochondrial protein coding genes from ad hoc sequence alignments of the Cc-AO-C turtle sequence (accession number KP256531.1) with sequences of other four loggerhead mitogenomes. These loggerhead mitogenomes were downloaded from the NCBI database and consisted of the Cc-AO-F (Florida-USA) (accession number JX454983), Cc-MS-G (Greece) (accession number NC_016923), Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 361 Otálora & Hernández-Fernández Cc-PO-P (Peru) (accession number JX454988), Cc-PO-H (Hawaii) (accession number JX454977). All genetic variation estimators were obtained with DNAsp v5.10 (Librado & Rozas, 2009). A similar approach was also applied to the D-Loop region of the Cc-AO-C and the afore mentioned four loggerhead mitogenomes and a set of 92 loggerhead D-Loop haplotype stretches of the Archi Carr Center for Sea Turtle Research at the University of Florida (accstr.uﬂ.edu). tRNA structure analysis Prediction of tRNA 2D structures were done with ARWEN (http://mbio-serv2.mbioekol.lu.se/ARWEN/) (Laslett & Canbäck 2008) and 3D structures were predicted with the 3D RNA composer Program (Popenda et al. 2012) (http://rnacomposer.cs.put.poznan.pl/Home/Compose). All structures were visualized using Geneious R6 in pdb format. The 3D structure of the tRNAs of Cc-AO-C was compared to those described for the loggerhead turtles of Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H. These tRNA data were also used to perform intraspeciﬁc analyzes. Phylogenetic analysis Universitas Scientiarum Vol. 23 (3): 355-381 Phylogenetic analysis Phylogenetic inferences were made for the superfamily Chelonioidae using data from individual genes and complete mitochondrial genomes. The inference was made with mitogenome sequence data from seven sea turtle species: C. mydas (Cm-AO) (accession number NC_000886.1), N. depressus (Nd-PO-A) (accession number NC_018550.1), E. imbricata (Ei-AO-C) (accession number KP2218061), C. caretta (Cc-MS-G) (accession number NC_016923), L. olivacea (Lo-PO-CR) (accession number NC_028634.1), L. kempi (Lk-AO-US) (accession number JX_454982.1), and the mitogenome described in this study. The mitochondrial genome of D. coriacea (Dc-AO-US) (accession number JX_454989.1) was used as an outgroup. Phylogenetic analyses were performed using three approaches, Maximum Likelihood (ML), Bayesian Inference algorithms (BI), and Maximum Parsimony (MP). ML and BI analyses were made with Geneious R6 (Biomatters Ltd., Auckland, New Zealand) and MP with MEGA 5.2. (Tamura et al. 2011). The models of nucleotide substitution of Tamura-Nei (TN), Hasegawa, Kishino and Yano (HKY), and the Generalized Time Reversible (GTR) model were used in the construction of phylogenetic trees. These models were chosen based on the lowest scores of the Bayesian Information Criterion (BIC) implemented in MEGA 5.2. (Nei & Kumar 2000, Tamura et al. 2011). For ML- and MP-based phylogenetic analyses, 1 000 bootstrap replicates were performed to generate a good statistical support. Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 362 Mitochondrial genome of Caretta caretta Bootstrap values above 70 % were accepted as strong enough support for the different branches, according to Hillis & Bull (1993). 10 000 iterations were performed for the BI analysis. A consensus tree, with posterior probability values expressed in percentages, was obtained. Results and discussion The Cc-AO-C mitogenome sequence The complete mitogenome sequence (16 362 bp in length) of the loggerhead turtle individual Cc-AO-C was obtained and deposited in the GenBank under accession number KP256531.1. Upon analysis of this mitogenome sequence, we conﬁrmed that the sampled turtle was indeed a member of the Caretta caretta species. Since hybrids between sea turtles have been frequently reported (Bowen & Karl, 2007; Drosopoulou et al. 2012; Duc ^ hene et al. 2012), it was necessary to ascertain that the captured mitogenome was indeed from the loggerhead species. In addition, attention was paid during primers design to avoid unintended ampliﬁcation and sequencing of nuclear paralogs of some mitogenome genes. Moreover, obtained sequence reads were inspected for double peaks, as seen in diploid nuclear sequences, before mitogenome assembly. As revealed by its base composition, the sampled mitogenome was A-rich (35 %) and had a low content of G (12.2 %), with intermediate C and T contents of 27 % and 25.7 %, respectively. The mitogenome of the Cc-AO-C turtle contains 13 protein-coding genes (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COI, COII, COIII, ATP6, ATP8, Cytb) two rRNA genes (12S and 16S), 22 tRNA genes and one non-coding control region (D-Loop). Regarding protein-coding, rRNA, and tRNA genes, the obtained sequence of the Cc-AO-C mitogenome corresponded well to functional and codon usage annotations reported for other marine turtles (Kumazawa & Nishida 1999, Duc ^ hene et al. 2012, Drosopoulou et al. 2012) (Suppl. 1) Universitas Scientiarum Vol. 23 (3): 355-381 Sequence variation across mitogenome protein-coding genes and D-Loop Sequence variation across mitogenome protein-coding genes and D-Loop The degree of sequence identity for all genes and functional units of the obtained loggerhead mitogenome was assessed against each of the other four loggerhead mitogenome sequences. Across all protein-coding genes, the average pair-wise sequence conservation degree was 98.49 % (Table 2). Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 363 Otálora & Hernández-Fernández Mitogenome ATP8 ATP6 COI COII COIII ND1 ND2 ND3 ND4 ND4L ND5 ND6 CyTb D-Loop Cc-AO-C Cc-AO-F 100 100 100 94.2 99.0 99.7 100 98.3 99.8 99.7 97.6 99.6 99.7 94.4 Cc-MS-G 100 100 99.9 94.2 99.0 99.8 100 98.3 99.8 100 97.6 99.6 99.7 94.6 Cc-PO-P 99.4 99.6 99.4 93.5 98.7 98.6 99.2 96.0 99.7 98.3 97.0 99.0 99.5 96.9 Cc-PO-H 90.0 99.6 99.4 93.5 98.7 98.6 98.8 96.0 99.7 98.3 97.0 99.0 99.5 96.1 Table 2. Identity percentages for all mitochondrial protein-coding genes between the Cc-AO-C and each mitogenome sequence of the four loggerhead mitogenomes: Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H. 23 (3): 355-381 364 Mitochondrial genome of Caretta caretta The parts of the mitogenome that showed the highest average number of differences between pairs of sequences (Π), and thus the greatest genetic variation (Table 3) and the lowest sequence identity values (Table 2) were the D-Loop and protein-coding genes COII and ND5. Compared to other mitochondrial functional units, the D-Loop has been reported as the stretch with the highest levels of genetic diversity among sea turtle populations (Abreu-Grobois et al, 2006; Novelletto et al. 2016) as a non-coding and likely neutrally evolving DNA stretch, the D-Loop is possibly one of the top informative mitogenome fragments to perform gene ﬂow analyses in populations of the species C. caretta. Based on the current results, the genes COII and ND5 could be equally useful when employed for this type of analyses. The parts of the mitogenome that showed the highest average number of differences between pairs of sequences (Π), and thus the greatest genetic variation (Table 3) and the lowest sequence identity values (Table 2) were the D-Loop and protein-coding genes COII and ND5. Compared to other mitochondrial functional units, the D-Loop has been reported as the stretch with the highest levels of genetic diversity among sea turtle populations (Abreu-Grobois et al, 2006; Novelletto et al. 2016) as a non-coding and likely neutrally evolving DNA stretch, the D-Loop is possibly one of the top informative mitogenome fragments to perform gene ﬂow analyses in populations of the species C. caretta. Based on the current results, the genes COII and ND5 could be equally useful when employed for this type of analyses. In contrast, the gene ATP8 was devoid of any sequence variation in the studied sequence set, thus having the highest degree of conservation. The availability of sequence data for the D-Loop from a broader sample of loggerhead turtles, allowed further investigation on D-Loop haplotype sequence identity across speciﬁc geographic ranges. The D-Loop haplotype of the Cc-AO-C mitogenome was most identical (99.2 %) with the CC-A1 haplotype, which is the most frequent (> 80 %) in nesting colonies along the North American east coast (North Carolina to South Florida) The CC-A1 haplotype has also been found in loggerhead turtles in the Colombian Caribbean (Franco & Hernandez, 2012, 2017). A total of 13 sites account for the differences between the Cc-AO-C and CC-A1 haplotype sequences. Mitogenome ATP8 ATP6 COI COII COIII ND1 ND2 ND3 ND4 ND4L ND5 ND6 CyTb D-Loop Cc-AO-C Cc-AO-F 100 100 100 94.2 99.0 99.7 100 98.3 99.8 99.7 97.6 99.6 99.7 94.4 Cc-MS-G 100 100 99.9 94.2 99.0 99.8 100 98.3 99.8 100 97.6 99.6 99.7 94.6 Cc-PO-P 99.4 99.6 99.4 93.5 98.7 98.6 99.2 96.0 99.7 98.3 97.0 99.0 99.5 96.9 Cc-PO-H 90.0 99.6 99.4 93.5 98.7 98.6 98.8 96.0 99.7 98.3 97.0 99.0 99.5 96.1 Table 2. Identity percentages for all mitochondrial protein-coding genes between the Cc-AO-C and each mitogenome sequence of the four loggerhead mitogenomes: Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H. Mitogenome ATP8 ATP6 COI COII COIII ND1 ND2 ND3 ND4 ND4L ND5 ND6 CyTb D-Loop Cc-AO-C Cc-AO-F 100 100 100 94.2 99.0 99.7 100 98.3 99.8 99.7 97.6 99.6 99.7 94.4 Cc-MS-G 100 100 99.9 94.2 99.0 99.8 100 98.3 99.8 100 97.6 99.6 99.7 94.6 Cc-PO-P 99.4 99.6 99.4 93.5 98.7 98.6 99.2 96.0 99.7 98.3 97.0 99.0 99.5 96.9 Cc-PO-H 90.0 99.6 99.4 93.5 98.7 98.6 98.8 96.0 99.7 98.3 97.0 99.0 99.5 96.1 Table 2. Identity percentages for all mitochondrial protein-coding genes between the Cc-AO-C and each mitogenome sequence of the four loggerhead mitogenomes: Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H. The lowest level of sequence similarity between the Cc-AO-C mitogenome and the set of four loggerhead turtle mitogenome sequences was observed at the COII gene (93-94 %); and across functional units, the Cc-AO-C mitogenome was most similar with the mitogenomes of the Cc-PO-F and Cc-PO-G turtles. Furthermore, it is interesting to see how the levels of genetic identity between the mitochondrial genes of Cc-AO-C and these Atlantic-Mediterranean turtles are higher than those between the mitochondrial genes of Cc-AO-C and the turtles of the Paciﬁc Ocean. This observation can substantiate the possibility of genetic ﬂow between Atlantic-Mediterranean turtles, which is further supported by various studies on their broad migratory routes and geographical range. For instance, Casale et al (2013) provided the ﬁrst evidence of a migratory connection of a loggerhead from the Mediterranean to North America. Besides, there is evidence that loggerhead turtles born on northwestern Atlantic beaches disperse as far as eastern Atlantic coasts, and some of them even enter the Mediterranean Sea (Bolten 2003) where they share foraging grounds with the Mediterranean population (Monzon-Arguello et al. 2010, Wallace et al. 2010, Carreras et al. 2011). Sequence analysis of the ND3 gene of the Cc-AO-C mitogenome reveladed an A insertion at position 175. This programed frameshift mutation has only been seen before in the Cc-MS-G mitogenome (Drosopoulou et al. 2012), and it is a likely neutral variant since it does not lead to protein sequence changes. Moreover, the same mutation has been described in other turtle species, reptiles and birds, and it is considered as relatively ancestral (Russel & Beckenbanch 2008). http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Universitas Scientiarum Vol. Mitogenome ATP8 ATP6 COI COII COIII ND1 ND2 ND3 ND4 ND4L ND5 ND6 CyTb D-Loop Cc-AO-C Cc-AO-F 100 100 100 94.2 99.0 99.7 100 98.3 99.8 99.7 97.6 99.6 99.7 94.4 Cc-MS-G 100 100 99.9 94.2 99.0 99.8 100 98.3 99.8 100 97.6 99.6 99.7 94.6 Cc-PO-P 99.4 99.6 99.4 93.5 98.7 98.6 99.2 96.0 99.7 98.3 97.0 99.0 99.5 96.9 Cc-PO-H 90.0 99.6 99.4 93.5 98.7 98.6 98.8 96.0 99.7 98.3 97.0 99.0 99.5 96.1 Table 2. Identity percentages for all mitochondrial protein-coding genes between the Cc-AO-C and each mitogenome sequence of the four loggerhead mitogenomes: Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H. Thus, the Cc-AO-C haplotype can be regarded as novel among those described for nesting loggerhead turtles in the Caribbean. The haplotype that showed 95 % identity with Cc-AO-C D-Loop was CC-A2. This haplotype has also been reported in the Colombian Caribbean (Franco & Hernandez, 2012, 2017), and it is the dominant haplotype in loggerhead turtles of samples in Quintana Roo (Mexico), southwestern Cuba (Ruiz-Urquiola et al. 2010), and the South Florida rookeries (SE and SW combined). Prior studies have reported opposite latitudinal gradients in the frequencies of these two main haplotypes in the Caribbean. The CC-A2 haplotype is most frequent in the north and becomes less common southward, whereas the opposite pattern is seen for haplotype CC-A1 (Encalada et al. 1998; Bowen et al. 2005 and Shamblin et al. 2011). Nesting aggregation in Colombia is related to nesting colonies in Southern Florida and Mexico. Loggerhead turtles from the foraging area around Don Diego beach (in the Colombian Caribbean) are grouped with other aggregations of feeding populations from the North Atlantic, Mediterranean Sea (Spain and Italy) and to sequences frequently reported from nesting populations in the North Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 365 Otálora & Hernández-Fernández Gene/ Region Fragment length (bp) Number of polymorphic sites (S) Number of Haplotypes (k) Haplotypic diversity (H) Nucleotide Diversity (π) Average number of pair-wise differences (Π) A ATP6 684 3 2 0.600 ± 0.175 0.02640 1.800 B ATP8 165 - 1 0.000 ± 0.000 0.00000 0.000 C COI 1548 10 3 0.800 ± 0.164 0.00375 5.800 D COII 687 38 3 0.800 ± 0.164 0.02438 16.600 E COIII 784 11 3 0.800 ± 0.164 0.00663 5.200 F Cytb 1145 8 3 0.800 ± 0.164 0.00385 4.400 G ND1 978 14 3 0.800 ± 0.164 0.00807 7.800 H ND2 1042 7 2 0.600 ± 0.175 0.00405 4.200 I ND3 352 14 3 0.800 ± 0.164 0.02057 7.200 J ND4 1381 7 3 0.800 ± 0.164 0.00304 4.200 K ND4L 297 5 3 0.800 ± 0.164 0.01010 3.000 L ND5 1797 56 3 0.800 ± 0.164 0.01436 25.000 M ND6 525 6 3 0.800 ± 0.164 0.00571 3.000 J D-Loop 681 36 3 0.800 ± 0.026 0.02891 19.400 Table 3. Universitas Scientiarum Vol. 23 (3): 355-381 Mitogenome ATP8 ATP6 COI COII COIII ND1 ND2 ND3 ND4 ND4L ND5 ND6 CyTb D-Loop Cc-AO-C Cc-AO-F 100 100 100 94.2 99.0 99.7 100 98.3 99.8 99.7 97.6 99.6 99.7 94.4 Cc-MS-G 100 100 99.9 94.2 99.0 99.8 100 98.3 99.8 100 97.6 99.6 99.7 94.6 Cc-PO-P 99.4 99.6 99.4 93.5 98.7 98.6 99.2 96.0 99.7 98.3 97.0 99.0 99.5 96.9 Cc-PO-H 90.0 99.6 99.4 93.5 98.7 98.6 98.8 96.0 99.7 98.3 97.0 99.0 99.5 96.1 Table 2. Identity percentages for all mitochondrial protein-coding genes between the Cc-AO-C and each mitogenome sequence of the four loggerhead mitogenomes: Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H. Genetic diversity estimators, for each mitochondrial proteincoding gene and the D-Loop region, for a loggerhead alignment consisting of the Cc-AO-C sequence and four loggerhead mitogenomes: Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H (see text for sequence name details). Atlantic and Mexico. This pattern suggests that individuals that use the Colombian Caribbean for feeding and reproduction are part of an Atlantic l i h h l CC A1 d CC A2 h f Table 3. Genetic diversity estimators, for each mitochondrial proteincoding gene and the D-Loop region, for a loggerhead alignment consisting of the Cc-AO-C sequence and four loggerhead mitogenomes: Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H (see text for sequence name details). Average number of pair-wise differences (Π) Atlantic and Mexico. This pattern suggests that individuals that use the Colombian Caribbean for feeding and reproduction are part of an Atlantic meta-population, where haplotypes CC-A1 and CC-A2 are the most frequent (Franco & Hernandez, 2012, 2017). The novel Cc-AO-C loggerhead haplotype may be endemic to the Colombian Caribbean rookery, and thus may suggest that Colombian loggerheads display natal homing. Atlantic and Mexico. This pattern suggests that individuals that use the Colombian Caribbean for feeding and reproduction are part of an Atlantic meta-population, where haplotypes CC-A1 and CC-A2 are the most frequent (Franco & Hernandez, 2012, 2017). The novel Cc-AO-C loggerhead haplotype may be endemic to the Colombian Caribbean rookery, and thus may suggest that Colombian loggerheads display natal homing. Atlantic and Mexico. This pattern suggests that individuals that use the Colombian Caribbean for feeding and reproduction are part of an Atlantic meta-population, where haplotypes CC-A1 and CC-A2 are the most frequent (Franco & Hernandez, 2012, 2017). The novel Cc-AO-C loggerhead haplotype may be endemic to the Colombian Caribbean rookery, and thus may suggest that Colombian loggerheads display natal homing. Atlantic and Mexico. This pattern suggests that individuals that use the Colombian Caribbean for feeding and reproduction are part of an Atlantic meta-population, where haplotypes CC-A1 and CC-A2 are the most frequent (Franco & Hernandez, 2012, 2017). The novel Cc-AO-C loggerhead haplotype may be endemic to the Colombian Caribbean rookery, and thus may suggest that Colombian loggerheads display natal homing. meta-population, where haplotypes CC-A1 and CC-A2 are the most frequent (Franco & Hernandez, 2012, 2017). The novel Cc-AO-C loggerhead haplotype may be endemic to the Colombian Caribbean rookery, and thus may suggest that Colombian loggerheads display natal homing. Universitas Scientiarum Vol. Mitogenome ATP8 ATP6 COI COII COIII ND1 ND2 ND3 ND4 ND4L ND5 ND6 CyTb D-Loop Cc-AO-C Cc-AO-F 100 100 100 94.2 99.0 99.7 100 98.3 99.8 99.7 97.6 99.6 99.7 94.4 Cc-MS-G 100 100 99.9 94.2 99.0 99.8 100 98.3 99.8 100 97.6 99.6 99.7 94.6 Cc-PO-P 99.4 99.6 99.4 93.5 98.7 98.6 99.2 96.0 99.7 98.3 97.0 99.0 99.5 96.9 Cc-PO-H 90.0 99.6 99.4 93.5 98.7 98.6 98.8 96.0 99.7 98.3 97.0 99.0 99.5 96.1 Table 2. Identity percentages for all mitochondrial protein-coding genes between the Cc-AO-C and each mitogenome sequence of the four loggerhead mitogenomes: Cc-AO-F, Cc-MS-G, Cc-PO-P and Cc-PO-H. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 366 Mitochondrial genome of Caretta caretta Universitas Scientiarum Vol. 23 (3): 355-381 tRNA variation The 22 tRNAs were distributed along the mitogenome (13 in the H-strand and 9 in the L-strand). When the 22 tRNAs sequences of the Cc-AO-C and Cc-MS-G turtles were compared, 9.1 % (2 out of 22, tRNATrp and tRNA Leu) revealed nucleotide differences (Fig. 1). The remainder 90.9 % (20) revealed a strong nucleotide conservation. Such level of conservation may be due to (Suzuki et al. 2011) (Suzuki et al. 2011) igure 1. Comparison of 2D structure of tRNATrp and tRNALeu bet oth, Cc-AO-C and Cc-MS-G that presented mutations (marked with cir Figure 1. Comparison of 2D structure of tRNATrp and tRNALeu between both, Cc-AO-C and Cc-MS-G that presented mutations (marked with circles). http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Universitas Scientiarum Vol. 23 (3): 355-381 367 Otálora & Hernández-Fernández the small size of these tRNAs and the selective pressure exerted on these important elements for the process of molecular translation (Florentz et al. 2003, Widmann et al. 2010) (Fig. 1). Secondary typology analysis of the Cc-AO-C tRNAs revealed unique features at tRNATrp and tRNALeu. These two tRNAs present typology II as deﬁned by Suzuki et al (2011) (Fig. 1). The unique features at Cc-AO-C tRNATrp and tRNALeu, however, did not lead to large changes in the predicted 3D structure of these tRNAs, with respect to other loggerhead structures of Cc-PO-H, Cc-PO-P, Cc-MS-G, and Cc-AO-F (Fig. 2). The tRNATrp was characterized by presenting tertiary interactions among positions 16 - 48, at the tRNA’s D-loop allowing the folding of the structure (Saks et al. 1998, Suzuki et al. 2011). Furthermore, this Cc-AO-C tRNATrp presented a transition in position 14 (Fig. 1) which does not allow for any interaction with position 48, leading to a modiﬁed 3D structure (Fig. 2). These structural changes might have a negative impact on the individual, and they should be studied at the population level to determine their actual frequency. Moreover, it is essential to verify if there are heteroplasmic mutations, and ﬁnally, study whether those changes can lead to a pathologic state of the turtles. Figure 2. 3D tRNA structures of the Cc-AO-C turtle with respect to the Cc-AO-F, Cc-PO-H, Cc-PO-P, and Cc-MS-G loggerhead turtle tRNA structures. A. tRNATrp B. tRNALeu. Regions of the tRNAs: (D) D-Loop, (T) T-loop, (A) anticodon are shown. Figure 2. 3D tRNA structures of the Cc-AO-C turtle with respect to the Cc-AO-F, Cc-PO-H, Cc-PO-P, and Cc-MS-G loggerhead turtle tRNA structures. A. tRNATrp B. tRNALeu. Universitas Scientiarum Vol. 23 (3): 355-381 tRNA variation Regions of the tRNAs: (D) D-Loop, (T) T-loop, (A) anticodon are shown. Figure 2. 3D tRNA structures of the Cc-AO-C turtle with respect to the Cc-AO-F, Cc-PO-H, Cc-PO-P, and Cc-MS-G loggerhead turtle tRNA structures. A. tRNATrp B. tRNALeu. Regions of the tRNAs: (D) D-Loop, (T) T-loop, (A) anticodon are shown. http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Universitas Scientiarum Vol. 23 (3): 355-381 368 Mitochondrial genome of Caretta caretta Universitas Scientiarum Vol. 23 (3): 355-381 Phylogenetic inference of marine turtles The individual markers that best explained the phylogeny of the sea turtles were ND5, ND4, and 16S when using the BI method. The ND5 gene has not been yet used as a molecular marker to do phylogenetic analysis in sea turtles (Fig. 3). However, in the present study the topology obtained with this gene is in full agreement with the currently accepted sea turtle phylogeny. These results support the analysis done by Dutton et al (1996) who used ND5 gene data to lay out a phylogenetic hypothesis for these organisms. Cases of phylogenetic incongruity among individual genes were found. For instance, trees based on data from the ATP8 and ND4L genes were not informative (results not shown), likely due to their small size and high level of nucleotide conservation (Table 2 and 3). These two genes are essential part of enzyme production in the mitochondria (Suzuki et al. 2011). The gene Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Figure 3. Phylogenetic inference of sea turtles. A: Tree based on data from gene ND4 employing Maximum Likelihood (ML), Maximum Parsomony (MP), and Bayesian Inference (BI) methods. B. Tree from complete mitogenome data following ML, MP, and BI methods. Following samples were included: L. kempii (LK), L. olivacea (LO), C. caretta- Colombian Atlantic Ocean (Cc-AO-C), C. mydas (CM), C. caretta- Greek Mediterranean Sea (Cc-MS-G), N. depressus (ND), E. imbricata (EI), and D. coriacea (DC). Figure 3. Phylogenetic inference of sea turtles. A: Tree based on data from gene ND4 employing Maximum Likelihood (ML), Maximum Parsomony (MP), and Bayesian Inference (BI) methods. B. Tree from complete mitogenome data following ML, MP, and BI methods. Following samples were included: L. kempii (LK), L. olivacea (LO), C. caretta- Colombian Atlantic Ocean (Cc-AO-C), C. mydas (CM), C. caretta- Greek Mediterranean Sea (Cc-MS-G), N. depressus (ND), E. imbricata (EI), and D. coriacea (DC). http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Universitas Scientiarum Vol. 23 (3): 355-381 369 Otálora & Hernández-Fernández ND1 resolved relations within the Cheloniidae family but was not useful in differentiating Cheloniidae from Dermochelyidae. Previous molecular studies have not established a coherent and statistically well-supported conclusion on the phylogeny of sea turtles (Kumazawa & Nishida 1999). In current phylogenetic analysis, the use of data from complete mitogenomes is gaining ground. Phylogenetic inference of marine turtles With full or partial mitogenome data, phylogenetic analyses become more robust and gain in phylogenetic resolution and greater precision compared to analysis based on data from individual markers (Duc ^ hene et al. 2011). The current results support previous relationships among sea turtle species, N. depressus as the sister taxon to Chelonia (Duch ne et al. 2012, Naro-Maciel et al. 2008) as well as the clade comprising Erecmochelys, Lepidochelys and Caretta (Fig. 3) (Dutton et al. 1999, Duc ^ hene et al. 2012). This result is important when explaining phylogenetic relationships within the family Cheloniidae, particularly the exclusion of N. ^e depressus from the subfamily Carettini (Dutton et al. 1999, Duch ne et al. 2012, Naro-Maciel et al. 2008). Out of the total number of mitochondrial markers, from which data were obtained to solve ancestry-descent relations among sea turtles, the ND5 gene produced highly supported trees. This marker can generate phylogenetic trees with a support comparable to that of a complete mitochondrial genome, and it conﬁrms the topology of the proposed phylogeny for these species. This study presents the use of mitochondrial genomes as an alternative to improve phylogenetic analysis to estimate the evolutionary relations among sea turtles. e^ Conclusions In this study, the complete mitochondrial genome of an individual of the endangered loggerhead marine turtle species, C. caretta, nesting in the Colombia Caribbean coast was sequenced. This has opened new possibilities to understand the extent of genetic variation and how matrilineal gene ﬂow happens within the loggerhead species across its broad distribution range. This loggerhead mitogenome is 16 362 bp long, comprises a non-coding region (D-Loop), 13 protein-coding genes, 22 tRNA genes and 2 rRNA (16S and 12S). This sampled nesting turtle harbors a new D-Loop haplotype, with thirteen sites differing from the closest previously reported Caribbean CC-A1 haplotype. The tRNATrp and tRNALeu presented speciﬁc mutations in Cc-AO-C. The other 20 tRNAs revealed a strong nucleotide conservation and tRNATrp presented modiﬁcation of its 3D structure. The phylogeny of sea turtles was revisited with this novel mitogenome. The entire mitogenome, and the loci ND5, ND4, and 16S provided sequence data to build well resolved trees that largely agreed with currently accepted Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 370 Mitochondrial genome of Caretta caretta sea turtle phylogenetic hypotheses. This study presents the use of complete mitogenomes as a feasible alternative to gather data useful to conduct thorough phylogenetic analysis in sea turtles. Research Permissions The samples were obtained under research permission granted by the Ministerio del Medio Ambiente y Desarrollo Territorial (No 24, June 22, 2012) and the Access Contract to Genetic Resources (No 64, April 23, 2013). Conﬂicts of interests The authors declare no conﬂict of interest and state that they are responsible for content and writing of the paper. Acknowledgements The authors would like to thank Jaime Rojas at the Ceiner Oceanarium for the loggerhead turtle blood sample collection and to the anonymous reviewers who have contributed their time and expertise to improve this manuscript. Financial support for this study was granted by the Research, Creativity, and Innovation Department at Universidad Jorge Tadeo Lozano. doi: 10.1111/j.1365-294X.2005.02598.x Bowen BW, Karl SA. 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Polymorphism in Mitochondrial Coding Regions of Mediterranean Loggerhead Turtles: Evolutionary Relevance and Structural Effects, Physiological and Biochemical Zoology, 89(6): 473- 486, 2016. doi: 10.1086/688679 doi: 10.1007/s10592-009-9975-0 Ruiz-Urquiola A, Riverón-Giró F, Pérez-Bermúdez E, Abreu-Grobois F, González-Pumariega M, James-Petric B, Díaz-Fernández R, Álvarez-Castro J, Jager M, Azanza-Ricardo J, Espinosa-López G. Population genetic structure of Greater Caribbean green turtles (Chelonia mydas) based on mitochondrial DNA sequences, with an emphasis on rookeries from Southwestern Cuba, Revista de Investigaciones Marinas, 31 (1): 33-52, 2010. Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 377 Otálora & Hernández-Fernández Russell RD, Beckenbach AT. Recoding of translation in turtle mitochondrial genomes: programmed frameshift mutations and evidence of a modified genetic code, Journal of Molecular Evolution, 67(6): 682-695, 2008. doi: 10.1146/annurev-genet-110410-132531 SWOT. The state of the world’s Sea turtles. The world’s most (and least) threatened sea turtles. SWOT Report VII. Editorial Team. Arlington, USA. 2011 doi: 10.1007/s00239-008-9179-0 Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. In Bioenergetics of the Cell: Quantitative Aspects pp. 81-100. Springer US. 1998. Shamblin BM, Dodd Mg, Bagley DN, Ehrhart LM, Tucker AD, Johnson C, Carthy RR, Scarpino RA, McMichael E, Addison DS, Williams KL, Frick MG, Ouellette S, Meylan AB, Godfrey MH, Murphy SR, Nairn CJ. Genetic structure of the southeastern United States loggerhead turtle nesting aggregation: evidence of additional structure within the peninsular Florida recovery unit, Marine Biology, 158: 571–587, 2011. doi: 10.1007/s00227-010-1582-6 Scotto C. Análisis filogenético comparativo entre secuencias codificadoras (Cytb y ATPasa8) y secuencias no codificadoras (D-Loop) del ADN mitocondrial de primates y sus implicancias evolutivas en los homínidos, Horizonte Médico, 6(2): 111-129, 2006. Suzuki T, Nagao A, Suzuki T. Human Mitochondrial tRNAs: Biogenesis, Function, Structural Aspects, and Diseases, Annual Review of Genetics, 45: 299-329, 2011. d i 10 1146/ 110410 132531 doi: 10.1146/annurev-genet-110410-132531 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Retrieved: http://seaturtlestatus.org/sites/swot/files/ report/030612_SWOT7_FinalA.pdf Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods, Molecular Biology and Evolution, 28(10): 2731- 2739, 2011. doi: 10.1093/molbev/msr121 Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 378 Mitochondrial genome of Caretta caretta Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic acids research, 22(22): 4673-4680, 1994. doi: 10.1093/nar/22.22.4673 Wallace BP, DiMatteo AD, Hurley BJ, Finkbeiner EM, Bolten AB, Chaloupka MY, Hutchinson BJ, Abreu-Grobois FA, Amorocho D, Bjorndal kA, Bourjea J, Bowen BW, Duenas RB, Casale P, Choudhury BC, Costa A, Dutton PH, Fallabrino A, Girard A, Girondot M, Godfrey MH, Hamann M, Lopez-Mendilaharsu M, Marcovaldi MA, Mortimer JA, Musick JA, Nel R, Pilcher NJ, Seminoff JA, Troeng S, Witherington B, Mast RB. Regional Management Units for Marine Turtles: A Novel Framework for Prioritizing Conservation and Research across Multiple Scales, PloS One, 5(12): e15465, 2010. http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Universitas Scientiarum Vol. 23 (3): 355-381 doi: 10.1371/journal.pone.0015465 Widmann J, Harris K, Lozupone C, Wolfson A, Knight R. Stable tRNA-based phylogenies using only 76 nucleotides, RNA, 16: 1469-1477, 2010. doi: 10.1261/rna.726010 doi: 10.1261/rna.726010 Universitas Scientiarum Vol. 23 (3): 355-381 379 Otálora & Hernández-Fernández Genoma mitocondrial completo de la tortuga caguama del Caribe colombiano: análisis de la estructura del tRNA y revisión de la filogenia de las tortugas marinas Resumen. La tortuga marina caguama, Caretta caretta, es una especie ampliamente distribuida pero que enfrenta una crítica reducción de su población en las colonias del Caribe colombiano. Los datos de las secuencias de DNA mitocondrial son de gran importancia para la descripción, monitoreo y análisis de la filogenia de las tortugas migratorias. En este estudio se secuenció y analizó por primera vez el genoma mitocondrial completo de la tortuga caguama que anida en el Caribe colombiano. Este genoma tiene un tamaño de 16.362 pb con una composición de nucleótidos de T: 25.7 %, C: 27 %, A: 35 % y G: 12 %. La anotación de la secuencia de la molécula reveló una organización y número de unidades codificantes y funcionales como los reportados para mitogenomas de otros vertebrados. Esta tortuga caguama colombiana (Cc-AO-C) mostró un nuevo haplotipo D-Loop que contiene trece nuevos sitios variables, que comparten el 99.2 % de identidad de secuencia con el haplotipo CC-A1 D-Loop previamente reportado para la tortuga caguama del Caribe. Los trece genes que codifican proteínas en el mitogenoma Cc-AO-C se compararon y alinearon con los de otras cuatro tortugas caguama de distintas localidades (Florida, Grecia, Perú y Hawái). Once de estos genes presentaron niveles moderados de diversidad genética, y los genes COII y ND5 mostraron las diversidades nucleotídicas más altas, con un número promedio de diferencias entre pares de secuencias de 6.6 y 25, respectivamente. Adicionalmente, se llevó a cabo la primera aproximación relacionada con el análisis de la estructura 2D y 3D de t-RNAs en este mitogenoma, lo cual condujo a la observación de características únicas en dos tRNAs (tRNATrp y tRNALeu). La filogenia de las tortugas marinas fue revisada a la luz de la nueva información mitogenómica. El mitogenoma, así como los genes individuales ND5, ND4 y 16S, proporcionan datos filogenéticamente informativos. En conclusión, este estudio resalta la importancia de los datos del mitogenoma para revelar procesos de flujo génico en las poblaciones naturales de tortuga caguama, así como para entender la historia evolutiva de las tortugas marinas. http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Universitas Scientiarum Vol. 23 (3): 355-381 doi: 10.1371/journal.pone.0015465 Palabras clave: mitogenome; Caretta caretta; cheloniidae; coding genes; sea turtle phylogeny. Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 380 Mitochondrial genome of Caretta caretta http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Universitas Scientiarum Vol. 23 (3): 355-381 Katherin Eliana Alejandra Otálora Acevedo j Her research interest is the analysis of evolutionary patterns in amphibians and reptiles, based primarily on the use of molecular tools and population genetics to delineate evolutionary units, management units in conservation processes, gene flow, determining connectivity patterns through the landscape and historical processes that could leave evolutionary marks on the demographics and the current geographical distribution of organisms. http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum Genoma mitocondrial de tartaruga-cabeçuda do Caribe colômbiano completo: análise de estrutura de tRNA e filogenia revisada de tartarugas marinhas Resumo. A tartaruga marinha Caretta caretta (Cc) é uma espécie amplamente distribuída e ameaçada de extinção que enfrenta um declínio crítico da população, especialmente nas colônias do Caribe colombiano. Marcadores moleculares, como sequências de DNA mitocondrial (mtDNA), são de grande importância para a descrição, monitoramento e análise filogenética de populações migratórias de tartarugas. Este estudo mostra a obtenção e análise do genoma mitocondrial de uma tartaruga-cabeçal Cc aninhada na costa Caribe da Colômbia. O genoma mitocondrial é constituído por 16.362 pb, com uma região não codificante (D-Loop), 13 genes codificadores de proteínas (13 PCG), 22 genes tRNA e 2 rRNA (16S e 12S) e uma frequência nucleotídica de T: 25.7 % , C: 27 %, A: 35 % e G: 12,2 %, todos organizados de forma semelhante à maioria dos mitogenomos de vertebrados. Esta tartaruga Cc colombiana apresentou um novo haplótipo D-Loop com treze sítios polimórficos quando comparado ao haplótipo CC-A1.1 (96 %). Além disso, onze genes codificadores de proteínas entre as tartarugas marinhas de diferentes origens apresentaram uma diversidade genética semelhante, exceto os genes COII e ND5 que apresentaram o maior número médio de diferenças entre pares de seqüências (16.600 e 25.000, respectivamente). Aqui relatase a primeira abordagem relacionada à análise de estruturas 2D e 3D para Cc e descrevese as diferenças em dois tRNAs (tRNATrp, tRNALeu). As inferências bayesianas e os métodos de máxima verossimilhança explicam melhor a filogenia das tartarugas marinhas quando utilizamse mitogenomes completos, assim como os genes ND5, ND4 e 16S. Os genes marcadores ATP8, ND4L e ND1 apresentaram relação filogenética pouco suportada. Como conclusão, este estudo apresenta o uso de mitogenomes completos como uma alternativa para melhorar a análise filogenética em tartarugas marinhas e é a primeira análise genética de mitogenomes completos de nidificação na Colômbia. Palavras-chave: mitogenoma; Caretta caretta; cheloniidae; genes codificadores; tartaruga marinha, filogenia. Universitas Scientiarum Vol. 23 (3): 355-381 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 381 Otálora & Hernández-Fernández Katherin Eliana Alejandra Otálora Acevedo Her research interest is the analysis of evolutionary patterns in amphibians and reptiles, based primarily on the use of molecular tools and population genetics to delineate evolutionary units, management units in conservation processes, gene flow, determining connectivity patterns through the landscape and historical processes that could leave evolutionary marks on the demographics and the current geographical distribution of organisms. Universitas Scientiarum Vol. 23 (3): 355-381 Javier Hernández Fernández Associate professor in the Department of Natural and Environmental Sciences at the Jorge Tadeo Lozano University in Bogota for 12 years. During these years he has taught genetics and molecular biology. Leader of the Research Group "Genetics, Molecular Biology and Bioinformatics". He is a PhD candidate at the Pontificia Universidad Javeriana and is currently working on the ecotoxitranscriptomics of the marine turtle Caretta caretta. Universitas Scientiarum Vol. 23 (3): 355-381
https://openalex.org/W4307517740	https://www.researchsquare.com/article/rs-1231062/latest.pdf	English	null	Design of a fully integrated VHF CP‐PLL frequency synthesizer with an all‐digital defect‐oriented built‐in self‐test	Journal of engineering	2,022	cc-by	8,928	Design of a Fully Integrated VHF CP-PLL Frequency Synthesizer with an All-Digital Defect-Oriented Built- In Self-Test. Benjamin Kommey (  bkommey.coe@knust.edu.gh ) Kwame Nkrumah University of Science and Technology Ernest Addo Kwame Nkrumah University of Science and Technology Jepthah Yankey Kwame Nkrumah University of Science and Technology Andrew Agbemenu Kwame Nkrumah University of Science and Technology Eric Tchao Kwame Nkrumah University of Science and Technology Bright Akowuah Kwame Nkrumah University of Science and Technology Benjamin Kommey (  bkommey.coe@knust.edu.gh ) Kwame Nkrumah University of Science and Technology Ernest Addo Kwame Nkrumah University of Science and Technology Jepthah Yankey Kwame Nkrumah University of Science and Technology Andrew Agbemenu Kwame Nkrumah University of Science and Technology Eric Tchao Kwame Nkrumah University of Science and Technology Bright Akowuah Kwame Nkrumah University of Science and Technology Research Article Design of a Fully Integrated VHF CP-PLL Frequency Synthesizer with an All-Digital Defect-Oriented Built-In Self-Test. Benjamin Kommey1, Ernest Ofosu Addo1, Jepthah Yankey1, Andrew Selasi Agbemenu1, Eric Tutu Tchao1, and Bright Yeboah Akowuah1 Benjamin Kommey1, Ernest Ofosu Addo1, Jepthah Yankey1, Andrew Selasi Agbemenu1, Eric Tutu Tchao1, and Bright Yeboah Akowuah1 1Department of Computer Engineering, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Kommey et al Abstract This paper presents the design of an on-chip charge pump phase-locked loop (CP-PLL) with a fully digital defect oriented built-in self-test (BIST) for very-high frequency (VHF) applications. The frequency synthesizer has a 40 to 100 MHz tuning range and uses a ring voltage-controlled oscillator for frequency synthesis. The PLL exhibits a phase noise of -132 dBc/Hz at 1 MHz and consumes 1.8 mW on a 3 V supply. The BIST implementation uses fewer external input or output, is capable of efficient fault diagnosis, and is compact, posing a low area overhead. The integrated circuit design was realized in the AMI 0.6µ complementary metal oxide-semiconductor process. Introduction The PLL is a very essential and ubiquitous component of modern electronics and finds a widespread use in communication systems where it is used for frequency synthesis or clock generation/synchronization/recovery. Other applications include frequency modulation and demodulation. Its use is indispensable for clock distribution, particularly in complex high performance synchronous systems where it is very difficult to distribute clean, low- skew high-frequency, phase synchronous, tunable clock signals reliably throughout various devices and package technologies (single-chip modules, multichip modules, cards etc.). A PLL-based system design allows the distribution of a relatively low-frequency reference clock to each or group of components. Each component’s PLL multiplies the reference to the required high frequency signals for use while maintaining proper phase alignment. 1 1 Kommey et al A PLL is described as a non-linear system that uses negative feedback to ’lock’ an output signal in phase (hence frequency) with a reference signal. The loop combines a voltage-controlled oscillator (VCO) and a phase/frequency comparator so connected that the oscillator maintains this constant relative phase angle relationship. Other basic components include a frequency divider, charge pump and a loop filter. The VCO generates an output signal based on the input reference clock. VCOs used in PLL frequency synthesizers are commonly realized using LC-tanks [1, 2] or ring topologies [3, 4]. Generally, LC-VCOs consist of an inductor L and voltage-tunable capacitor C forming a parallel tank which oscillates at 1 2𝜋√𝐿𝐶. They also feature active elements which compensate for losses in the inductor. Ring VCOs rely on an interconnection of amplifiers or inverters to achieve a 360° phase shift, which causes the structure to oscillate. In the designs, where single-ended inverters are used, the number of delay stages are required to be odd to achieve the ringing effect. LC VCOs have the lower in-band phase noise and power consumption of the two but has a rather limited tuning range and occupies large die area in fully integrated PLLs. On the other hand, ring oscillators offer easier design or integration, smaller area, and traditionally wider tuning ranges at the cost of higher phase noise and power expenditure. Some works have demonstrated the design of low noise ring VCOs. However, their power consumption remains higher than that of their LC counterparts [5]. The phase/frequency detector (PFD) is a tri-state device which compares the phase difference between the reference input and the VCOs output signal. Introduction The PFD produces feedback error pulses proportional to the phase difference, to correct the discrepancy. The generated error signal is smoothed out by the CP integrator (CP + loop filter) and applied to the VCO to generate the desired signal. The PFD structure may be a simple XOR gate, latch-based or of the pre-charge type [6]. Finally, a PLL may require a feedback divider, programmable or fixed, to bring the VCO output to the range of the reference signal. The VCO signal can be tuned in integer steps of the reference clock or in fractional amounts. Figure 1 shows the architecture of a basic CP-PLL. A PLL is described as a non-linear system that uses negative feedback to ’lock’ an output signal in phase (hence frequency) with a reference signal. The loop combines a voltage-controlled oscillator (VCO) and a phase/frequency comparator so connected that the oscillator maintains this constant relative phase angle relationship. Other basic components include a frequency divider, charge pump and a loop filter. The VCO generates an output signal based on the input reference clock. VCOs used in PLL frequency synthesizers are commonly realized using LC-tanks [1, 2] or ring topologies [3, 4]. Generally, LC-VCOs consist of an inductor L and voltage-tunable capacitor C forming a parallel tank which oscillates at 1 2𝜋√𝐿𝐶. They also feature active elements which compensate for losses in the inductor. Ring VCOs rely on an interconnection of amplifiers or inverters to achieve a 360° phase shift, which causes the structure to oscillate. In the designs, where single-ended inverters are used, the number of delay stages are required to be odd to achieve the ringing effect. LC VCOs have the lower in-band phase noise and power consumption of the two but has a rather limited tuning range and occupies large die area in fully integrated PLLs. On the other hand, ring oscillators offer easier design or integration, smaller area, and traditionally wider tuning ranges at the cost of higher phase noise and power expenditure. Some works have demonstrated the design of low noise ring VCOs. However, their power consumption remains higher than that of their LC counterparts [5]. The phase/frequency detector (PFD) is a tri-state device which compares the phase difference between the reference input and the VCOs output signal. The PFD produces feedback error pulses proportional to the phase difference, to correct the discrepancy. Introduction The generated error signal is smoothed out by the CP integrator (CP + loop filter) and applied to the VCO to generate the desired signal. The PFD structure may be a simple XOR gate, latch-based or of the pre-charge type [6]. Finally, a PLL may require a feedback divider, programmable or fixed, to bring the VCO output to the range of the reference signal. The VCO signal can be tuned in integer steps of the reference clock or in fractional amounts. Figure 1 shows the architecture of a basic CP-PLL. Fig. 1 Architecture of the charge-pump PLL Fig. 1 Architecture of the charge-pump PLL The basic BIST concept involves the design of test circuitry around or into a system to allow automatic measuring of system properties by generating and/or applying appropriate test stimuli and observing the response of the host system. This testing approach attempts to migrate parts of Automated Test Equipment (ATE) functionalities onto the chip for facilitated testing and reduced test cost. Various testing procedures are involved at three main phases of the hardware products design and manufacturing cycle: Pre-silicon verification, manufacturing tests, and post- 2 Kommey et al silicon validation. Fault diagnosis during manufacturing is very crucial and can greatly ease the workload on post- silicon validation. This is where BIST becomes relevant. A basic BIST structure introduces three hardware blocks into a host circuit: a test stimulus generator (TSG), a test controller and a response analyzer (Figure 2). Fig. 2 Typical BIST structure [27] Fig. 2 Typical BIST structure [27] The TSG produces a set of test vectors for a given circuit under test (CUT) depend on the specific faults being tested for and the required fault coverage. A TSG may come in the form of a read-only-memory (ROM) with stored patterns, a linear feedback shift register (LFSR), or a counter. The BIST controller manages all transactions in the self-testing procedure. BIST controllers typically have a single-input single-output external interface. An input signal activates self-test sequence after which the controller puts the CUT in test mode by allowing the TSG and the controller itself to drive the CUTs inputs directly though isolation circuitry like a multiplexer (MUX) or some other switching mechanism. Depending on the BIST paradigm, the controller feeds the TSG with signals required to generate test sequences. In some other cases, the TSG and the controller may even be fused into one block [7]. Introduction The controller also interacts with the response analyzer to ensure that the CUTs responses to the test suite are properly collected and compared with known outputs. The outcome of the comparison which informs whether the CUT is faulty or otherwise is compacted into a single pass/fail response using functions such as signature analysis or syndrome counts. Fault-free CUT responses are sometimes stored on- or off-chip for future reference. Hardware implementations of response analyzers include LFSRs, multiple-input shift registers (MISRs) or comparator logic with a ROM-based lookup table. BIST paradigms are broadly classified as either defect oriented or functional. Defect-oriented schemes focus on the detection of possible structural flaws in a chip [8] whereas functional methods target direct on-chip measurement of performances [9–11]. Furthermore, a BIST scheme may be designed and appropriate for generic use or as a one-off solution. That is, the scheme may be applicable to any circuits class [12, 13], specific to a given class [14, 15], or even tailored to individual intra-class circuit architectures [16, 17]. Generic defect-oriented paradigms employ topology transforming techniques by integrating oscillation-based mechanisms [10, 18] or using structures such as pull-up/pulldown transistors [8]. Embedding BIST schemes into integrated circuits/systems is quite a complex task as an effective strategy must satisfy some very challenging requirements. The integration of 3 Kommey et al the BIST infrastructure should not require significant redesign or reconfiguration and it must be transparent to the main circuitry without degrading its performance. Furthermore, the test infrastructure must pose minimal overhead in terms of die real-estate and power consumption. Intuitively, a small footprint of BIST within the chip die reduces manufacturing costs as well as the probability of defect occurrence, considerably. An elegant way of achieving this goal is by re-purposing existing IC blocks as part of the test circuit where possible. The test paradigm should also have a fast response time to enable low-latency error detection. The speed criterion is also useful during the design phase because it allows for large-scale defect simulations in reasonable time and enables performing simulations for multiple test design iterations. Next, the BIST circuitry should be more rugged than the IC to guarantee low failure probability. As a good strategy, some BIST designs even attempt to include checker facilities to test the infrastructure itself [12, 19]. Introduction Finally, the BIST should also interface easily with standard test access mechanisms and must be portable across technology nodes without requiring significant reconfiguration. In this paper, the design of a fully integrated PLL frequency synthesizer which operates from 40MHz to 100MHz is presented. The designed PLL exhibits phase noise of -132dBc/Hz at 1 MHz, which is low enough for a wide array of applications in the VHF band. An all-digital BIST is designed into the PLL core. The entire system is designed in a typical CMOS process using a 3 V power supply which is commonly found in today’s portable products. The remainder of this paper is organized as follows: Section 2 presents the related works; section 3 considers the design of the CP-PLL core and the BIST including a stepwise description of their building blocks as well as the PLL-BIST integration. Experimental verification of the proposed design is described and discussed in Section 4. The paper is concluded in Section 5. Literature Review PLL since its inception has been integral especially in communication technology. In recent times, its advancements in integrated circuit (IC) technology have made it an indispensable tool in areas such as wireless systems [20], micro-electro-mechanical systems (MEMS) [21], consumer electronics [22], motor control [23], and many more. PLL employs a negative feedback architecture that enables economic multiplication of crystal frequencies by larger variable numbers. Information obtained from this field prompted a few studies aimed at developing phase locked loops with a handful of models and testing techniques developed. Classical VLSI testing methods have been employed in testing PLLs in the past. Off-chip testing process by means of Automatic Testing Equipment (ATE) is no exception. However, these systems have proved to be time consuming, expensive and do not provide sufficient high fault coverage. On-chip testing techniques emerged with Design-for-Test (DFT) and Built-in-self test (BIST) as modern and efficient means for testing PLLs. DFT scan designs had considerable limitations such as high-power dissipation and slow clock problems. BIST designs have made advances over DFT even though not much work has been done in recent years. 4 Kommey et al [24] presented a method for parametric built-in-self-test with realistic design constraint. In this model, the N and M dividers were initialized, and the test control unit awaited PLL synchronization. This synchronization was achieved by a lock signal generated by the phase detector. Built-in-self-test was then performed by using LFSRs (Linear-Feedback Shift Registers) and MISRs (Multi-Input Signature Registers) of the circuit under test (CUT). The original response of the test was then compared to a golden signature. The value of N is stored in the output register and subsequently incremented, provided the test was passed, else the test was restarted as long as N does not exceed Nmax and the test does not fail. The maximum frequency of the circuit under test can then be determined. The PLL utilized in this system was however not optimized for a large bandwidth. Hence detection of faults through direct and indirect measurements were not fully exploited. [25] proposed a BIST technique which employs a two-step AC and DC testing technique. In this technique, the defective circuit under test is checked by monitoring fault signatures through output voltage signal characteristics. Literature Review Based on the output signal characteristics according to the equation 𝑣(𝑡) = 𝑉𝐷𝐶+ 𝑉𝑚sin⁡(𝜔𝑡+ Φ), the AC testing was achieved by detecting changes in Vm whereas DC testing by changes in VDC. Vm and VDC are tested consecutively hence the name. Though this strategy is cost effective, it is only aimed at achieving on-chip testing in the pre- screening of defective chips before higher precision testing is carried out which makes it less robust. CP-PLL Core Design The CP-PLL was designed as a fully integrated mixed signal core with digital blocks such as the PFD and purely analog parts like the VCO. The specifications for the integer-N design are listed below in Table 1. The division ratio N was fixed as 32. The design is based on a linearized model of the second-order CP-PLL. An in-depth non- linear analysis of the CP-PLL is rigorously discussed in [20]. The linearized form however suffices for this design. Table 1: CP-PLL specifications. Parameter Value Lock range 40 MHz - 100MHz Phase noise (at 1 MHz) < -100 dBc/Hz Lock time < 30 µs Supply voltage 3 V Power budget 2 mW Table 1: CP-PLL specifications. As was established in Section 1, a simple odd chain of inverters oscillates at a fixed frequency fvco = fout. To create a variable fvco oscillator, a simple tuning mechanism is to control the current through the inverters. Such a current source is driven by a control voltage Vcntl which determines an inverter’s charge-discharge times. This current- 5 Kommey et al starved inverter topology was used in the VCO design. The VCO used seven-stage inverter network to produce signals in the 40 - 100 MHz frequency range as shown in Figure 3. Fig. 3 Schematic of complete VCO Fig. 3 Schematic of complete VCO The oscillator was designed for a wider 30 - 105 MHz band to accommodate the extremes of the PLL’s tuning range (fmin and fmax). The range was designed by appropriately sizing the inverter’s transistors. The VCO’s output waveform was improved by loading each inverter stage with a 220 pF capacitor. However, such as load increases the charge-discharge times of the stage and ultimately diminishes fvco. An optimum trade-off was struck via simulations. A sweep of the VCO output signal’s frequency is shown in Figure 4. The oscillator was designed for a wider 30 - 105 MHz band to accommodate the extremes of the PLL’s tuning range (fmin and fmax). The range was designed by appropriately sizing the inverter’s transistors. The VCO’s output waveform was improved by loading each inverter stage with a 220 pF capacitor. However, such as load increases the charge-discharge times of the stage and ultimately diminishes fvco. An optimum trade-off was struck via simulations. A sweep of the VCO output signal’s frequency is shown in Figure 4. 6 Kommey et al Fig. CP-PLL Core Design 4 VCO output frequency plot [27] From the graph, the Vcntl range was found to be approximately 1.3 V. The VCO’s gain Kvco is found to be: From the graph, the Vcntl range was found to be approximately 1.3 V. The VCO’s gain Kvco is found to be: From the graph, the Vcntl range was found to be approximately 1.3 V. The VCO’s gain Kvco is found to be: 𝐾𝑣𝑐𝑜= 2𝜋(𝑓𝑚𝑎𝑥−𝑓𝑚𝑖𝑛) 𝑉𝑐𝑛𝑡𝑙 ≈290𝑀𝑟𝑎𝑑/𝑉𝑠 (1) 𝐾𝑣𝑐𝑜= 2𝜋(𝑓𝑚𝑎𝑥−𝑓𝑚𝑖𝑛) 𝑉𝑐𝑛𝑡𝑙 ≈290𝑀𝑟𝑎𝑑/𝑉𝑠 (1) Kvco may be calculated by averaging the gradients of multiple shorter linear sections in the plot’s non-linear operating region. The PFD was designed using a sequential phase and frequency detecting logic illustrated in Figure 5. 5. 5. Fig. 5 Implemented dead-zone free phase detector Fig. 5 Implemented dead-zone free phase detector The detector generates short length UP and DOWN pulses for controlling the charge pump by comparing the input reference and feedback clock. For perfectly synchronized input waveforms, the PFD produces identical output pulses, causing their respective currents at the charge pump’s output to cancel out. Contrarily, unequal UP and DOWN pulses produce a nonzero charge pump current which charges/discharges the loop filter to adjust the feedback clock phase or frequency. The charge-pump used in this design is shown in Figure 6. 7 7 Kommey et al Fig. 6 Schematic of charge pump. Fig. 6 Schematic of charge pump. The presented PFD eliminates the dead-zone non-ideality by introducing delays into the loop, which work to turn both sources on simultaneously. It also has a lowered sensitivity to duty cycle variation. The critical path of the PFD circuitry is limited to three gate delays: one from the 4-input reset NAND and two from the two cross-coupled gate pairs (SR latches). With such a strategy, there is the risk of spurious noise injection to the output control node in the case of a current mismatch due to the PFD’s pulses. To remedy the possible Icp fluctuations, high impedance cascode current sources were used. In some cases, the switching UP or DOWN pulses may result in extra charge injections, a sluggish response and further Icp fluctuations (due to finite output impedances of current sources). To solve this problem, the charge pump’s output node is buffered. This technique was not applied to this design for simplicity because the current topology sufficed. CP-PLL Core Design Finally, to keep the PFD’s gain constant during charge/discharge of the loop filter, Icp sources were designed to supply equal currents. For proper functioning of the CP-PLL, the CP’s output voltage should keep the current generator transistors in the saturation region. To achieve this, a very low impedance to ground (GND) or a dc rail is required. For a purely passive solution, the loop filter should have a GND-referenced capacitor. In this design, a second order passive filter network was used (Figure 7). 8 Kommey et al Fig. 7 Low Pass Loop Filter Kommey et al Fig. 7 Low Pass Loop Filter Fig. 7 Low Pass Loop Filter The series RC arm dampens the resonance of the overall filter response without degrading the bypass effectiveness at high frequencies. The series resistor R introduces a zero in the LPF’s transfer function which makes it possible to control the damping of the PLL HF response separately from its speed. On the other hand, the so-called secondary capacitor C2 has a reduced effect on the loop’s dynamics and as such is sometimes ignored in the mathematical modeling of the LPF behavior [27]. To determine the values of the LPF elements, the desired loop dynamics should be known or determined. This dynamic is studied in the form of the CP-PLL’s natural frequency ωn or the loop bandwidth ω3dB. The ωn of a CP-PLL, employing a passive LPF, is related to ω3dB by 𝜔𝑛= 0.075𝜔3𝑑𝐵 [2𝜁2+1+√(2𝜁2+1)2+1] 0.5 (2) (2) where ζ is the damping factor (ζ ∈ [0.5,2] for generally good dynamics). A good speed-stability compromise is usually achieved with ζ = 0.707. To uphold the continuous time approximations of the CP-PLL’s dynamics, ω3dB should be at most a tenth of ωREF. However, ω3dB is preferred to be as wide as possible to aid suppression of the VCO’s phase noise. A good noise-stability trade-off is to design ω3dB to be 7.5% of ωREF. Therefore, designing for ζ = 0.707, ωn becomes approximately 0.036ωREF. Designing for a 3 MHz resolution, ωn was 0.69 Mrad/s. By setting Icp, the values of C1 and R, are found using Equations 3 and 4 respectively. Conventionally, the value of C2 is chosen to be about 10% that of C1. This allows for reducing R values to reasonable levels. Furthermore, the setup requires a large dc blocking C1 to avoid excessive dissipation of the unwanted ripple energy on R. CP-PLL Core Design Generally, a high Icp is desirable for higher loop gain and hence a more stable CP-PLL. However, it produces a large C1 requirement (Equation 3) which translates into large die areas. Icp for this design was chosen as 25 µA through a short calculative iteration for a decent loop gain and C1 𝐶1 = 𝐾𝑣𝑐𝑜𝐼𝑐𝑝 𝜋𝜔2𝑁 (3) 9 Kommey et al (4) Therefore, the design values of the LPF elements were: C1 = 76.9 pF, C2 = 7.5 pF, and R = 26.9 kΩ. Finally, the fixed-ratio N-divider was designed. The N = 32 frequency divider was constructed as a chain of five power of-2 integer dividers as illustrated in Figure 8. Fig. 8 N = 32 feedback divider Each divider cell was an asynchronous binary counter created from a D-FF whose inverted output Q was tied to the data input D. The signal to be divided is fed to the clock input. The FFs were implemented using true single phase clocking latches for low power high speed operation. The topology required minimum sized transistor and hence, was also efficient in terms of die area. Fig. 8 N = 32 feedback divider Fig. 8 N = 32 feedback divider Fig. 8 N = 32 feedback divider Each divider cell was an asynchronous binary counter created from a D-FF whose inverted output Q was tied to the data input D. The signal to be divided is fed to the clock input. The FFs were implemented using true single phase clocking latches for low power high speed operation. The topology required minimum sized transistor and hence, was also efficient in terms of die area. Each divider cell was an asynchronous binary counter created from a D-FF whose inverted output Q was tied to the data input D. The signal to be divided is fed to the clock input. The FFs were implemented using true single phase clocking latches for low power high speed operation. The topology required minimum sized transistor and hence, was also efficient in terms of die area. BIST Paradigm A fully digital charge-based frequency measurement BIST scheme was designed with focus on and embedded into the CP-PLL. The BIST strategy presented here is defect-oriented and an improved version of the scheme first presented in [28]. The test strategy is implemented with minimal hardware, requires fewer external signals, and is highly integrable. An all-digital architecture was chosen due to their well-known robustness [12]. The PLL-under- test (PUT) is subjected to testing by undertaking some fundamental checks. Although the BIST is defect-oriented, it takes advantage of component/system functionality and dynamics to achieve testing. The checks are: A. START sequence check: A. START sequence check: This check verifies that the PUT can be tuned up to frequency within a specified time from system startup. It also includes the demonstration of phase locking. B. Normal mode check: Here, checks ascertain if output signal produced by the PUT, post-lock, is of the desired or correct frequency. It also involves PUT reprogramming to effect and study the PUT response to dynamic frequency changes. B. Normal mode check: C. Mode-switch check: This check establishes the ability of the PUT to be operated in and switched from its various operation modes: normal mode, system test mode, etc. 10 Kommey et al D. STOP sequence check: This verifies that the PUT can be stopped when a specific sequence of signals is applied to the appropriate inputs. Together with the START sequence check, it can be demonstrated that the PUT would correctly cease and resume operation in tandem The test strategy is conceptually straightforward. The scheme is initialized, and the PUT is exercised to stimulate all its blocks and register their interactions. The responses are collected in the form of frequency variations. That is, the PUT is charged from a very low frequency fo towards a predetermined maximum frequency fmax threshold, preferably the top of the synthesizer’s tuning range. The final frequency attained is measured and compared to the known standard, f. The PUT is fault-free if fmax = f (allowing a little error margin), otherwise the device is faulty. A similar process is carried out in the discharge phase of the PLL as a foolproof check. The test procedure occurs in five successive steps and is described as follows (Figure 9): Fig. 9 A high-level abstraction of the BIST procedure and related test stimuli. Fig. 9 A high-level abstraction of the BIST procedure and related test stimuli. I. BIST Paradigm Therefore, provided fREF < fFDBK, the PLL remains in a continuous charge state. Conversely, the PLL is in a continuous discharge state if fREF > fFBK. Test stimuli were designed based on this concept to be fed to the PFD inputs which charges and discharges the PUT for specified periods. These BIST patterns are illustrated in Figure 9 alongside their related test phases. The lines TCK and TFB represent the test clock and test feedback signals respectively. Additionally, this version of BIST features a new signal SEL_MODE for controlling the state of the response analyzer. A transition on this line controls the state of the response analyzer. The three signals are generated by the built-in TSG. BIST Paradigm Initialization and STOP sequence check: This PUT undergoes an initial discharge phase where the device is brought to fo to ensure that CP-integrator is drained, and the charge stage commences from a frequency as close to the minimum VCO output frequency fout as possible. These ushers the PUT into a state where the output current is dc. If successful, this stage essentially stops the normal operation, establishing the STOP sequence check described above. II. Charge: In this stage, the PUT frequency is raised from fo to fmax by a steady increase Vcntl, an action which corresponds to the START sequence. The PUT’s frequency is held constant for a specified time afterwards to verify phase locking. 11 Kommey et al III. Measurement of fmax: The value of the post-charge frequency is measured by employing a counter to register the number of cycles within a given time interval. The count value is then shifted out and compared to f. This frequency measurement constitutes the normal mode check. III. Measurement of fmax: The value of the post-charge frequency is measured by employing a counter to register the number of cycles within a given time interval. The count value is then shifted out and compared to f. This frequency measurement constitutes the normal mode check. IV. Discharge: Here fout is gradually decreased from to a predefined frequency fmin using by steadily decreasing Vcntl. Here fout is gradually decreased from to a predefined frequency fmin using by steadily decreasing Vcntl. V. Measurement of fmin: The obtained frequency after step IV is determined and studied using the same method as in step III. The access and control mechanism of the test is achieved by applying appropriate digital stimuli to specific PUT blocks. Some of the stimuli are generated internally while others are supplied via external pins. To start the test procedure, a START_BIST signal is used to switch from the PUT’s normal operation mode to the test mode and back, post-test. A low (logic 0) on the START_BIST line triggers the BIST mode whereas a high (logic 1) selects the normal mode. To generate the waveforms for the charge and discharge test phases, we refer to the basic operational principle of the PLL as described in Section 1. That is, the PLL is a feedback control system which compares the feedback signal to reference and adjusts 𝑓𝐹𝐵𝐾= 𝑓𝑣𝑐𝑜 𝑁⁡ to meet fREF. VHF CP-PLL with BIST To make the frequency synthesizer testable, the CP-PLL architecture was modified slightly by introducing a few extra hardware whilst maintaining the core circuitry intact. The modified CP-PLL structure is illustrated in Figure 11 12 Kommey et al Fig. 11 Designed PLL with BIST. Fig. 11 Designed PLL with BIST. Extra (digital) hardware introduced for the BIST integration include two identical 3-input switches, a 2×1 MUX, a response collector, and a TSG. The PLL’s feedback divider is repurposed as the BIST controller, hence the need for the MUX. Here, and additional D-FF stage is added as the BIST controller requires six asynchronous counter stages: A, B, C, D, E, and F. A state machine in the controller block manages the overall self-test procedure. The reference switch SW1 selects between the PLL’s reference clock (RCK) and the test clock (TCK) for the respective operational modes. Similarly, the feedback switch SW2 selects between the PLL’s feedback (RCK) and test feedback (TFB) signals respectfully. The two switches are connected to the PFD input and have a common START_BIST input. For START_BIST = X, RCK/FBK = Y and TCK/TFB = Z, the switch output is related to its inputs as XY + 𝑍̅. This setup requires lesser number of gates (two 2-input NANDs) than a traditional 2×1 MUX, which also suffices for this function (with START_BIST as the selector). This switching mechanism implies that the inverse of TCK/TFB is always supplied. The switching mechanism can only be used if it can be guaranteed that the circuit never enters the state: START_BIS = 1, RCK/FBK = 0, TCK/TFB = 1. The TSG is a combinational circuit which generates the test patterns based on the output of the controller, which operates on a 6.25 MHz test input clock. Since the controller generates 26 = 64 unique states, one state per BIST clock cycle, each test runs for 10.24 µs. The design of the TSG and hence the generated test waveforms were based on the following Boolean relations: The START_BIST signal remains low throughout the entire test run. As discussed in Section 1, classic response analyzers employ compaction. For a given test strategy, the functional CUT is first exercised through computer simulations or pre-tests to generate response sequences. These sequences are compacted and stored. During a BIST run, actual responses are compacted and then compared against the stored pattern for test evaluations. VHF CP-PLL with BIST Although a practical solution for GHz range PLLs, the approach can be quite lossy and susceptible to aliasing. For relatively 13 Kommey et al lower frequency applications, the combination of a digital counter and shift register suffices as a response collector. In our test paradigm, the counter measures the frequency whereas the later shifts the result out. In this design, a simpler and lossless D-FF-based circuit that operates conjointly as both a counter and a shift register was used to reduce hardware cost (Figure 10). Fig. 10 Schematic of response collector. The 6 FF network, which has a potential for wider fault coverage, exploits the inherent operability of FFs as counters or shift register, building both configurations into one device albeit separable using MUXs (Figure 10). Together, the 12 MUXs use the SEL_MODE line to select a given configuration (SEL_MODE = 0: counter, SEL_MODE = 1: shift register). Fig. 10 Schematic of response collector. Fig. 10 Schematic of response collector. Fig. 10 Schematic of response collector. The 6 FF network, which has a potential for wider fault coverage, exploits the inherent operability of FFs as counters or shift register, building both configurations into one device albeit separable using MUXs (Figure 10). Together, the 12 MUXs use the SEL_MODE line to select a given configuration (SEL_MODE = 0: counter, SEL_MODE = 1: shift register). The 6 FF network, which has a potential for wider fault coverage, exploits the inherent operability of FFs as counters or shift register, building both configurations into one device albeit separable using MUXs (Figure 10). Together, the 12 MUXs use the SEL_MODE line to select a given configuration (SEL_MODE = 0: counter, SEL_MODE = 1: shift register). The collector is configured as a shift register during most of the test runtime. For each test cycle, the device switches configuration twice. First, all the FFs are reset low by tying the data input to VSS. During the measurement phase, the device switches to the ’count-mode’ and holds for a predefined 0.32µs after which it reverts to serially shift out the result in ’shift-mode’. The delay element on the shift register’s clock SR_CLK line is to remedy an inherent timing problem of the devices. This malfunction arises during the second configuration switch where the rising edges of the SEL_MODE and SR_CLK signal coincide since the two are ultimately fed from the same source. VHF CP-PLL with BIST Here, the device is expected to operate as shift register even before SEL_MODE fully asserts. Thus, for the first cycle, the shift register does not function properly, leading to unpredictable results. Nonetheless, the device has a wide fault coverage potential and remains practical even for ultra-high frequency applications. I. A and B are in-phase but have the different frequencies. II. A and B oscillate at the same frequency but are out-of-phase. III. A and B have the different frequencies and are out-of-phase. Results and Discussions The proposed CP-PLL with built-in self-test was designed and tested in AMI 600 nm CMOS process technology using Cadence Design Environment. All building blocks of both PLL and BIST were verified through simulations 14 Kommey et al and the optimized circuits was laid out regarding good high frequency techniques. The entire design was then simulated post-layout to verify system-level functionality. The system was powered by ± 1.5 V supply rails. The tuning sensitivity Kvco of the optimized VCO was calculated from a frequency sweep of its output against Vcntl as 302 Mrad/Vs using Equation 1 and a fine averaging of the effective region as described in Section 3.1. Fig. 12 Simulated VCO output at different Vcntl: -0.5 V, -0.312 V, -0.125 V, 0.0625 V, and 0.25 V (in order). Figure 12 show the VCO outputs for five Vcntl values: -0.5 V, -0.321 V, -0.125 V, 0.0625 V, and 0.25 V. The short- term stability of the VCO was also determined in the form of the oscillator’s phase noise. The CP-PLL had a simulated phase noise of -71 dBc/Hz at 1 kHz and consumed 1.80 mW of power at full operation. To test the PFD, two ± 1.5 V square pulses A and B for the reference clock and the feedback signal respectively, were applied to the PFD input. Three distinct scenarios were studied: Fig. 12 Simulated VCO output at different Vcntl: -0.5 V, -0.312 V, -0.125 V, 0.0625 V, and 0.25 V (in order). Figure 12 show the VCO outputs for five Vcntl values: -0.5 V, -0.321 V, -0.125 V, 0.0625 V, and 0.25 V. The short- term stability of the VCO was also determined in the form of the oscillator’s phase noise. The CP-PLL had a simulated phase noise of -71 dBc/Hz at 1 kHz and consumed 1.80 mW of power at full operation. To test the PFD, two ± 1.5 V square pulses A and B for the reference clock and the feedback signal respectively, were applied to the PFD input. Three distinct scenarios were studied: I. A and B are in-phase but have the different frequencies. III. A and B have the different frequencies and are out-of-phase. For brevity, only case III’s results are shown in this paper. A case where the feedback has a higher frequency feedback signal with respect to A is simulated. For B’s frequency to match that of A, PFD must cause the VCO to adjust the feedback frequency downwards. It is seen in Figure 13 that the PFD generates DOWN pulses, which when fed to the charge pump, results in a discharge of the loop filter. The VCO control voltage Vcntl and the feedback signal’s frequency ultimately reduces. The PFD-CP-LPF blocks were simulated together to study the performances of the CP and loop filter, and to verify their interactions. 15 Kommey et al Fig. 13 Simulated PFD for case III Fig. 13 Simulated PFD for case III Fig. 13 Simulated PFD for case III The average Icp was measured over a relative long time for both charge and discharge phases of the loop filter. The CP pumped an average of 24.9 µA and drew 24.5 µA from the filter in the respective phases, as was expected from the design. On the other hand, the filter produced an output voltage that increased periodically provided the frequency of the feedback signal remain lower than the references. Plots capturing the PFD’s input waveforms and the corresponding Vcntl output of the filter captured in a 10 µs window is shown in Figure 14. Fig. 13 Simulated PFD for case III The average Icp was measured over a relative long time for both charge and discharge phases of the loop filter. The CP pumped an average of 24.9 µA and drew 24.5 µA from the filter in the respective phases, as was expected from the design. On the other hand, the filter produced an output voltage that increased periodically provided the frequency of the feedback signal remain lower than the references. Plots capturing the PFD’s input waveforms and the corresponding Vcntl output of the filter captured in a 10 µs window is shown in Figure 14. Fig. 14 Vcntl at loop filter output node and corresponding PFD inputs Fig. 14 Vcntl at loop filter output node and corresponding PFD inputs 16 Kommey et al The verification of the feedback divider was quite straightforward. A clock signal was applied to its input and the output of each of its five counter stages were measured to verify proper frequency division. Next, the CP-PLL was simulated to verify design correctness and proper operation. The PLL was used to synthesize five signals with frequencies sampled from the loop’s operating range: fout = 40 MHz, 55 MHz, 70 MHz, 85 MHz, and 100 MHz. Since the CPPLL uses a N = 32 divider: fout/32 references. Figures 15 and 16 show the synthesis of the 40 MHz and 85 MHz signals. Fig. 15. A 40 MHz signal synthesized by CP-PLL from a 1.25 MHz reference Fig. 16. An 85 MHz signal synthesized by the CP-PLL from a 2.66 MHz reference Fig. 15. A 40 MHz signal synthesized by CP-PLL from a 1.25 MHz reference Fig. 15. A 40 MHz signal synthesized by CP-PLL from a 1.25 MHz reference Fig. 15. A 40 MHz signal synthesized by CP-PLL from a 1.25 MHz reference Fig. 16. An 85 MHz signal synthesized by the CP-PLL from a 2.66 MHz reference Fig. 16. An 85 MHz signal synthesized by the CP-PLL from a 2.66 MHz reference Fig. 16. An 85 MHz signal synthesized by the CP-PLL from a 2.66 MHz reference 17 17 Kommey et al Kommey et al Kommey et al The CP-PLL was able to generate the required fout signals. For the 40 MHz case, the signal was acquired starting from under an initial 2 Hz frequency in 11.6 µs. The acquisition times of all frequency locks are listed in Table 2 The CP-PLL was able to generate the required fout signals. For the 40 MHz case, the signal was acquired starting from under an initial 2 Hz frequency in 11.6 µs. The acquisition times of all frequency locks are listed in Table 2 Table 2: Frequency syntheses and corresponding lock times fout [MHz] fREF [MHz] Lock time [µs] 40 1.25 11.6 55 1.72 12.2 70 2.18 10.9 85 2.66 15.7 100 3.25 25.1 Table 2: Frequency syntheses and corresponding lock times Table 2: Frequency syntheses and corresponding lock times Finally, the BIST hardware was checked, and fault simulations were conducted. We present the results from the TSG and the response analyzer only since the BIST controller is the same as the feedback divider whiles the remaining hardware are trivial. Kommey et al The BIST area overhead was estimated to be between 10% and 15%. Fig. 18 Layout of CP-PLL with BIST in 600 nm CMOS Fig. 18 Layout of CP-PLL with BIST in 600 nm CMOS Kommey et al To test the TSG, the BIST controller was connected to a 6.25 MHz pulse source and the TSG output, alongside Vcntl and resulting fout changes were observed for one test cycle. But for the exception of a few negligible glitches, the test stimuli generated were as expected. The counter/shift register operation was also verified. Simulations revealed that the BIST circuitry can generate the exact patterns of required test stimuli. It also performs all the unique checks of the strategy efficiently. The final test output is very consistent and produces the same results for a few different runs of the simulation. The overall check of the BIST scheme is summarized in Figure 17. Fig. 17 Simulation results of BIST scheme The PUT charges to fmax ≈ 84 MHz. Then, there is a 0.48 µs hold state where the fmax measurement is taken. The loop discharges to fmin ≈ 74 MHz and fmin is measured in the next hold state. Consistent 011010 (26 decimal) and 010111 (23 decimal) values corresponding to fmax and fmin were obtained respectively for different test runs. Since Fig. 17 Simulation results of BIST scheme Fig. 17 Simulation results of BIST scheme The PUT charges to fmax ≈ 84 MHz. Then, there is a 0.48 µs hold state where the fmax measurement is taken. The loop discharges to fmin ≈ 74 MHz and fmin is measured in the next hold state. Consistent 011010 (26 decimal) and 010111 (23 decimal) values corresponding to fmax and fmin were obtained respectively for different test runs. Since 18 Kommey et al the response analyzer counts in a 0.32 µs time window, the measured outputs correspond to 𝑓𝑚𝑎𝑥= 26 0.32 = 81.3⁡𝑀𝐻𝑧 and 𝑓𝑚𝑖𝑛= 23 0.32 = 71.8⁡𝑀𝐻𝑧. With a tolerable error margin, the designed and simulated CP-PLL can be declared fault-free. Transistor level fault models developed in [28] were also used for both digital and analog components during simulations. These included transistor stuck-on, source open, drain open, gate-to-source short and gate-to-drain short faults. The described BIST scheme was very effective in detecting faults. Of simulated 316 faults simulated, only 8 remained undetected. All 8 undetected faults were found to be redundant. Figure 18 shows the final layout of the CP-PLL with BIST in one IC. The BIST area overhead was estimated to be between 10% and 15%. Figure 18 shows the final layout of the CP-PLL with BIST in one IC. In this paper, the design of a VHF CP-PLL with built-in self-test has been presented. The PLL is implemented as a frequency synthesizer with a 40 MHz - 100 MHz tuning range. The building blocks of the PLL core as well as that of the embedded defect-oriented test scheme were designed and tested through simulations in 600 nm CMOS using the Cadence Design Environment. Experiments showed good synthesis of various frequencies within the Conclusions In this paper, the design of a VHF CP-PLL with built-in self-test has been presented. The PLL is implemented as a frequency synthesizer with a 40 MHz - 100 MHz tuning range. The building blocks of the PLL core as well as that of the embedded defect-oriented test scheme were designed and tested through simulations in 600 nm CMOS using the Cadence Design Environment. Experiments showed good synthesis of various frequencies within the 19 Kommey et al loop’s operating range. The generated signals are obtained at the exact frequencies, have clean waveforms with no spikes/glitches whatsoever, and are acquired within reasonably short lock times. The CP-PLL exhibited a phase noise of -132 dBc/Hz at 1 MHz and with a 3 V supply, consumed 1.8 mW. The designed BIST is a fully digital architecture with an output which can be read serially on a single pin. The simple digital output allows for a seamless interfacing of the test process with standard ATEs and makes it easier to integrate the final IC into larger electronic hardware. The design also features a very simple yet effective response collector on the BIST output which uses dynamic FFs for fast, lossless, and aliasing-free compaction of test results. The overhead posed by the BIST hardware was also kept minimal for practical and low defect-risk integration. Fault simulations revealed very high fault coverage for the BIST scheme, with most undetected faults being redundant. The implemented test scheme was designed to be as generic as possible and is easily portable across similar PLL architectures and technology nodes without requiring significant reconfiguration. References 1. J. Chen, W. Zhang, Q. Sun, and L. Liu, “An 812.5-ghz lc pll with dual vco and noise reduced ldo regulator for multilane multiprotocol serdes in 28-nm cmos technology,” Electronics, vol. 10, no. 14, 2021. [Online]. Available: https://www.mdpi.com/2079-9292/10/14/1686 2. J. Kim, J. Park, and J.-G. 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Gall, N. Bhargava, and A. Bal, “A fully- digital bist wrapper based on ternary test stimuli for the dynamic test of a 40 nm cmos 18-bit stereo audio σδ adc,” IEEE Transactions on Circuits and Systems I: Regular Papers, vol. 63, no. 11, pp. 1876–1888, 2016. References 20 Kommey et al 11. Pavlidis, M.-M. Louërat, E. Faehn, A. Kumar, and H.-G. Stratigopoulos, “Symbist: Symmetry based analog and mixed-signal built-in self-test for functional safety,” IEEE Transactions on Circuits and Systems I: Regular Papers, vol. 68, no. 6, pp.2580–2593, 2021. 12. S. Dasnurkar and J. Abraham, “Closed-loop built in self-test for pll production testing with minimal tester resources,” in 2009 IEEE 15th International Mixed-Signals, Sensors, and Systems Test Workshop, 2009, pp. 1–5. 13. Y. Kim, K. Kim, I. Kim, and S. Kang, “A new built-in self-test scheme for phase-locked loops using internal digital signals,” IEICE Trans. Electron., vol.91-C, pp. 1713–1716, 2008. 14. L. Xia and J. 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Wimol San-Um, “Development of built-in self test and calibration of analog mixed-signal LSI for catastrophic failures and paramentric variations,” PhD dissertation, Graduate School of Engineering, Kochi University of Technology Academic Resource Repository, Kochi, Sep 2010, Available: http://hdl.handle.net/10173/757 y Jephthah, “A 40-100 MHz phase-locked loop frequency synthesizer with built-in self-test,” M.S. thesis, of Engineering, KNUST, Kumasi, Aug 2012, Available: http://hdl.handle.net/123456789/5357 28. K. O. Boateng, “A new defect-oriented method for testing the pll,” Journal of Engineering and Technology, vol. 1, pp. 37–42, 2007. 21 Kommey et al List of Abbreviations CP charge pump PLL phase lock loop VHF very high frequency BIST built-in self-test CMOS complementary metal oxide semiconductor VCO voltage-controlled oscillator PFD phase frequency detector PUT pll under test ATE automated test equipment TSG test stimulus generator CUT circuit under test ROM read only memory LFSR linear feedback shift register MISR multiple input shift register IC integrated circuit MEMS micro-electro-mechanical system VLSI very large-scale integration DFT design for test Availability of data and material Availability of data and material Data sharing not applicable to this article as no datasets were generated or analyzed during the current study Competing interests Authors' contributions BK – idea, proposed design, and specifications and test and evaluation, read and approved final manuscrip EOA – Simulations and Verification, read and approved final manuscript JY – idea, introduction, and experimental strategies, read and approved final manuscript ASA – Experimental set ups, read and approved final manuscript ETT – coordinator, abstract and conclusion, read and approved final manuscript BYA – related works, drawings, read and approved final manuscript BK – idea, proposed design, and specifications and test and evaluation, read and approved final manuscript EOA – Simulations and Verification, read and approved final manuscript JY – idea, introduction, and experimental strategies, read and approved final manuscript ASA – Experimental set ups, read and approved final manuscript ETT – coordinator, abstract and conclusion, read and approved final manuscript BYA – related works, drawings, read and approved final manuscript Funding There was no funding for this project Competing interests 22 Kommey et al Kommey et al The authors declare that they have no competing interests. Acknowledgements Not applicable 23 23
https://openalex.org/W3196973715	https://www.frontiersin.org/articles/10.3389/fonc.2021.712041/pdf	English	null	RhoC Modulates Cell Junctions and Type I Interferon Response in Aggressive Breast Cancers	Frontiers in oncology	2,021	cc-by	10,694	ORIGINAL RESEARCH published: 26 August 2021 doi: 10.3389/fonc.2021.712041 Edited by: Edited by: San-Gang Wu, First Afﬁliated Hospital of Xiamen University, China Edited by: San-Gang Wu, First Afﬁliated Hospital of Xiamen University, China Reviewed by: Steven G Allen, University of Michigan, United States Kathleen Gallo, Michigan State University, United States Reviewed by: Steven G Allen, University of Michigan, United States Kathleen Gallo, Michigan State University, United States Reviewed by: Steven G Allen, University of Michigan, United States Kathleen Gallo, Michigan State University, United States Correspondence: Soﬁa D. Merajver smerajve@umich.edu Correspondence: Soﬁa D. Merajver smerajve@umich.edu †Present address: Peter J. Ulintz, Diamond Age Data Science, Somerville, MA, United States Andrew C. Little, Boehringer Ingelheim Oncology, Ridgeﬁeld, CT, United States †Present address: Peter J. Ulintz, Diamond Age Data Science, Somerville, MA, United States Andrew C. Little, Boehringer Ingelheim Oncology, Ridgeﬁeld, CT, United States Specialty section: This article was submitted to Breast Cancer, a section of the journal Frontiers in Oncology Specialty section: This article was submitted to Breast Cancer, a section of the journal Frontiers in Oncology Received: 19 May 2021 Accepted: 05 August 2021 Published: 26 August 2021 Received: 19 May 2021 Accepted: 05 August 2021 Published: 26 August 2021 Keywords: RhoC, breast cancer, junctions, interferon, TNBC Metastases are the leading cause of death in cancer patients. RhoC, a member of the Rho GTPase family, has been shown to facilitate metastasis of aggressive breast cancer cells by inﬂuencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. A common phenotypic characteristic of metastatic cells inﬂuenced by these mechanisms is dysregulation of cell-cell junctions. Thus, we set out to study how RhoA- and RhoC- GTPase inﬂuence the cell-cell junctions in aggressive breast cancers. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM 149 and MDA 231 breast cancer cells results in increased normalization of junctional integrity denoted by junction protein expression/ colocalization. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity and increased cell-cell adhesion compared to wild-type cells. Whole exome RNA sequencing and targeted gene expression proﬁling demonstrate decreased expression of Type I interferon-stimulated genes in RhoC knockout cells compared to wild-type, and subsequent treatment with interferon-alpha resulted in signiﬁcant increases in adhesion and decreases in invasiveness of wild-type cells and a dampened response to interferon-alpha stimulation with respect to adhesion and invasiveness in RhoC knockout cells. We delineate a key role of RhoC-GTPase in modulation of junctions and response to interferon, which supports inhibition of RhoC as a potential anti-invasion therapeutic strategy. Abraham HG, Ulintz PJ, Goo L, Yates JA, Little AC, Bao L, Wu Z and Merajver SD (2021) RhoC Modulates Cell Junctions and Type I Interferon Response in Aggressive Breast Cancers. Front. Oncol. 11:712041. doi: 10.3389/fonc.2021.712041 Citation: Cancer metastases are the leading cause of death in cancer patients, and yet details of the cellular processes that drive the early metastases in aggressive cancers are not fully understood. RhoC, a member of the Rho GTPase family, has been linked to the metastatic potential of a variety of cancers including inﬂammatory breast cancer, pancreatic cancer, and melanoma (1–5). In breast cancer, RhoC expression correlates with increasing breast cancer stage and grade (as a histologic surrogate for aggressiveness), and higher RhoC expression was associated with higher patient mortality (6). Moreover, in this historical cohort, high RhoC was a predictor of poor response to standard Abraham HG, Ulintz PJ, Goo L, Yates JA, Little AC, Bao L, Wu Z and Merajver SD (2021) RhoC Modulates Cell Junctions and Type I Interferon Response in Aggressive Breast Cancers. Front. Oncol. 11:712041. doi: 10.3389/fonc.2021.712041 August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org RhoC Modulates Junctions, Interferon Signaling Abraham et al. Ham’s F12 (+) L-glutamine, 0.5% penicillin-streptomycin, 2.5 mg/mL Amphotericin B, 5 mg/mL gentamycin, 5 mg/mL insulin, and 1 mg/mL hydrocortisone. SUM 149 cells were additionally supplemented with 5% FBS, while SUM 190 cells were supplemented with 0.1% bovine serum albumin. MCF7 cells were acquired from ATCC and maintained in DMEM, 10% FBS, 5 mg/mL gentamycin, and 1X anti-anti. MCF10A cells were acquired from ATCC and maintained in 50:50 DMEM:F12, 5% horse serum, 10 mg/mL insulin, 0.02 mg/mL epidermal growth factor, 0.5 mg/mL hydrocortisone, 0.1 mg/mL cholera toxin, 5 mg/ mL gentamycin, and 1X anti-anti. All cells were maintained at 5% CO2, except for SUM 149 and SUM 190 which were maintained at 10% CO2. Interferon-alpha 2a (IFN-a) was obtained from GenScript (# Z03003-1), reconstituted in ddH2O, and used to treat cells at either 100 IU/ml or 1000 IU/ml. chemotherapy regimens, increasing the likelihood that patients would experience metastasis and relapse (6). RhoC is overexpressed in the majority of cases of inﬂammatory breast cancer, the most aggressive and metastatic form of breast cancer (7). Animal and in vitro studies demonstrated that RhoC is necessary speciﬁcally for facilitating metastasis, primarily through protecting metastatic cells from apoptosis, modulating cell motility, and inﬂuencing chemokine secretion (8, 9). These studies led us to postulate that a possible cellular effect of RhoC-driven metastatic progression is through modulation of cell junctions that would signal to motility and evasion of apoptosis. Citation: The Rho family GTPases regulate actin cytoskeleton organization (10), and thereby interact directly or indirectly with components of adherens junctions (AJs) and tight junctions (TJs) (11). RhoA, whose amino acid sequence is 90% homologous to RhoC (12), speciﬁcally is important for both the initial formation and the structural maintenance of AJs and TJs (13). Indeed, studies using both dominant negative and constitutively active forms of RhoA result in AJ and TJ instability (13, 14), suggesting that the stability of epithelial junctions is dependent on balanced activation of Rho GTPases. Antibodies The following primary antibodies were used: anti-E-cadherin rabbit polyclonal antibody (ThermoFisher #PA5-32178) at 1:500 dilution, anti-b-catenin mouse monoclonal antibody (Invitrogen #MA1-300) at 1:500 dilution, anti-ZO-1 mouse monoclonal antibody (Invitrogen #33-9100) at 1:150 dilution for immunoﬂuorescent staining and 1:200 for Western Blot, anti- Occludin rabbit polyclonal antibody (Zymed #71-1500) at 1:300 dilution for immunoﬂuorescent staining and Western Blot, anti- p-STAT1 rabbit monoclonal antibody (CST #9167) at 1:1000 dilution, anti-STAT1 rabbit monoclonal antibody (CST #9172) at 1:1000 dilution, anti-p-STAT2 rabbit antibody (CST #4441) at 1:500 dilution, anti-STAT2 rabbit antibody (CST #4594) at 1:500 dilution, anti-IRF9 rabbit monoclonal antibody (CST #76684) at 1:500 dilution, anti-IFI27 rabbit polyclonal antibody (ThermoFisher #PA5-68038) at 1:1000 dilution, anti-IFITM1 Generation of CRISPR-Cas9 Knockout Cells As described in Allen et al. (24), SUM 149, MDA 231, VARI068, MCF7, MCF10A, and SUM 190 cells were transfected with pSpCas9(BB)-2A-GFP (PX458), provided by Feng Zhang (Addgene plasmid # 48138), containing the sequence GCCCTGATAGTTTAGGTGAG targeting RhoA for RhoA knockout lines or the sequence AGGAAGACTATGAT CGACTG targeting RhoC for the RhoC knockout lines. Transfection was accomplished using the Nucleofactor II system (Lonza). 48 hours post-transfection, single cells were sorted by GFP expression and seeded into 96-well plates, and clonal expansion was carried out. Genomic DNA was then harvested from clones and screened for RhoA or RhoC mutations via SURVEYOR reactions (IDT) with the primer pair Forward-GTTTTAGACCGTCTGCCATTTC and Reverse- AATCTCCACCTACCAGGTTCAA for RhoA and Forward- CTGTCTTTGCTTCATTCTCCCT and Reverse-CCAGAGC AGTCTTAGAAGCCAT for RhoC. Clones that screened positive were subsequently sequenced to characterize their RhoA or RhoC mutations and were also immunoblotted for RhoA and RhoC. When considering pathological settings of junction instability, the metastatic process itself is a prime example. Multiple studies of diverse cancer types demonstrate a loss of junction markers in malignant vs normal tissue; however, these studies differ on the prognostic value derived from the loss or dysregulation of junctions (15, 16). In a study of colorectal cancers, decreased E-cadherin and ZO-1 expression in primary tumors predicted which tumors went on to have liver metastases (17). In addition to the observed dysregulation of cell-cell junctions in the metastatic process, junction proteins are also known to be downregulated in settings of increased inﬂammatory interferon signaling (18, 19) and treating cells with interferon-alpha (IFN-a) leads to increased RhoA activation (20, 21). Furthermore, breast cancer tumors with high interferon signaling pathway expression are nearly twice as likely to metastasize compared to tumors with low levels of expression (22). This study aims to investigate the role of RhoC in regulating cell-cell junction stability and interferon signaling in aggressive breast cancer cell lines. We assess the hypothesis that RhoC ampliﬁes interferon signaling and thereby increases junction dysregulation, consequently promoting cancer cells’ motility and invasiveness; this work supports inhibition of RhoC as a potential therapeutic strategy in aggressive cancers. Centrifugation Adhesion Assay Centrifugation Adhesion Assay Adapted from Weetall et al (25). V-bottom 96-well plates were seeded with 2 x 104 cells/well and left in 37°C overnight. Calcein- AM-labeled cells (2uM Calcein-DMSO solution, Invitrogen #C3100MP) were subsequently seeded at 1.5 x 104 cells/well to the plate (negative control: wells with overnight-seeded cells but no Calcein-labeled cells; positive control: empty wells with Calcein-labeled cells added). Plates were incubated at 37°C for 2 hours, then centrifuged at 75 g for 10 minutes. Nonadherent cells accumulated at the bottom of the wells and ﬂuorescence at the bottom of the well was quantiﬁed. Log fold change in ﬂuorescence between test wells and positive control wells was recorded. Assay was repeated with media containing 100 IU/ml IFN-a; overnight-seeded cells were treated with IFN-a for 48 hours prior to seeding in v-bottom plates, then were seeded in media with IFN-a for 24 hours, while Calcein-labeled cells were treated with IFN-a for 72 hours prior to Calcein labeling, seeding, and incubation in v-bottom plate for 2 hours (they were also seeded in media containing IFN-a). MATERIALS AND METHODS Cell Culture and Reagents g MDA-MB 231 (MDA 231) cells were acquired from ATCC and maintained in Gibco RPMI-1640 (+) L-glutamine, 10% fetal bovine serum (FBS), 5 mg/mL gentamycin, and 1X anti-anti. VARI068 cells, sourced from a patient-derived xenograft (23), were maintained the same way. SUM 149 cells and SUM 190 cells were provided by Dr. Steve Ethier and were maintained in Gibco August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 2 RhoC Modulates Junctions, Interferon Signaling Abraham et al. Western Blot Protein lysates were mixed with loading dye and boiled at 95°C, then loaded into a 4-15% polyacrylamide gel and run at 130-160 V for about 90 minutes. For blotting proteins smaller than 90 kDa, gel was subsequently removed from chamber and soaked in 20% methanol at RT for 5 minutes, then transferred to a PVDF membrane using the iBlot 2 Dry Blotting System. For proteins larger than 90 kDA, gel was removed from chamber and soaked in 20% methanol transfer buffer for 5 minutes, then transferred to a PVDF membrane using a BioRad Wet Transfer chamber running at 80 V for 75 minutes. After transfer, the membrane was blocked in 5% milk/TBST at RT for 1 hr, rinsed with TBST thrice for 5 minutes each, then incubated in primary antibody solution at 4°C overnight on shaker. The next day, the membrane was again rinsed with TBST, then incubated in secondary antibody solution (in 5% milk-TBST) at RT for 1 hr. Once again, the membrane was rinsed with TBST, and then incubated in developing reagent at RT for 2 minutes. Finally the membrane was placed in a chemilluminescence reader and the blot was recorded. FITC-Dextran Assay mouse monoclonal antibody (Proteintech #60074-1-IG) at 1:20,000 dilution, anti-MX1 rabbit polyclonal antibody (Proteintech #13750-1-AP) at 1:1000, anti-ISG15 rabbit polyclonal antibody (Proteintech #15981-1-AP) at 1:1000, and anti-actin antibody (Sigma #A3854) at 1:15,000 dilution. The following secondary antibodies were used: Alexa Fluor 488- conjugated anti-mouse secondary antibody (Molecular Probes) at 1:1000 for immunoﬂuorescent staining, Alexa Fluor 647- conjugated anti-rabbit secondary antibody (Molecular Probes) at 1:1000 for immunoﬂuorescent staining, HRP-conjugated anti- mouse secondary antibody (Santa Cruz) at 1:4000 for Western Blot, and HRP-conjugated anti-rabbit secondary antibody (CST) at 1:2500 for Western Blot. Cells were seeded into Transwell plates and grown for 36 hours, until they reached conﬂuency. FITC-Dextran solution was prepared at 1mg/ml, and 0.5ml of this solution was added to the apical chambers of the Transwells, with normal media in the basal chambers. After 24 hours, 50ul was removed from the basal chambers and transferred to a 96-well plate, then ﬂuorescence was measured in a ﬂuorescent plate reader. The ratio of ﬂuorescence from the apical chamber to the basal chamber was recorded. Immunoﬂuorescent Staining Cells were seeded on 4-well chamber slides and grown to a conﬂuent monolayer. Slides were ﬁxed in 4% paraformaldehyde for 10 minutes at room temperature, washed with 100 mM PBS- glycine for 10 minutes at room temperature, then permeabilized in 0.1% Triton X-100 in PBS for 10 minutes at 4°C. Samples were washed thrice with 100mM PBS-glycine, then incubated in blocking solution containing IF Buffer (0.2% Triton X-100, 0.05% Tween-20, 0.1% BSA, 7.7mM NaN3 in PBS) and 10% goat serum for 1.5 hr at room temperature. Subsequently, samples were incubated in a primary antibody solution overnight at 4°C. The samples were then washed four times in IF Buffer for 15 minutes each at room temperature, then incubated in a secondary antibody solution (all secondary antibodies used at 1:1000 dilution), followed by one wash with IF Buffer for 20 minutes and two washes with PBS for 10 minutes each, at room temperature. Slides were mounted in Prolonged Gold Antifade reagent with DAPI (4′,6- diamidino-2- phenylindole) for nuclear counterstaining (Molecular Probes). Images were acquired on a Nikon A1B confocal microscope at 40X magniﬁcation. siRNA Knockdown of Junction Proteins siTJP1 (ZO-1) and non-targeting control siRNA were ordered from Dharmacon (siTJP1 5 nmol #L-0077-46-00-0005) and transfected in SUM 149s using 5.2 ml DharmaFECT 2/well in 6-well plates, while in MDA 231s transfection used 2 ml DharmaFECT 4/well in 6-well plates (Dharmacon). Protein was harvested from cells 2-5 days after transfection and immunoblotted for ZO-1 to conﬁrm transient knockdown. RNAseq d seq In order to assess the impact of RhoC knockout on gene expression in breast cancers, a panel of cell lines was assembled: SUM 149 (triple-negative inﬂammatory breast cancer), MDA 231 (triple-negative non-inﬂammatory breast cancer), VARI068 (triple-negative non-inﬂammatory breast cancer), SUM 190 (hormone receptor negative, HER2 positive inﬂammatory breast cancer), MCF7 (estrogen receptor positive, progesterone receptor positive, HER2 negative non-inﬂammatory breast cancer), and MCF10A (normal-like breast epithelial cells). These cells all express varying levels of RhoC at baseline and vary in phenotype, from highly metastatic to noninvasive, and were chosen in order to assess whether RhoC knockout would induce gene expression changes that would be consistent across different cell contexts. Four biological replicates of all cell lines, wild-type and RhoC knockouts, were incubated at 37°C overnight. Normal growth media for each cell line was replaced with DMEM for 24 hrs, and RNA was harvested. RNA was sequenced via the Illumina HiSeq 4000 as paired 51bp reads to a targeted depth of 75M paired reads per sample. Read data in FASTQ format were quality assessed with FastQC/MultiQC (v.0.11.3) and contamination checked with fastq_screen (v.0.11.1). Reads were adapter-trimmed using CutAdapt (v.1.8.1) and aligned to the GRCh37 hg19 human genome using Tophat/Bowtie2 (v.2.0.13/v.2.2.1, options –b2_very_sensitive and the default max intron length of 500000). Raw read counts were extracted for each gene using HTSeq (v.0.6.0). DESeq2 (v1.14.1), run within the R (v.3.3.3) Bioconductor package (Biobase v.2.34) was used to model differential expression in genes between modeled conditions. The main factors used in the model were cell line and CRISPR knockout status (cRhoC or WT). DESeq2 utilizes generalized linear models for each gene and infers a log2 fold change between conditions using maximum likelihood estimation and (by default) a Wald test for signiﬁcance. Default parameters for DESeq2 were used, specifying a standardized normal prior on the non-intercept coefﬁcients (betaPrior=TRUE). QC plotting was performed in R using ggplot. Genes were annotated with NCBI Entrez GeneIDs and text descriptions. Data has been deposited in GEO, accession ID GSE175787 In addition to classic enrichment, a prediction of upstream regulators was also performed by iPG based on the differentially expressed gene set and a network of regulatory (activation/ inhibition) interactions from a proprietary knowledge base compiled from StringDB (32) (Version 11.0. Jan 19th, 2019) and BioGrid (33) (v3.5.171. March 25th, 2019) data. Targeted Gene Expression Proﬁling Targeted Gene Expression Proﬁling Three biological replicates of MDA 231 and SUM 149 wild-type, RhoA knockout, and RhoC knockout cells were treated with 100 IU/ml IFN-a for 72 hours, then RNA was harvested and run on nanoString Pan Cancer Immune Proﬁling panels (nanoString Technologies, Inc.). The expression of 730 immune-related genes and 40 housekeeping genes was measured, and the nSolver 4.0 software (nanoString Technologies, Inc.) was used to normalize expression values and conduct differential expression analysis. Genes were considered differentially expressed between treated and untreated cells if they had FDR-adjusted p-value < 0.05. To compare the change in expression with IFN-a treatment in wild- type, RhoA knockout, and RhoC knockout cells, p-values were calculated as per Kaye et al. (35). In the crRhoC vs WT dataset comprising data from all cell lines listed above, 1293 differentially expressed genes were identiﬁed out of a total of 20,978 with detected expression based on an adjusted p-value threshold of 0.05 and a minimum absolute log2 fold change of 0.585. Gene set enrichment was performed on these data using the commercial iPathwayGuide software (Advaita Bioinformatics, Ann Arbor, MI). iPathwayGuide (iPG) scores pathways using a custom enrichment method (26–28) that is composed of two primary sub-methods: i) the over-representation of differentially expressed (DE) genes in a given pathway, and ii) the perturbation of that pathway computed by propagating the measured expression changes across the pathway topology. These two sub-methods each produce p-values (pORA and pAcc, Transwell Invasion Assay y 100,000 cells/well were seeded into Matrigel Transwell Invasion chambers (Corning #354480) in serum-free media, with serum- containing media in bottom chambers. Cells were incubated at 37°C for 24 hours, then the top chambers were scrubbed to remove cells that had not invaded. Chambers were then ﬁxed in 70% ethanol for 10 minutes, stained in 0.2% Crystal Violet for 10 minutes, and left to dry overnight. Brightﬁeld images of each insert were acquired at 2X magniﬁcation on an Olympus IX51, and the ImageJ Color Inspector 3D plugin was used to quantify the percent coverage of purple pixels per insert image. Assays were performed in technical triplicate and biological triplicate. Multiple comparisons ANOVA was conducted on the data in GraphPad Prism 9. For assays with siRNA-treated cells, cells were seeded into chambers 48 hours post-transfection. For assays with IFN-a-treated cells, cells were seeded into chambers either August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 3 RhoC Modulates Junctions, Interferon Signaling Abraham et al. with no prior IFN-a treatment or with 48 hours pre-treatment with IFN-a, and were seeded in serum-free media containing 100 IU/ml of IFN-a. respectively) that are combined using Fisher’s method into a pathway-speciﬁc p-value, which is then corrected for multiple comparisons using an FDR correction. The tool searched KEGG pathways (Release 90.0+/05-29, May 19) utilizing directional information in gene-relationships (29). An enrichment against GO terms (30, 31) was also performed, utilizing the ORA method (i) above. Frontiers in Oncology \| www.frontiersin.org RNAseq d The activation/inhibition network is polled using gene expression information to consider hypotheses that upstream regulators of genes are either activated or inhibited. A z-score for each upstream regulator is computed by iterating over connected downstream genes and their incoming edges, as well as a p- value corresponding to the z-score as the one-tailed area under the probability density function for a normal distribution, N(0,1). An over-representation approach is also used to compute the statistical signiﬁcance of observing at least a given number of consistent DE genes, with an associated p-value computed using the hypergeometric distribution (34). Finally, these two p-values are combined using the Fisher’s method to rank the upstream regulators and test the hypothesis that the upstream regulators are predicted as activated or inhibited in the experimental condition (crRhoC vs WT). Loss of RhoA and RhoC Expression in Breast Cancer Cells Results in Signiﬁcant Morphological Changes In order to investigate the effect of RhoC expression on cell-cell junctions, we created MDA 231 and SUM 149 cell lines where RhoA and RhoC had been independently knocked-out via CRISPR-Cas9 (cell lines denoted crRhoA and crRhoC, respectively). We found that the crRhoC cells exhibited August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 4 RhoC Modulates Junctions, Interferon Signaling Abraham et al. crRhoA cells demonstrated similar or decreased expression of junction proteins as compared to wild-type via Western Blot. In contrast, crRhoC cells exhibited increased junction marker expression compared to wild-type (Supplementary Figure 1). Out of the four junction markers, this pattern of Rho-modulated expression was most evident in ZO-1, both in SUM 149 and in MDA 231 cells. Immunoﬂuorescent staining for junction proteins (Figure 2A) highlighted an increase in the amount of junction markers localizing to areas of cell-cell contact in the crRhoC cells, as well as increased colocalization of junction markers in crRhoC cells. Moreover, the SUM 149 crRhoA cells were observed to consistently assemble in loose or disordered clusters, characterized by variable spaces between cells, and had decreased tight junctions and cell-cell projections compared to both wild-type and crRhoC cells (Figures 2B, C). compensatory increases in RhoA expression, while crRhoA cells had smaller magnitude increases in RhoC expression (Figure 1A). Furthermore, the crRhoA cells assumed a more spindlelike shape compared to their wild-type counterparts, and the crRhoC cells were more cuboidal compared to wild- type (Figure 1B). RhoA and RhoC Expression Modulate Junctional Protein Expression and Colocalization (A) Representative images of immunoﬂuorescence staining of junction markers in wild-type, crRhoA, and crRhoC cells. White arrows point to areas of junction marker localization to cell-cell borders. Scale bars = 50 mm. Quantiﬁcation from 3 ﬁelds of view per cell type of (B) adherens junction marker and (C) tight junction marker localization to cell-cell borders in wild-type, crRhoA and crRhoC cells, with area of peak intensity corresponding to areas of cell-cell border localization. Solid bars are MDA 231, striped bars are SUM 149; black bars are wild type, light grey bars are crRhoA cells, and dark grey bars are crRhoC cells. p < 0.01; p < 0.001. RhoA and RhoC Expression Modulate Junctional Protein Expression and Colocalization Due to the marked changes in morphology, we sought to characterize the role of RhoC and RhoA in epithelial junctions. We assessed the junctions structurally by the expression of the tight junction proteins ZO-1 and Occludin, and the adherens junction proteins E-cadherin and b-catenin. We found that A B FIGURE 1 \| Rho knockout changes expression and morphology of cells. (A) Validation of CRISPR-Cas9 knockout of RhoA and RhoC via Western Blot. (B) Brightﬁeld images of wild-type, crRhoA, and crRhoC cells. RhoA knockout markedly changes the morphology of both MDA 231 and SUM 149 cells, leading to a consistent “triangle” shape in the MDA 231s and a rounded shape in the SUM 149s. RhoC knockout changes cell morphology more subtly, leading to a consistent “crescent” shape in the MDA 231s and a more cuboidal shape in the SUM 149s. Scale bars = 200mm. A A A B B B FIGURE 1 \| Rho knockout changes expression and morphology of cells. (A) Validation of CRISPR-Cas9 knockout of RhoA and RhoC via Western Blot. (B) Brightﬁeld images of wild-type, crRhoA, and crRhoC cells. RhoA knockout markedly changes the morphology of both MDA 231 and SUM 149 cells, leading to a consistent “triangle” shape in the MDA 231s and a rounded shape in the SUM 149s. RhoC knockout changes cell morphology more subtly, leading to a consistent “crescent” shape in the MDA 231s and a more cuboidal shape in the SUM 149s. Scale bars = 200mm. August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 5 RhoC Modulates Junctions, Interferon Signaling Abraham et al. A B C RE 2 \| Rho expression changes junction marker localization to cell-cell borders. (A) Representative images of immunoﬂuorescence staining of junction markers -type, crRhoA, and crRhoC cells. White arrows point to areas of junction marker localization to cell-cell borders. Scale bars = 50 mm. Quantiﬁcation from 3 of view per cell type of (B) adherens junction marker and (C) tight junction marker localization to cell-cell borders in wild-type, crRhoA and crRhoC cells, with f peak intensity corresponding to areas of cell-cell border localization. Solid bars are MDA 231, striped bars are SUM 149; black bars are wild type, light grey re crRhoA cells, and dark grey bars are crRhoC cells. p < 0.01; *p < 0.001. A B B C C FIGURE 2 \| Rho expression changes junction marker localization to cell-cell borders. crRhoC Cells Have Altered Interferon-a Signaling Compared to Wild-Type crRhoC cells have signiﬁcantly increased adhesiveness compared to wild type. (B) Quantiﬁcation of the ratio of ﬂuorescent intensity in apical vs basal chambers in FITC-Dextran barrier permeability assay (n = 3 biological replicates); higher ratio corresponds to increased barrier integrity. crRhoA cells have decreased barrier integrity compared to wild-type, whereas crRhoC cells have increased barrier integrity. Solid bars are MDA 231, striped bars are SUM 149; black bars are wild-type, light grey bars are crRhoA, and dark grey bars are crRhoC cells. p < 0.05; **p < 0.001. FIGURE 3 \| Rho expression changes cell-cell adhesiveness and junction stability. (A) Quantiﬁcation of difference in ﬂuorescent intensity between positive control (non-adhering) and test wells in centrifugation adhesion assay (n = 3 biological replicates); decreases in ﬂuorescent intensity correspond to increases in cell-cell adhesiveness. crRhoC cells have signiﬁcantly increased adhesiveness compared to wild type. (B) Quantiﬁcation of the ratio of ﬂuorescent intensity in apical vs basal chambers in FITC-Dextran barrier permeability assay (n = 3 biological replicates); higher ratio corresponds to increased barrier integrity. crRhoA cells have decreased barrier integrity compared to wild-type, whereas crRhoC cells have increased barrier integrity. Solid bars are MDA 231, striped bars are SUM 149; black bars are wild-type, light grey bars are crRhoA, and dark grey bars are crRhoC cells. p < 0.05; **p < 0.001. FIGURE 3 \| Rho expression changes cell-cell adhesiveness and junction stability. (A) Quantiﬁcation of difference in ﬂuorescent intensity between positive control (non-adhering) and test wells in centrifugation adhesion assay (n = 3 biological replicates); decreases in ﬂuorescent intensity correspond to increases in cell-cell adhesiveness. crRhoC cells have signiﬁcantly increased adhesiveness compared to wild type. (B) Quantiﬁcation of the ratio of ﬂuorescent intensity in apical vs basal chambers in FITC-Dextran barrier permeability assay (n = 3 biological replicates); higher ratio corresponds to increased barrier integrity. crRhoA cells have decreased barrier integrity compared to wild-type, whereas crRhoC cells have increased barrier integrity. Solid bars are MDA 231, striped bars are SUM 149; black bars are wild-type, light grey bars are crRhoA, and dark grey bars are crRhoC cells. p < 0.05; **p < 0.001. these cells. crRhoC Cells Have Altered Interferon-a Signaling Compared to Wild-Type In both crRhoC SUM 149 and crRhoC MDA 231 cells, there was a signiﬁcant increase in the barrier integrity of the cell monolayer compared to wild-type, and in crRhoA SUM 149 and crRhoA MDA 231 there was a signiﬁcant decrease in the barrier integrity of the cell monolayer compared to wild-type (Figure 3B). These changes imply that tight junction stability increases with RhoC knockout, and decreases with RhoA knockout, which is consistent with the changes observed via immunoﬂuorescent staining. RhoA and RhoC Expression Modulate Cell-Cell Adhesion and Barrier Impermeability subjected to centrifugal shear stress in order to measure the perturbation of adhesion between different cell types. Both crRhoC SUM 149 and crRhoC MDA 231 cells had a greater reduction in ﬂuorescent signal compared to their positive controls than did wild-type cells, suggesting a functionally stronger cell-cell adhesion when RhoC is knocked out (Figure 3A). To further assess the functional signiﬁcance of the junction changes induced by reducing RhoA and RhoC expression, we undertook a FITC-Dextran barrier integrity assay to determine the effectiveness of the tight junctions in Having observed a qualitative change in junction protein expression and localization, we sought to determine whether this change translated into functional differences in adhesion between wild-type and crRhoC cells. We measured cell-cell adhesion using a ﬂuorometric centrifugation assay, wherein ﬂuorescently-labeled cells were added to wells with previously- seeded cells of the same type, incubated for 2 hours, and then August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 6 RhoC Modulates Junctions, Interferon Signaling Abraham et al. A B FIGURE 3 \| Rho expression changes cell-cell adhesiveness and junction stability. (A) Quantiﬁcation of difference in ﬂuorescent intensity between positive control (non-adhering) and test wells in centrifugation adhesion assay (n = 3 biological replicates); decreases in ﬂuorescent intensity correspond to increases in cell-cell adhesiveness. crRhoC cells have signiﬁcantly increased adhesiveness compared to wild type. (B) Quantiﬁcation of the ratio of ﬂuorescent intensity in apical vs basal chambers in FITC-Dextran barrier permeability assay (n = 3 biological replicates); higher ratio corresponds to increased barrier integrity. crRhoA cells have decreased barrier integrity compared to wild-type, whereas crRhoC cells have increased barrier integrity. Solid bars are MDA 231, striped bars are SUM 149; black bars are wild-type, light grey bars are crRhoA, and dark grey bars are crRhoC cells. p < 0.05; **p < 0.001. control siRNA for 72 hrs to achieve transient ZO-1 knockdown, following which transwell invasion was assessed. ZO-1 knockdown did not signiﬁcantly change invasiveness in wild- type, crRhoA, or crRhoC cells (data not shown). A crRhoC Cells Have Altered Interferon-a Signaling Compared to Wild-Type Seeking to understand the molecular mechanisms of Rho-driven junction regulation, we conducted whole transcriptome RNAseq analysis of SUM 149, MDA 231, VARI068, MCF7, MCF10A, and SUM 190 wild-type and crRhoC cells. Analysis detected 1,293 genes differentially expressed between crRhoC cells and wild-type at an adjusted p-value of 0.05 and a minimum log2 fold change threshold of 0.585. A number of interferon-stimulated genes (ISGs) had signiﬁcantly decreased expression in crRhoC cells compared to wild-type. Inferring the upstream regulation of genes from the overall differential expression result set yielded IRF9 and STAT2 as the two leading inhibitory regulators (Supplementary Table 1), with 17 consistent genes each listed as inhibited (out of a total of 38 and 43 target genes, p-values 7.9e- 11 and 3.9e-10, respectively) (Figure 4A and Supplementary Table 2). The expression of IRF9 and STAT2 themselves were not signiﬁcantly differentially altered between crRhoC cells and wild-type cells. B B As type I interferon signaling is known to inﬂuence junction protein expression in a context-dependent manner (18, 36), we sought to investigate speciﬁcally whether the predicted inhibition of type I interferon signaling in crRhoC cells was borne out at the protein level, and whether any changes in junctional behavior would result. SUM 149 and MDA 231 wild-type and crRhoC cells were subsequently treated with IFN-a at two doses (100 and 1000 IU/ml) for 2 hours and 72 hours, and expression of proteins in the type I interferon signaling pathway was assessed via Western Blot. In response to interferon treatment, we observed that RhoC modiﬁed the cells’ responses: MDA 231 crRhoC cells had increased p-STAT2 and IRF9 expression compared to wild type (Figure 4B), whereas SUM 149 crRhoC cells had decreased p-STAT2 and total STAT2 expression compared to wild type (Figure 4C). There were no signiﬁcant differences between the two doses tested. The difference in interferon response expression between crRhoC and wild-type cells were evident at both the 2 hour and 72 hour time points, consistent with the 17 ISGs identiﬁed by RNAseq that are downstream of short-term phosphorylated STAT1-STAT2-IRF9-complex(ISGF3)-driven signaling as well as long-term unphosphorylated-ISGF3-driven signaling (37–39) (Figure 4D). FIGURE 3 \| Rho expression changes cell-cell adhesiveness and junction stability. (A) Quantiﬁcation of difference in ﬂuorescent intensity between positive control (non-adhering) and test wells in centrifugation adhesion assay (n = 3 biological replicates); decreases in ﬂuorescent intensity correspond to increases in cell-cell adhesiveness. RhoC Modulation of Interferon Signaling Leads to Functional Changes in Junction Behavior and Cell Invasiveness To investigate whether RhoA and RhoC expression affect the invasive potential of breast cancer cells, we conducted transwell invasion assays. Compared to wild-type and crRhoA cells, crRhoC cells had signiﬁcantly less transwell invasion in MDA 231 cells and trended to less invasion in SUM 149 cells (Supplementary Figure 2). To assess whether transiently modulating expression of ZO-1 would contribute to invasive capability, cells were treated with ZO-1 siRNA or scrambled To assess the impact of RhoA and RhoC expression on long-term ISG expression, SUM 149 and MDA 231 wild-type, crRhoA and crRhoC cells were treated with IFN-a at 100 IU/ml for 72 hours, then RNA was harvested and a relevant array of cancer related genes was assessed by the nanoString Pan-Cancer Immune August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 7 RhoC Modulates Junctions, Interferon Signaling Abraham et al. anel. Out of the genes that were signiﬁcantly differentially both crRhoA and crRhoC cells compared to wild type, and A B D C FIGURE 4 \| RhoC expression inﬂuences gene and protein expression of Type I interferon signaling response. (A) Type I interferon signaling pathway and the genes signiﬁcantly downregulated in crRhoC knockout cells compared to wild-type, as measured in RNAseq. Genes in blue circles were downregulated in crRhoC compared to wild-type; genes in grey circles were not signiﬁcantly differentially expressed between crRhoC and wild-type. (B) Western blot of interferon signaling markers in MDA 231 wild-type and crRhoC cells—crRhoC cells have increased p-STAT2 and IRF9 compared to wild type. (C) Western blot of interferon signaling markers in SUM 149 wild-type and crRhoC cells—crRhoC cells have decreased p-STAT2 and STAT2 compared to wild type. (D) Short-term and long-term signaling through type I interferon signaling pathways. Short-term interferon signaling is driven by phosphorylated STAT1 and STAT2 complexed with IRF9 that translocates to the nucleus, binds to interferon stimulated response elements (ISREs), and promotes transcription of interferon stimulated genes (ISGs); phosphorylation of STAT1 and STAT2 peak about 2 hours after treatment with a type I interferon. Long-term signaling is driven by unphosphorylated STAT1 and STAT2 complexed with IRF9, and peaks around 72 hours after treatment with type I interferons. Genes listed in order of decreasing magnitude of log fold-change. A B C A B C B C B C D FIGURE 4 \| RhoC expression inﬂuences gene and protein expression of Type I interferon signaling response. RhoC Modulation of Interferon Signaling Leads to Functional Changes in Junction Behavior and Cell Invasiveness (A) Type I interferon signaling pathway and the genes signiﬁcantly downregulated in crRhoC knockout cells compared to wild-type, as measured in RNAseq. Genes in blue circles were downregulated in crRhoC compared to wild-type; genes in grey circles were not signiﬁcantly differentially expressed between crRhoC and wild-type. (B) Western blot of interferon signaling markers in MDA 231 wild-type and crRhoC cells—crRhoC cells have increased p-STAT2 and IRF9 compared to wild type. (C) Western blot of interferon signaling markers in SUM 149 wild-type and crRhoC cells—crRhoC cells have decreased p-STAT2 and STAT2 compared to wild type. (D) Short-term and long-term signaling through type I interferon signaling pathways. Short-term interferon signaling is driven by phosphorylated STAT1 and STAT2 complexed with IRF9 that translocates to the nucleus, binds to interferon stimulated response elements (ISREs), and promotes transcription of interferon stimulated genes (ISGs); phosphorylation of STAT1 and STAT2 peak about 2 hours after treatment with a type I interferon. Long-term signaling is driven by unphosphorylated STAT1 and STAT2 complexed with IRF9, and peaks around 72 hours after treatment with type I interferons. Genes listed in order of decreasing magnitude of log fold-change. Panel. Out of the genes that were signiﬁcantly differentially expressed in treated cells compared to untreated controls, the interferon-stimulated gene IFITM1 had decreased expression in both crRhoA and crRhoC cells compared to wild type, and additional interferon-stimulated genes like MX1 and ISG15 were signiﬁcantly decreased in only the crRhoC cells compared August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 8 RhoC Modulates Junctions, Interferon Signaling Abraham et al. to wild type. IFI27 was the only interferon-stimulated gene that had signiﬁcantly increased expression in treated crRhoC cells compared to both treated crRhoA cells and treated wild-type (Figure 5A and Supplementary Figure 3A). treatment has similar effects on wild-type cells as the effect of RhoC knockout—the increased adhesion and decreased invasion induced by 72 hours of IFN-a treatment in wild-type cells (Figures 5C, D) is comparable in magnitude to the increased adhesion and decreased invasion seen in crRhoC cells compared to wild-type. crRhoC cells treated with IFN-a exhibit dampened response in terms of changes in adhesion and invasion, compared to treated wild-type cells, but it is important to highlight that IFN- a does increase adhesion and decrease invasion in both crRhoC and wild-type cells. RhoC Modulation of Interferon Signaling Leads to Functional Changes in Junction Behavior and Cell Invasiveness Taken together, these data point to IFN-a and RhoC inhibition as being capable of reducing cancer cell invasion in a cumulative fashion—a potential combination strategy that could be more effective in RhoC-driven phenotypes, such as inﬂammatory breast cancer, as there was clearly a larger effect on adhesion and invasion in SUM 149 crRhoC cells treated with IFN-a compared to MDA 231 crRhoC cells. In order to determine whether these differences in mRNA expression between crRhoC cells and wild-type cells were borne out at the protein level, cells were again treated with IFN-a at 100 IU/ml for 72 hours, and protein was harvested for Western Blot of MX1, ISG15, IFITM1 and IFI27 (Supplementary Figure 3B). There was no expression of these proteins in the untreated SUM 149 cells, whereas in MDA 231 the untreated cells all expressed ISG15 and the untreated crRhoA and crRhoC cells expressed IFI27, with crRhoC cells expressing the highest amount of these two proteins at baseline. The treated cells all had similar protein expression of MX1, ISG15, and IFI27, with MDA 231 cells having slightly increased protein expression compared to SUM 149 cells. However IFN-a treatment elicited higher IFITM1 protein expression in crRhoC cells than in wild-type. SUM 149 crRhoA cells had decreased expression of IFITM1 with IFN-a treatment compared to wild-type, whereas MDA 231 crRhoA cells had increased expression of IFITM1 with IFN-a treatment compared to wild-type. p A corollary interpretation of these results is that RhoC knockout blunts cellular response to IFN-a overall. This interpretation is further supported by the smaller increase in expression of interferon-stimulated genes in crRhoC cells post- IFN-a treatment compared to wild-type cells, in which IFN-a treatment robustly increased interferon-stimulated gene expression (Figure 5A). The potential for RhoC contributing to normal IFN-a signaling is a novel ﬁnding. Expression of interferon signaling proteins IFI27 and ISG15 was higher in untreated MDA 231 crRhoC cells than in wild-type, and IFITM1 expression was higher in treated crRhoC cells than in wild-type. Expression of IFI27 in some studies is correlated with decreased proliferation and migration (40, 41), and in others with increased tumorigenesis and migration and decreased patient survival (42–44). ISG15 expression is correlated with increased invasion, induction of M2-like macrophages, and decreased patient survival (45, 46). IFITM1 is also correlated with increased tumorigenesis and invasion (47). RhoC Modulation of Interferon Signaling Leads to Functional Changes in Junction Behavior and Cell Invasiveness The increased expression of these invasion-associated proteins and the overall decreased ISG expression in crRhoC cells compared to wild-type, in the context of IFN-a treatment reducing cell invasion without signiﬁcantly affecting cell viability, adds complexity to the understanding of RhoC as primarily a promoter of metastasis. We further sought to assess the impact of RhoA and RhoC expression on functional responses to IFN-a treatment. The centrifugation adhesion assay was repeated with media containing IFN-a at 100 IU/ml. 72 hours of IFN-a treatment increased cell-cell adhesion for all cell types. However, the change in adhesion between untreated and IFN-a treated cells was greater in magnitude in wild-type cells compared to crRhoC cells (Figure 5B and Table 1). Transwell invasion assays were also repeated with media containing IFN-a at 100 IU/ml, with cells treated for 24 or 72 hours. Cells treated for 72 hours had reduced invasion compared to untreated cells and 24-hour- treated cells. In MDA 231, the magnitude of invasion reduction was greater in wild-type and crRhoA cells compared to crRhoC cells, whereas in SUM 149s the reverse was demonstrated—crRhoC cells had a larger reduction in invasion than wild-type or crRhoA cells (Figures 5C, D and Table 2). There were no signiﬁcant differences in proliferation or viability between treated and untreated or between wild-type and Rho knockout cells (data not shown). g y Previous studies from our lab have found that macrophage- conditioned media, speciﬁcally from M2a macrophages, promotes cancer cell invasion, and that functional RhoC is necessary to achieve the full extent of macrophage-promoted invasion (24, 48). Interestingly, IFN-a treatment has been demonstrated to promote a shift in macrophage polarization from M2 to M1 (49, 50). Our current study posits that functional RhoC contributes to increased IFN signaling in cancer cells, which would conﬂict with the logical conclusion from previous studies that RhoC is positively associated with M2 macrophages and M2 macrophages are negatively associated with IFN-a. Further study is therefore necessary to determine why cells with functional high RhoC expression have reduced junction functionality and increased invasion in the absence of IFN-a, and the opposite effect in the presence of IFN-a. DISCUSSION In investigating the cellular and molecular basis of the impact of RhoC on metastasis, we demonstrate that RhoC affects both cell- cell junction behavior as well as IFN-a response. Knocking out RhoC results in a trend towards increased tight and adherens junction protein expression (Supplementary Figure 1) and membrane localization (Figure 2) that resembles normal junctions, while also signiﬁcantly increasing the functionality of these junctions with respect to adhesiveness and impermeability (Figure 3). crRhoC cells also have decreased cell invasion (Supplementary Figure 2). Interestingly, low-dose IFN-a IFN-a has been recognized as an anti-tumor compound since 1970 (51). High-dose IFN-a (>1000 IU/ml) is FDA-approved as August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 9 A B D C FIGURE 5 \| RhoC expression modulates cells’ functional response to interferon. (A) RNA expression of interferon stimulated genes in response to 72 hours of IFN-a treatment (100 IU/ml) in MDA 231 and SUM 149 cells. Wild-type cells had larger increases in gene expression with IFN-a treatment compared to crRhoC cells. Black bars are expression in wild-type cells, dark grey bars are expression in crRhoC cells. (B) Quantiﬁcation of difference in ﬂuorescent intensity with IFN-a treatment between positive control (non-adhering) and test wells in centrifugation adhesion assay (n = 3 biological replicates); decreases in ﬂuorescent intensity correspond to increases in cell-cell adhesiveness. Treatment with IFN-a at 100 IU/ml for 72 hours led to increased adhesion for all cells, but the increases were larger and more signiﬁcant in wild-type and crRhoA cells compared to crRhoC cells. (C) Quantiﬁcation of transwell invasion with IFN-a treatment (n = 3 biological replicates) in MDA 231 and in (D) SUM 149. Treatment with IFN-a led to decreased invasion for all cells; in MDA 231 wild-type and crRhoA cells had larger and more signiﬁcant decreases than in crRhoC cells, whereas in SUM 149 crRhoC cells had the largest relative decrease in invasion. Downward-slanting stripes represent IFN-a treatment; black bars are wild-type, light grey bars are crRhoA cells, and dark grey bars are crRhoC cells. p < 0.05; p < 0.01; p < 0.001. Abraham et al. RhoC Modulates Junctions, Interferon Signaling Abraham et al. RhoC Modulates Junctions, Interferon Signaling A B B D C FIGURE 5 \| RhoC expression modulates cells’ functional response to interferon. DISCUSSION Fold change between the percent invading cells for untreated and treated cells was calculated. Signiﬁcance determined by p < 0.05 and indicated by . \| RhoC knockout modulates IFN-a-driven inhibition of cell invasio variation in clinical efﬁcacy of IFN-a can be attributed to differing ISG induction at differing concentrations of IFN-a; low-dose IFN- a tends to induce anti-viral ISGs, whereas high- dose induces proliferation and inﬂammation-related ISGs (54). Our ﬁndings that low-dose IFN-a modulates breast cancer invasion and adhesion is notable in that it posits a potential anti-tumor clinical beneﬁt through multiple mechanisms of action, without the morbidity of high-dose treatment. Our overall hypothesis—that RhoC ampliﬁes interferon signaling and thereby increases junction dysregulation, consequently promoting cancer cells’ motility and invasiveness— is borne out insofar as RhoC contributes to Type I interferon cellular response and also contributes to regulation of junction behavior. However, we ﬁnd that IFN-a signaling itself results in increased cell-cell adhesion and decreased invasion. Our current work supports that the role of RhoC in metastases of certain aggressive cancers appears to be a result of intrinsic modulation of the cancer cells’ junctions and invasiveness, and potential ampliﬁcation of interferon signaling; other effects on the tumor microenvironment, such as a shift in macrophage population abundance, may cooperate to produce highly aggressive phenotypes. As such, via multiple mechanisms, our data indicate that the inhibition of RhoC in aggressive breast cancers could provide anti-invasion therapeutic beneﬁt. y g Higher ISG expression is associated with estrogen receptor negative breast cancers (22). The cell lines we focused on in this study are both triple-negative breast cancers, and the RNAseq results of decreased ISG expression in RhoC knockout cells compared to wild-type were more signiﬁcant in our triple- negative breast cancer cell lines than in other breast cancer cell lines(SupplementaryTable1).ArecentstudybyDohertyetal.(55) alsoexaminedtheeffectoflow-doseIFN-aontriple-negativebreast cancer and found that chronic, weeks-long exposure to low-dose IFN-a led to increased epithelial morphology, decreased stemness markers, and decreased migration (55). This is consistent with our results of decreased invasion with 3 days of low-dose IFN-a treatment, and comparable to our results of both increased epithelial morphology and decreased invasion in RhoC knockout cells compared to wild-type. Previous work from our lab has identiﬁed RhoC as a modulator of stemness markers in breast cancer cells, and moreover identiﬁed RhoC as necessary for lung metastasis from orthotopic xenografts while increased stemness markers modulated the number of metastases (56). DISCUSSION (A) RNA expression of interferon stimulated genes in response to 72 hours of IFN-a treatment (100 IU/ml) in MDA 231 and SUM 149 cells. Wild-type cells had larger increases in gene expression with IFN-a treatment compared to crRhoC cells. Black bars are expression in wild-type cells, dark grey bars are expression in crRhoC cells. (B) Quantiﬁcation of difference in ﬂuorescent intensity with IFN-a treatment between positive control (non-adhering) and test wells in centrifugation adhesion assay (n = 3 biological replicates); decreases in ﬂuorescent intensity correspond to increases in cell-cell adhesiveness. Treatment with IFN-a at 100 IU/ml for 72 hours led to increased adhesion for all cells, but the increases were larger and more signiﬁcant in wild-type and crRhoA cells compared to crRhoC cells. (C) Quantiﬁcation of transwell invasion with IFN-a treatment (n = 3 biological replicates) in MDA 231 and in (D) SUM 149. Treatment with IFN-a led to decreased invasion for all cells; in MDA 231 wild-type and crRhoA cells had larger and more signiﬁcant decreases than in crRhoC cells, whereas in SUM 149 crRhoC cells had the largest relative decrease in invasion. Downward-slanting stripes represent IFN-a treatment; black bars are wild-type, light grey bars are crRhoA cells, and dark grey bars are crRhoC cells. p < 0.05; p < 0.01; *p < 0.001. monotherapy for Kaposi’s sarcoma, follicular non-Hodgkin lymphoma, melanoma, and hairy-cell leukemia, and for adjuvant therapy of melanoma; overall clinical response rates are modest, and high-dose IFN-a toxicity is high, thus oncological use has diminished in recent times (52). On the other hand, IFN-a is also used clinically as an anti-viral agent, and achieves sustained anti-virologic responses for signiﬁcant populations of Hepatitis B and C patients (53). Some of the August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 10 RhoC Modulates Junctions, Interferon Signaling Abraham et al. TABLE 1 \| RhoC knockout dampens IFN-a-driven increase in cell-cell adhesion. Cell Line \|Log fold change\| with IFN-a tx p value with IFN-a tx MDA 231 WT 3.63 0.250 MDA 231 crRhoA 1.30 0.982 MDA 231 crRhoC 1.12 0.864 SUM 149 WT 5.69 0.037 SUM 149 crRhoA 4.59* 0.007 SUM 149 crRhoC 2.02 0.120 Adhesion was measured using the ﬂuorimetric centrifugation adhesion assay. Log fold change in ﬂuorescence corresponds to change in adhesiveness. Signiﬁcance determined by p < 0.05 and indicated by . TABLE 1 \| RhoC knockout dampens IFN-a-driven increase in cell-cell adhesion. DISCUSSION Of note, we created crRhoA and crRhoC breast cancer cell lines and found that knocking out RhoC resulted in increased expression of RhoA while knocking out RhoA resulted in a smaller magnitude increase in RhoC expression (Figure 1A). Thus, our results in our crRhoC cells could be due to mixed effects from increases in RhoA as well as a lack of RhoC signaling. Practically speaking, however, a compensatory feedback loop of RhoA and RhoC is most likely active in vivo, so any therapeutic trials of RhoC inhibitors would need to also show beneﬁt in the setting of increased RhoA expression. We also ﬁnd that knocking out RhoC increased adhesion and junction stability to a similar extent in both MDA 231 and SUM 149 (Figure 3), but had differing effects in modifying IFN-a-driven inhibition of invasion in MDA 231 and SUM 149—transwell invasion was more inhibited by IFN-a treatment in crRhoC compared to wild-type SUM 149, while in MDA 231 IFN-a treatment inhibited transwell invasion to a greater extent in wild-type compared to crRhoC cells (Figure 5B). We propose that this differing effect on invasion but not on junction function may be due to a difference in STAT2 and pSTAT2 regulation, as we ﬁnd that in SUM 149 IFN- a-treated cells RhoC knockout decreases STAT2 and pSTAT2 expression compared to wild-type while in MDA 231 IFN-a- treated cells RhoC knockout increases STAT2 and pSTAT2 expression compared to wild-type (Figures 4B, C). Thus, cells with increased STAT2 and pSTAT2 upon stimulation with IFN- a were more resistant to IFN-a-driven transwell invasion inhibition, and furthermore RhoC expression affects STAT2 and pSTAT2 expression in different ways in inﬂammatory and non-inﬂammatory breast cancer cells. Further studies are needed to validate these ﬁndings in other inﬂammatory and non- inﬂammatory breast cancer cell lines and to assess the mechanism of action by which RhoC expression may modulate STAT2 expression. TABLE 2 \| RhoC knockout modulates IFN-a-driven inhibition of cell invasion. TABLE 2 \| RhoC knockout modulates IFN-a-driven inhibition of cell invasion. Cell Line Fold change with IFN-a tx 72 hr p value with IFN-a tx 72 hr MDA 231 WT 0.559 0.431 MDA 231 crRhoA 0.645 0.012 MDA 231 crRhoC 0.289 0.809 SUM 149 WT 0.447* 2E-4 SUM 149 crRhoA 0.514* 1E-4 SUM 149 crRhoC 0.604* 9E-4 Cells were treated with IFN-a for 72 hours and transwell invasion assays were conducted. DISCUSSION This study suggests that these previously discovered links to epithelial character, stemness and invasion in both IFN-a and RhoC may be, at least in part, related to RhoC’s contribution to IFN response. Frontiers in Oncology \| www.frontiersin.org REFERENCES Patients With Breast Cancer. Eur J Cancer (2004) 40:2717–25. doi: 10.1016/ j.ejca.2004.08.008 1. Kondo T, Sentani K, Oue N, Yoshida K, Nakayama H, Yasui W. Expression of RhoC Is Associated With Metastasis of Gastric Carcinomas. Pathobiology (2004) 71(1):19–25. doi: 10.1159/000072958 16. Hoover KB, Liao SY, Bryant PJ. 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Genome Res (2007) 17 (10):1537–45. doi: 10.1101/gr.6202607 13. Citi S, Guerrera D, Spadaro D, Shah J. Epithelial Junctions and Rho Family GTPases: The Zonular Signalosome. Small GTPases (2014) 5:1–15. doi: 10.4161/21541248.2014.973760 14. Harris TJC, Tepass U. Adherens Junctions: From Molecules to Morphogenesis. Nat Rev Mol Cell Biol (2010) 11(7):502–14. doi: 10.1038/ nrm2927 27. FUNDING HA was partially supported by the University of Michigan Medical ScientistTrainingProgramandthe ShethResearchFund. SDMwas also supported by the Breast Cancer Research Foundation, the Ravitz Foundation, and the GreaterGood Charities. HA was partially supported by the University of Michigan Medical ScientistTrainingProgramandthe ShethResearchFund. SDMwas also supported by the Breast Cancer Research Foundation, the Ravitz Foundation, and the GreaterGood Charities. AUTHOR CONTRIBUTIONS HA: Performed experiments, analyzed data, wrote ﬁrst draft of manuscript. PU: analyzed bioinformatics data, wrote bioinformatics methods, edited manuscript. LG: performed experiments JY: troubleshooted experiments, edited manuscript, assisted with ﬁgures. AL: performed experiments, analyzed data. LB: performed experiments. ZW: performed experiments. SM: suggested topic, funded work, mentored and directed experiments, edited manuscript. All authors contributed to the article and approved the submitted version. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2021. 712041/full#supplementary-material Patients With Breast Cancer. Eur J Cancer (2004) 40:2717–25. doi: 10.1016/ j.ejca.2004.08.008 DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and August 2021 \| Volume 11 \| Article 712041 Frontiers in Oncology \| www.frontiersin.org 11 RhoC Modulates Junctions, Interferon Signaling Abraham et al. accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/geo/, GSE175787. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2021. 712041/full#supplementary-material REFERENCES Khatri P, Draghici S, Tarca AL, Hassan SS, Romero R. A System Biology Approach for the Steady-State Analysis of Gene Signaling Networks. In: Lecture Notes in Computer Science (Including Subseries Lecture Notes in Artiﬁcial Intelligence and Lecture Notes in Bioinformatics). Springer, Berlin, Heidelberg (2007). 15. Martin TA, Watkins G, Mansel RE, Jiang WG. 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https://openalex.org/W2970947975	https://www.aclweb.org/anthology/W19-5341.pdf	English	null	Baidu Neural Machine Translation Systems for WMT19	null	2,019	cc-by	5,052	Proceedings of the Fourth Conference on Machine Translation (WMT), Volume 2: Shared Task Papers (Day 1) pages 374–381 Florence, Italy, August 1-2, 2019. c⃝2019 Association for Computational Linguistics 1 Introduction The Transformer model (Vaswani et al., 2017), which exploits self-attention mechanism both in the encoder and decoder, has signiﬁcantly im- proved the translation quality in recent years. It is also adopted by most participants as the ba- sic Neural Machine Translation (NMT) system in the previous translation campaigns (Bojar et al., 2018; Niehues et al., 2018). In this year’s transla- tion task, we focus on the improvement of single system, and propose three novel Transformer vari- ants: The remainder of paper is structured as follows: Section 2 describes the detailed overview of our training strategy. Section 3 shows the experimen- tal settings and results. Finally, we conclude our work in Section 4. • Pre-trained Transformer: We train a big Transformer language model (Radford et al., 2018; Devlin et al., 2018; Dai et al., 2019; Sun et al., 2019) on monolingual corpora, and use the language model as the encoder of the Transformer model. Abstract Moreover, the sequence-level knowledge distillation (Hassan et al., 2018) mechanism is employed to boost the performance by means of using the model decod- ing from right to left (Right-to-Left) and the afore- mentioned Transformer variants to generate syn- thetic data for training the NMT model (Wang et al., 2018). Baidu Neural Machine Translation Systems for WMT19 ng Sun, Bojian Jiang, Hao Xiong, Zhongjun He, Hua Wu, Haifeng Wang Baidu Inc. No. 10, Shangdi 10th Street, Beijing, 100085, China {sunmeng09,jiangbojian,xionghao05, hezhongjun,wu hua,wanghaifeng}@baidu.com Meng Sun, Bojian Jiang, Hao Xiong, Zhongjun He, Hua Wu, Haifeng Wang Baidu Inc. No. 10, Shangdi 10th Street, Beijing, 100085, China {sunmeng09,jiangbojian,xionghao05, hezhongjun,wu hua,wanghaifeng}@baidu.com g Sun, Bojian Jiang, Hao Xiong, Zhongjun He, Hua Wu, Haifeng Wang Baidu Inc. No. 10, Shangdi 10th Street, Beijing, 100085, China {sunmeng09,jiangbojian,xionghao05, hezhongjun,wu hua,wanghaifeng}@baidu.com Abstract • Bigger Transformer: According to the pre- vious experiments, the performance of the Transformer model is largely dependent on the dimensions of feed forward network. To further improve the performance, we increase the inner dimension of feed-forward network from 4,096 to 15,000 for big version. • Bigger Transformer: According to the pre- vious experiments, the performance of the Transformer model is largely dependent on the dimensions of feed forward network. To further improve the performance, we increase the inner dimension of feed-forward network from 4,096 to 15,000 for big version. In this paper we introduce the systems Baidu submitted for the WMT19 shared task on Chinese↔English news translation. Our sys- tems are based on the Transformer architec- ture with some effective improvements. Data selection, back translation, data augmenta- tion, knowledge distillation, domain adap- tation, model ensemble and re-ranking are employed and proven effective in our ex- periments. Our Chinese→English system achieved the highest case-sensitive BLEU score among all constrained submissions, and our English→Chinese system ranked the sec- ond in all submissions. In addition, we develop effective approaches to exploit additional monolingual data and generate augmented bilingual data. To use the monolingual data, back translation (Sennrich et al., 2015a) is employed on large corpora including News Cor- pus and Gigaword. We also use an iterative ap- proach (Zhang et al., 2018) to extend the back translation method by jointly training source-to- target and target-to-source NMT models. For bilingual data augmentation, a target-to-source baseline system is used to translate the target of the bilingual corpus as the synthetic data. Moreover, the sequence-level knowledge distillation (Hassan et al., 2018) mechanism is employed to boost the performance by means of using the model decod- ing from right to left (Right-to-Left) and the afore- mentioned Transformer variants to generate syn- thetic data for training the NMT model (Wang et al., 2018). In addition, we develop effective approaches to exploit additional monolingual data and generate augmented bilingual data. To use the monolingual data, back translation (Sennrich et al., 2015a) is employed on large corpora including News Cor- pus and Gigaword. We also use an iterative ap- proach (Zhang et al., 2018) to extend the back translation method by jointly training source-to- target and target-to-source NMT models. For bilingual data augmentation, a target-to-source baseline system is used to translate the target of the bilingual corpus as the synthetic data. 2 System Overview • Deeper Transformer: We increase the en- coder layers to better learn the representation of the source sentences. Speciﬁcally, we in- crease the number of encoder layers from 6 to 30 for the base version, and from 6 to 15 layers for the big version. Figure 1 depicts the overall process of our sub- missions in this year’s evaluation task, in which we train our advanced Transformer models on the bilingual corpus together with synthetic corpora, ﬁne-tune them on the well-selected in-domain data, and generate the ensemble model for the ﬁnal 374 Figure 1: Architecture of Baidu NMT system Figure 1: Architecture of Baidu NMT system re-ranking strategy. In this section, we will intro- duce each step in details. glish language model, we use the concatenation of English Gigaword, English News Crawl and the English part of CWMT and UN corpus. There are 45 million Chinese sentences and 170 million En- glish sentences in our pre-training corpora. It is worth noting that our advanced Trans- former model requires larger GPU memory to train due to the large number of training parame- ters. Hence we train our models on machines with 8 NVIDIA V100 GPUs each of which has 32 GB memory, to avoid out-of-memory issues. In train- ing phase, we limit the number of source and target tokens per batch to 4,096 per GPU for deeper and bigger Transformer models (at most 526,052,128 parameters), while the token batch size is 3,072 for pre-trained Transformer model due to GPU mem- ory limitation. To use the pre-trained language model as en- coder of NMT and enable the open-vocabulary translation, we learn a BPE (Sennrich et al., 2015b) model with 30K merge operations. We use Adam with learning rate of 1e-4, β1 = 0.9, β2 = 0.999, L2 weight decay of 0.001, and learn- ing rate warmup over the 10,000 steps. We train the big Transformer language model with 24 lay- ers, setting the hidden size to 1,024 and the num- ber of self-attention heads to 16. Both Chinese and English pre-training took 7 days to complete. 2.1 Pre-trained Transformer In the ﬁne-tuning procedure of the translation task, we employ a pre-trained language model as encoder of NMT, and the parameters of decoders are learned during ﬁne-tuning. The decoder has 6 self-attention layers, and the hidden size is 1024, which is same with the decoder of standard big Transformer. During ﬁne-tuning, we only ﬁx the parameters of the language model for the ﬁrst 10,000 steps. Recent empirical improvements with language models have showed that unsupervised pre- training (Peters et al., 2018; Radford et al., 2018; Devlin et al., 2018; Dai et al., 2019; Sun et al., 2019) on very large corpora is an integral part of many NLP tasks. We implement a big Trans- former language model using PaddlePaddle1, an end-to-end open source deep learning platform de- veloped by Baidu. It provides a complete suite of deep learning libraries, tools and service plat- forms to make the research and development of deep learning simple and reliable. The language model is pre-trained only with masked language model task (Taylor, 1953; Devlin et al., 2018; Sun et al., 2019) on a monolingual corpus of the source language. 1https://github.com/paddlepaddle/ paddle 2.2 Deeper Transformer According to the previous literatures, the model tends to specialize in word sense disambiguation and tends to focus on local dependencies in lower layers but ﬁnds long dependencies on higher ones while increasing the size of layers in the encoder (Tang et al., 2018; Domhan, 2018; Raganato and Tiedemann, 2018). Meanwhile, inspired by the success of pre-trained Transformer, that transla- tion results can beneﬁt from very deep architec- tures of encoder, we introduce the deeper Trans- former. But vanishing-gradient problem is en- countered by just increasing the encoder depth, We use all the available resources of WMT19 as the pre-training corpus. For the Chinese language model, we use the concatenation of Chinese Gi- gaword, Chinese News Crawl, XMU and the Chi- nese part of CWMT and UN corpus. For the En- 375 large amount of monolingual data according to the perplexity score. the standard Transformer failed to train. To alle- viate the vanishing-gradient problem, we design a particular residual connections. Speciﬁcially, the outputs of all preceding layers are used as inputs for each layer, as opposed to the standard Trans- former model in which the residual connection is employed between two adjacent layers. After training language models on different types of monolingual data (i.e., News crawl, Giga- word), we select 96M English sentences and 23M Chinese sentences according to LM scores, since Chinese monolingual corpus provided by WMT 19 is much less than that of English. The selected English sentences are translated and divided into 12 portions. For the 23M Chinese sentences, we translate and divide the sentences into 3 portions, resulting in 8M synthetic parallel sentence pairs in each portion. We further evaluate the performance of the similar model training on a different bilin- gual corpus which consists of the original bilin- gual corpus and the generated synthetic bitext. Ac- cording to the BLEU score of translation results on the WMT 18 news translation dev set, we se- lect the top 4 most effective portions for training Chinese→English system and the top 2 portions for training English→Chinese system. In the ﬁ- nal submission, the selected synthetic portions are used to enhance individual baseline models by the following joint training technique, respectively. 2.2 Deeper Transformer In our experiments, both the big Transformer with 15 encoder layers and the base trans- former with 30 encoder layers obtain signiﬁ- cant improvements compared with the standard big Transformer on Chinese→English translation task, whereas the improvement is not remarkable on English→Chinese translation task. 2.3 Bigger Transformer Motivated by the success of increasing the model size on the language modeling (Devlin et al., 2018) and NMT (Vaswani et al., 2017) tasks, we propose bigger Transformer which has larger inner dimen- sion of feed-forward network than the standard big Transformer. Speciﬁcally, we increase the inner dimension of feed-forward network from 4,096 to 15,000 constrained by the GPU memory capacity. To overcome the overﬁtting problem, we set at- tention dropout and relu dropout from 0.1 to 0.3, increasing the value of label smoothing from 0.1 to 0.2. Note that the speciﬁc settings are only em- ployed for the bigger Transformer. 2.5 Joint Training and Data Augmentation In the work of Zhang et al. (2018), they proposed a novel method for better usage of monolingual data from both source side and target side by jointly optimizing a source-to-target (S2T) model and a target-to-source (T2S) model, training with sev- eral iterations. In each iteration, the T2S model is responsible for generating synthetic parallel train- ing data for S2T model using target-side monolin- gual data, while S2T model is employed to gener- ate synthetic parallel training data for T2S model using source-side monolingual data. After train- ing on the additional synthetic data, the perfor- mance of both T2S model and S2T model can be further improved. In the next iteration, the two im- proved models can potentially generate better syn- thetic parallel data. This procedure can be applied in several iterations until no further improvement can be obtained. In addition, we explore the effectiveness of in- creasing hidden size with respect to the Trans- former model. However, the results indicate that the model with increased hidden size performs worse than the model with big feed-forward net- work. Nevertheless, we retain the model with dif- ferent hidden size as one diverse system for the generation of the ﬁnal ensemble model, which has shown effective performance in our further exper- iments. 2.6 Knowledge Distillation The early adoption of knowledge distillation (Kim and Rush, 2016) is for model compression, where the goal is to deliver a compact student model that matches the accuracy of a large teacher model or the ensemble of models. In our knowledge dis- tillation approach, we translate the source side of the bilingual data with a Right-to-Left (R2L) (Liu et al., 2016) model teacher and different architec- ture NMT teachers to use the translations as addi- tional training data for the student network. Con- sidering that distillation from a bad teacher model is likely to hurt the student model and thus result in inferior accuracy, we selectively use distillation in the training process. In particular, the sentences generated by a teacher model are ﬁltered if BLEU scores are below a threshold τ. According to our previous empirical results, we select English trans- lations with BLEU score higher than 30 and Chi- nese translations with BLEU score higher than 42. Table 1: Statistics of the bilingual training data (Chn indicates Chinese while En indicates English). Table 1: Statistics of the bilingual training data (Chn indicates Chinese while En indicates English). data. The dominant approach for domain adapta- tion is training on large-scale out-of-domain data and then ﬁne-tuning on the in-domain data (Lu- ong and Manning, 2015). Thus the effectiveness of the domain adaptation depends on the selected in-domain data. According to our previous empirical results, us- ing the WMT 18 dev set to ﬁne-tune the models straightforwardly achieves the best results. In our ﬁnal submission, we set the batch size to 1,024 and ﬁne-tune the model for a few iterations on the WMT 18 dev set. It is surprising to ﬁnd a gain of almost +2 BLEU improvement on WMT 18 Chinese→English test set. However, on WMT 18 English→Chinese test set, the improvement is not signiﬁcant. There are two kinds of teacher models to help a student model improve translation performance: In WMT 17 and 18, the source side of both dev set and test set are composed of two parts: documents created originally in Chinese and doc- uments created originally in English. We split both the dev set and test set into original Chi- nese part and original English part according to tag attributes of SGM ﬁles. Finally, we trans- late each speciﬁc test part with the model ﬁne- tuned on the corresponding dev set. 2.4 Large-scale Back-Translation In recent work, Edunov et al. (2018) proposed an effective approach to improve the translation quality by exploiting back-translation mechanism on the large-scale monolingual corpus. Follow- ing their work, we also train our model on the synthetic bilingual corpus to further improve the performance. However, the provided monolingual data contains a certain amount of noise and out-of- domain data which may affect the translation qual- ity implicitly. Therefore, we use a language model to select high-quality and in-domain data from the In addition, we also augment the training data by exploring the bilingual corpus rather than the monolingual corpus. Speciﬁcally, we translate the sentences in the target language back into the source language by diverse training models, such as Left-to-right model and Right-to-left model. This procedure can be viewed as one alternative 376 Source Chn→En En→Chn CWMT 6.7M 6.7M UN 9M 3.5M Wiki Titles - 0.6M Total 15.7M 10.8M Table 1: Statistics of the bilingual training data (Chn indicates Chinese while En indicates English). Source Chn→En En→Chn CWMT 6.7M 6.7M UN 9M 3.5M Wiki Titles - 0.6M Total 15.7M 10.8M Table 1: Statistics of the bilingual training data (Chn indicates Chinese while En indicates English). solution for alleviating the exposure bias problem (Ranzato et al., 2016). 2.6 Knowledge Distillation Experi- ments show signiﬁcant improvement with this method, that is, 2.23 BLEU improvements on Chinese→English test set and 0.5 BLEU improve- ments on English→Chinese test set. This indi- cates that the translation quality is affected by the original sources of the language. Consider the English→Chinese task, if the English sentences are created from native English corpus, then the corresponding Chinese sentences are translation style, so the model ﬁne-tuned on these parallel sentences is more inclined to decode with transla- tion style. Similarly, if the Chinese sentences are created from native Chinese corpus, the ﬁne-tuned English→Chinese model decodes with more na- tive style. • R2L Teacher: The idea is to reverse the tar- get sentences of bilingual corpus and train a R2L model. Then we employ R2L model to translate the source sentences of the bilin- gual corpus and reverse the translated sen- tences. The pseudo corpus is added to the real bilingual corpus in order to enhance the L2R model. The paradigm can be regarded as a kind of knowledge transfer method which provides complementary information for stu- dent model to learn. • Hybrid Heterogeneous Teacher: Pre-trained Transformer, deeper Transformer and bigger Transformer represent a source sentence at different granularities, therefore it is intuitive that each model can learn effective knowl- edge from other models. For each individual model, we use the other two models as the teacher model to further improve the perfor- mance. 2.7 Fine-tuning with In-domain Data Domain adaptation plays an important role in im- proving the performance towards given testing In the ﬁnal submission, we take the following steps to avoid overﬁtting: 1) We employ the en- 377 Settings Big Transformer Pre-trained Transformer Deeper Transformer Bigger Transformer Baseline 25.86 - - - + Back Translation 26.72 27.68 26.83 27.54 + Joint Training 26.95 27.79 27.01 27.61 + Knowledge Distillation 27.4 28.11 27.43 27.88 + Fine-tuning 29.39 29.87 29.82 30.11 + Ensemble 31.59 + Re-ranking* 31.83 Table 2: BLEU evaluation results on the WMT 2018 Chinese→English test set (* denotes the submitted system). • Weighted Voting Features: Average of BLEU scores calculated between each hypothesis and the other hypotheses. semble models to translate the WMT 19 test set, and use the translations as additional synthetic ﬁne-tuning corpus. 2) We ﬁne-tune the ﬁnal sys- tem on the mixture of the additional synthetic cor- pus and the selected in-domain corpus. 2.8 Model Ensemble Model ensemble is a widely used technique to boost the performance by combining the predic- tions of several models at each decoding step. In our previous experiments, we ﬁnd that the im- provement is slight while integrating the predic- tions of multiple models with similar model archi- tecture. Instead, we train our models with different model architectures training on different versions of training data, increasing the model diversity for the model ensemble. The experimental results in- dicate that this method achieves absolute improve- ments over the single system (at most a 1.7 BLEU point improvements). 3.1 Pre-processing and Post-processing The Chinese data has been tokenized using the Jieba tokenizer3. For English data, punctuation normalization, aggressive tokenization and true- casing are applied orderly to all sentences with the scripts provided in Moses. We also ﬁlter the paral- lel sentences which are duplicated or bad align- ment scores obtained by fast-align (Dyer et al., 2013), and then we have a preprocessed bilingual training data consisting of 18M parallel sentences. The Chinese data has been tokenized using the Jieba tokenizer3. For English data, punctuation normalization, aggressive tokenization and true- casing are applied orderly to all sentences with the scripts provided in Moses. We also ﬁlter the paral- lel sentences which are duplicated or bad align- ment scores obtained by fast-align (Dyer et al., 2013), and then we have a preprocessed bilingual training data consisting of 18M parallel sentences. In post-processing phase, the English transla- tions are true-cased and de-tokenized with the scripts provided in Moses. We use simple rules to normalize the punctuations and Arabic numer- als in the Chinese translations. 3 Experiments and Results All of our experiments are carried out on 32 ma- chines with 8 NVIDIA V100 GPUs each of which have 32 GB memory. For all models, we average the last 20 checkpoints to avoid overﬁtting. We use cased BLEU scores calculated with Moses2 mteval-v12a.pl script as evaluation metric. Fol- lowing the organizers’ suggestion, News dev 2018 is used as the development set and News test 2018 as the test set. 2.9 Re-ranking In order to get better translation results, we gener- ate n-best hypotheses with an ensemble model and then train a re-ranker using k-best MIRA (Cherry and Foster, 2012) on the validation set. K-best MIRA is a version of MIRA (Chiang et al., 2008) that works with a batch tuning to learn a re-ranker for the n-best hypotheses. The features we use for re-ranking are: In post-processing phase, the English transla- tions are true-cased and de-tokenized with the scripts provided in Moses. We use simple rules to normalize the punctuations and Arabic numer- als in the Chinese translations. • NMT Features: Ensemble model score and Right-to-Left model score. 3https://github.com/fxsjy/jieba 2http://www.statmt.org/moses/ 3.2 Chinese→English For Chinese→English task, we do not use all of the 18M preprocessed parallel sentences, in that there is much out-of-domain data in UN cor- pus. Table 1 shows that the 6.7M CWMT cor- pus and 9M UN corpus which are selected ran- • Language Model Features: Multiple n-gram language models and backward n-gram lan- guage models. • Length Features: Length ratio and length dif- ference between source sentences and hy- potheses. 378 Settings Big Transformer Pre-trained Transformer Deeper Transformer Bigger Transformer Baseline 39.2 - - - + Back Translation 43.33 43.7 42.19 44 + Joint Training 43.86 44.12 42.5 44.78 + Knowledge Distillation 44.25 44.6 42.86 45.27 + Fine-tuning 44.72 44.75 42.94 45.79 + Ensemble 46.42 + Re-ranking* 46.51 Table 3: BLEU evaluation results on the WMT 2018 English→Chinese test set (* denotes the submitted system). evaluation results on the WMT 2018 English→Chinese test set (* denotes the submitted system). best single model by 1.49 BLEU score. How- ever, the improvement of re-ranking is relatively slight, and we attribute this to the strong per- formance of ensemble models. Our WMT 2019 Chinese→English submission achieves a cased BLEU score of 38.0, winning the ﬁrst place among all submissions. domly are used as our bilingual training set for Chinese→English task. We learn a BPE (Sen- nrich et al., 2015b) model with 30K merge oper- ations, in which 46.4K and 31K sub-word tokens are adopted as Chinese and English vocabularies separately. We set beam size to 12 and alpha to 1.1 during decoding. 12 portions of sentences are selected from huge volumes of English monolingual data, and we carry out a large number of experiments in which the Transformer models are trained with each por- tion. And then 4 most effective portions are se- lected. Due to the extensive training time and the approaching deadline for submissions, pre-trained transformer, deep Transformer(base Transformer with 30 encoder layers) and bigger Transformer are trained on the combination of real bilingual data and the synthetic data directly. For each dif- ferent architecture model, we train 4 more sys- tems with different portions of monolingual data and different parameters in order to obtain more diverse models. For comparison, we only report results on the WMT 2018 test set with the same portion of monolingual data. 3.3 English→Chinese As listed in the Table 1, the parallel training data for English→Chinese translation task con- sists of about 6.7M sentence pairs from the ﬁl- tered CWMT Corpus, 3.5M sentence pairs from the UN Parallel Corpus, 0.6M sentence pairs from the Wiki Titles Corpus. For the UN data, we train a 5-gram KN language model on the Chinese sides of the CWMT data and select 3.5M sentence pairs according to their perplexities. The size of the En- glish vocabulary and the Chinese vocabulary are 31K and 48.6K respectively after BPE operation. We use beam search with a beam size of 12, and set alpha 0.8. From the Table 3, we can observe: 1) We obtain +4.13 BLEU score when adding the synthetic par- allel data to the training set of the Transformer. 2) We further gain +0.92 BLEU score after applying joint training and knowledge distillation for the Transformer system. 3) The improvement from the ﬁne-tuning technique is relative slight for the pre-trained Transformer and deeper Transformer, whereas it is effective for the Transformer and big- ger Transformer, with about 0.5 BLEU score im- provements. Table 2 shows that the translation quality is largely improved using proposed techniques. We observe solid improvement of 0.86 BLEU for the baseline system after back translation. Joint train- ing and knowledge distillation yield improvements over all the different architecture models, approx- imating 0.34-0.68 BLEU improvements toward single models. It is also clear that the ﬁne-tuning technique brings substantial improvements com- pared with the baseline systems. Notably, the ensemble models consist of pre- trained Transformers and bigger Transformers. We omit the deeper Transformer model due to its worse performance on this translation task. On the WMT 2019 English→Chinese task, our submis- sion achieves 42.4 cased BLEU score, winning the In our experiments, the ensemble models con- sists of 8 single models: 1 Transformer, 2 pre- trained Transformers, 2 deeper Transformers and 3 bigger Transformers. As shown in the Ta- ble 2, the ensemble models also outperform the 379 second place in the translation task. bidirectional transformers for language understand- ing. arXiv preprint arXiv:1810.04805. 5 Acknowledgements Hany Hassan, Anthony Aue, Chang Chen, Vishal Chowdhary, Jonathan Clark, Christian Feder- mann, Xuedong Huang, Marcin Junczys-Dowmunt, William Lewis, Mu Li, et al. 2018. Achieving hu- man parity on automatic chinese to english news translation. arXiv preprint arXiv:1803.05567. 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https://openalex.org/W3207481263	https://www.frontiersin.org/articles/10.3389/fcimb.2021.736186/pdf	English	null	Microbial Biofilm Decontamination on Dental Implant Surfaces: A Mini Review	Frontiers in cellular and infection microbiology	2,021	cc-by	22,733	Microbial Bioﬁlm Decontamination on Dental Implant Surfaces: A Mini Review Jagjit Singh Dhaliwal 1, Nurul Adhwa Abd Rahman 1, Long Chiau Ming 1, Sachinjeet Kaur Sodhi Dhaliwal 1, Joe Knights 1 and Rubens Ferreira Albuquerque Junior 2 Jagjit Singh Dhaliwal 1, Nurul Adhwa Abd Rahman 1, Long Chiau Ming 1, Sachinjeet Kaur Sodhi Dhaliwal 1, Joe Knights 1 and Rubens Ferreira Albuq 1 Pengiran Anak Puteri Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei, Darussalam, Gadong, Brunei, 2 Faculdade de Odontologia, Universidade de São Paulo, Butantã, Brazil Keywords: dental implant, decontamination, titanium, bacteria, bioﬁlm Introduction: After insertion into the bone, implants osseointegrate, which is required for their long-term success. However, inﬂammation and infection around the implants may lead to implant failure leading to peri-implantitis and loss of supporting bone, which may eventually lead to failure of implant. Surface chemistry of the implant and lack of cleanliness on the part of the patient are related to peri-implantitis. The only way to get rid of this infection is decontamination of dental implants. Edited by: Jean-Paul Motta, U1220 Institut de Recherche en Sante´ Digestive (INSERM), France Reviewed by: Ferdinand Xiankeng Choong, Karolinska Institutet (KI), Sweden Andrea Cochis, University of Eastern Piedmont, Italy Reviewed by: Ferdinand Xiankeng Choong, Karolinska Institutet (KI), Sweden Andrea Cochis, University of Eastern Piedmont, Italy Objective: This systematic review intended to study decontamination of microbial bioﬁlm methods on titanium implant surfaces used in dentistry. Correspondence: Jagjit Singh Dhaliwal jagjit.dhaliwal@ubd.edu.bn Methods: The electronic databases Springer Link, Science Direct, and PubMed were explored from their inception until December 2020 to identify relevant studies. Studies included had to evaluate the efﬁciency of new strategies either to prevent formation of bioﬁlm or to treat matured bioﬁlm on dental implant surfaces. Specialty section: This article was submitted to Bioﬁlms, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 04 July 2021 Accepted: 09 September 2021 Published: 08 October 2021 Results and Discussion: In this systematic review, 17 different groups of decontamination methods were summarized from 116 studies. The decontamination methods included coating materials, mechanical cleaning, laser treatment, photodynamic therapy, air polishing, anodizing treatment, radiation, sonication, thermal treatment, ultrasound treatment, chemical treatment, electrochemical treatment, antimicrobial drugs, argon treatment, and probiotics. MINI REVIEW published: 08 October 2021 doi: 10.3389/fcimb.2021.736186 Data Sources A systematic search was performed in Science Direct, PubMed, and Springer Link from inception up until December 2020. The search was limited to studies published in the English language due to the authors’ inability to translate other published languages in the literature. The following search terms were used: “dental” AND “implant” AND “titanium” AND “bacteria” AND “bioﬁlm” AND (“treatment” OR “decontamination” OR “eradication” OR “cleaning” OR “remove”). Wildcards, e.g., asterisk (), a question mark ()?, or other symbols that designate the selected database, were applied. Original papers comprising of experimental studies that used titanium materials for the studies were included in the present review. The studies included should have reported the presence of microbial bioﬁlm both by bacteria and fungi. After date was extracted, it was entered into Microsoft Excel in a data extraction form for enabling summarization of data and ﬁnal report writing. Data analysis was completed using SPSS Software Version 23 and PRISM Software Version 7 (GraphPad Inc.). Implant loss often triggers a devastating psychological impact on patients’ lives, with signiﬁcantly high ﬁnancial losses to families and healthcare systems (Alzahrani and Gibson, 2018; Frazadmoghadam et al., 2018; Hoeksema et al., 2018). Peri-implantitis is reported to occur at different levels of severity, with about 10% of them being lost within a 5-year period (Koldsland et al., 2010; Caton J. et al., 2018). It has been reported that peri‐implantitis takes place in 3–47% of dental implants (Koldsland et al., 2010; Matarazzo et al., 2018; French et al., 2019). Peri-implantitis is frequently related to periodontal pathogens and usually treated with antibiotics (Ting et al., 2018; Hussain et al., 2018). Bacterial bioﬁlm has been advocated to be the leading etiological factor in the causation of peri-implantitis. Bioﬁlm is a highly structured, matrix-enclosed bacterial community in a sessile state (Magana et al., 2018). Bioﬁlm formation is a typical feature of bacterial growth, causing evasion from host cells and avoiding competition with other microbial communities (Guilhen et al., 2017). Several steps are needed to form bioﬁlms, which include (i) planktonic bacteria adhesion on the surface, (ii) adhesion on the surface to establish a ﬁrm platform, (iii) co-aggregation with other bacteria, which further stabilizes their buildup architecture, (iv) growth by absorbing nutrition from the environment until maturation phase, and (v) detachment of a portion of bioﬁlms to invade another vulnerable site (Guilhen et al., 2017). Data Sources It is more challenging to eradicate established bioﬁlm colonization than to eradicate the circulating contamination itself (i.e., planktonic microbiota). This signiﬁcantly increases resistance to antibiotic treatment (Arciola et al., 2018). Resident microbiota and matrix in bioﬁlm may hinder access of antimicrobials locally, preventing their penetration into deeper layers of bioﬁlm (Kuang et al., 2018). Citation: Dhaliwal JS, Abd Rahman NA, Ming LC, Dhaliwal SKS, Knights J and Albuquerque Junior RF (2021) Microbial Bioﬁlm Decontamination on Dental Implant Surfaces: A Mini Review. Front. Cell. Infect. Microbiol. 11:736186. doi: 10.3389/fcimb.2021.736186 Conclusion: The ﬁndings suggest that most of the decontamination methods were effective in preventing the formation of bioﬁlm and in decontaminating established bioﬁlm on dental implants. This narrative review provides a summary of methods for future research in the development of new dental implants and decontamination techniques. October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. INTRODUCTION Therefore, it is deemed imperative that studies in the future focus on the prevention or reduction of bioﬁlm formation. Dental implants are number one choice for replacement of teeth. It is a valuable option in several treatment scenarios, e.g., (i) when teeth are lost to non-periodontal diseases, infection due to caries, traumatic injuries; (ii) loss of teeth due to periodontal diseases but the remaining teeth can be maintained; and (iii) combined scenarios requiring replacement of missing teeth (Greenwell et al., 2019). An implant is typically composed of titanium or titanium alloys because of its biocompatibility and mechanical properties. Recent studies show that titanium dental implants survived for more than 20 years, regardless of their long-term exposure to the oral environment (Chen et al., 2017; Albrektsson et al., 2019). After insertion into the bone, implant osseointegration is expected to occur and is fundamental for its long-term success (Alghamdi, 2018). However, dental implants cannot be used for treatment of periodontal or dental disease (Greenwell et al., 2019). In addition, inﬂammatory reaction and infection around the implants may occur and can lead to implant failure, a phenomenon called peri-implantitis (Berglundh et al., 2019). Peri-implantitis is caused by plaque bacteria, leading to inﬂammation of mucosa and subsequent bone loss surrounding the implant, which may ultimately result in total implant failure (Berglundh et al., 2019). Surface chemistry and lack of cleanliness are intimately associated with peri-implantitis (Rupp et al., 2018). Several techniques for decontamination of implants have been used over the years. However, none of them have successfully produced optimal results. Mechanisms underpinning these processes are still not understood fully and decontamination of implants remains challenging. A previous systematic review was performed a few years ago (Grischke et al., 2016). Thus, an updated review is necessary. Additionally, other systematic reviews have only evaluated the ability of a selected type of method for decontamination of the implant surface (Louropoulou et al., 2015; Chouirfa et al., 2018; Dutra et al., 2018; Mishra and Chowdhary, 2018). The primary objective of this systematic review was to study and report evidence on all currently investigated methods for decontaminating pathogenic microbiota on dental implant surfaces. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org RESULTS AND DISCUSSION 2014; Ciandrini et al., 2014; Drago et al., 2014; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Kaliaraj et al., 2014; Kang et al., 2014; Lv et al., 2014; Massa et al., 2014; Sahrmann et al., 2014; Schmage et al., 2014; Schmidt et al., 2014; Yamada et al., 2014; Abdulkareem et al., 2015; Charalampakis et al., 2015; Cruz et al., 2015; de Avila et al., 2015; Duske et al., 2015; Janković et al., 2015; Jennings et al., 2015; Lewandowska et al., 2015; Narendrakumar et al., 2015; Park et al., 2015; Yucesoy et al., 2015; Wood et al., 2015; Zhang et al., 2015; Ayre et al., 2016; Chen et al., 2016; Ciandrini et al., 2016; Cochis et al., 2016; Cotolan et al., 2016; Cunha et al., 2016; Giannelli et al., 2016; Godoy-Gallardo et al., 2016; Gopal et al., 2016; Guan et al., 2016; John et al., 2016; Kuehl et al., 2016; Kotsakis et al., 2016; Mang et al., 2016; Preissner et al., 2016; Rodrı́guez-Contreras et al., 2016; Shi et al., 2016; Verardi et al., 2016; Al-Hashedi et al., 2017; Batsukh et al., 2017; Canullo et al., 2017; Ciandrini et al., 2017; Cometa et al., 2017; Dostie et al., 2017; Eick et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Granick et al., 2017; Hirschfeld et al., 2017; Kim et al., 2017; Kulkarni Aranya et al., 2017; Li et al., 2017; Macpherson et al., 2017; Matthes et al., 2017; Prieto-Borja et al., 2017; Schmidt et al., 2017; Strever et al., 2017; Ramesh et al., 2017; Wang et al., 2017; Wiedmer et al., 2017; Ye et al., 2017; Zhang et al., 2017; Al-Hashedi et al., 2018; Akhavan et al., 2018; Atefyekta et al., 2018; Azizi et al., 2018; Ferraris et al., 2018; Fukushima et al., 2018; Hidalgo-Robatto et al., 2018; Hoyos- Nogués et al., 2018; Lee et al., 2018; Montelongo-Jauregui et al., 2018; Pantaroto et al., 2018; Pissinis et al., 2018; Santos-Coquillat et al., 2018; Schneider et al., 2018; Souza et al., 2018; Trobos et al., 2018; Vilarrasa et al., 2018; Wang et al., 2018; Zhang et al., 2018; Huang et al., 2019; Schmidt et al., 2019). titanium materials, method of decontamination, and type of decontamination method. RESULTS AND DISCUSSION In this systematic review, 63 studies focused on bioﬁlm- prevention methods (n = 63, 54.3%) (Duarte et al., 2009; Ewald and Ihde, 2009; Größner-Schreiber et al., 2009; Sennhenn-Kirchner et al., 2009; Tamai et al., 2009; Baffone et al., 2011; Ercan et al., 2011; Fröjd et al., 2011; Giordano et al., 2011; Cortizo et al., 2012; Rehman et al., 2012; Subramanian et al., 2012; Trujillo et al., 2012; Alcheikh et al., 2013; De Giglio et al., 2013; Holmberg et al., 2013; Roberts et al., 2013; Godoy-Gallardo et al., 2014; Lv et al., 2014; Kaliaraj et al., 2014; Kang et al., 2014; Massa et al., 2014; Yamada et al., 2014; Abdulkareem et al., 2015; de Avila et al., 2015; Janković et al., 2015; Jennings et al., 2015; Lewandowska et al., 2015; Narendrakumar et al., 2015; Wood et al., 2015; Yucesoy et al., 2015; Zhang et al., 2015; Ayre et al., 2016; Cochis et al., 2016; Cotolan et al., 2016; Cunha et al., 2016; Godoy-Gallardo et al., 2016; Gopal et al., 2016; Guan et al., 2016; Kuehl et al., 2016; Rodrı́guez-Contreras et al., 2016; Shi et al., 2016; Ciandrini et al., 2017; Cometa et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Hirschfeld et al., 2017; Kulkarni Aranya et al., 2017; Macpherson et al., 2017; Schmidt et al., 2017; Ye et al., 2017; Akhavan et al., 2018; Atefyekta et al., 2018; Ferraris et al., 2018; Hidalgo-Robatto et al., 2018; Hoyos-Nogués et al., 2018; Pantaroto et al., 2018; Pissinis et al., 2018; Santos-Coquillat et al., 2018; Trobos et al., 2018; Vilarrasa et al., 2018; Wang et al., 2018; Zhang et al., 2018), whereas 51 studies focused on bioﬁlm-treatment methods (n = 51, 44.0%) (Schwarz et al., 2005; Gonçalves et al., 2010; Mohn et al., 2011; Ntrouka et al., 2011; Bürgers et al., 2012; Cochis et al., 2013; Diab Al-Radha et al., 2013; Eick et al., 2013; Idlibi et al., 2013; Chen et al., 2014; Drago et al., 2014; Hauser-Gerspach et al., 2014; Sahrmann et al., 2014; Schmage et al., 2014; Schmidt et al., 2014; Charalampakis et al., 2015; Cruz et al., 2015; Duske et al., 2015; Park et al., 2015; Chen et al., 2016; Ciandrini et al., 2016; Mang et al., 2016; Giannelli et al., 2016; John et al., 2016; Kotsakis et al., 2016; Preissner et al., 2016; Verardi et al., 2016; Al-Hashedi et al., 2017; Batsukh et al., 2017; Canullo et al., 2017; Dostie et al., 2017; Eick et al., 2017; Granick et al., 2017; Kim et al., 2017; Li et al., 2017; Matthes et al., 2017; Prieto-Borja et al., 2017; Ramesh et al., 2017; Strever et al., 2017; Wang et al., 2017; Wiedmer et al., 2017; Zhang et al., 2017; Al-Hashedi et al., 2018; Azizi et al., 2018; Fukushima et al., 2018; Lee et al., 2018; Montelongo-Jauregui et al., 2018; Schneider et al., 2018; Souza et al., 2018; Huang et al., 2019; Schmidt et al., 2019), while two studies offered their novel method to work for both bioﬁlm-prevention and bioﬁlm-treatment measures (n = 2, 1.7%) (Lilja et al., 2012; Ciandrini et al., 2014). RESULTS AND DISCUSSION The electronic exploration yielded six hundred and forty-three results (n = 643). Duplicates were removed, and on additional screening, one hundred and sixteen studies comprised this review (n =116) (Schwarz et al., 2005; Duarte et al., 2009; Ewald and Ihde, 2009; Größner-Schreiber et al., 2009; Sennhenn-Kirchner et al., 2009; Tamai et al., 2009; Gonçalves et al., 2010; Baffone et al., 2011; Ercan et al., 2011; Fröjd et al., 2011; Giordano et al., 2011; Mohn et al., 2011; Ntrouka et al., 2011; Bürgers et al., 2012; Cortizo et al., 2012; Lilja et al., 2012; Rehman et al., 2012; Subramanian et al., 2012; Trujillo et al., 2012; Alcheikh et al., 2013; Cochis et al., 2013; De Giglio et al., 2013; Diab Al-Radha et al., 2013; Eick et al., 2013; Holmberg et al., 2013; Idlibi et al., 2013; Roberts et al., 2013; Chen et al., Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org October 2021 \| Volume 11 \| Article 736186 2 Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. RESULTS AND DISCUSSION In Vivo Studies A ﬂowchart (Supplementary Information 1) demonstrates the procedure of inclusion and exclusion of pertinent articles. We excluded review papers, non-English language studies, and those not relevant to the focus of this review. Our analysis revealed that the topic of our systematic review has been deeply researched for the previous 10 years, especially in the years 2017–2018. The interest on this topic is mainly because of titanium being an inert material with the properties of encouraging tissue healing and bacterial colonization resistance, as compared with other implant materials (Chen et al., 2017). Numerous studies have attempted to evaluate different methods to decontaminate dental bioﬁlms. The decontamination methods included in this systematic review were categorized into 19 groups, namely, the coating of titanium materials (47.4%), mechanical cleaning (10.3%), laser treatment (9.5%), photodynamic therapy (4.3%), air polishing (3.4%), anodizing treatment (3.4%), radiation (3.4%), sonication (0.9%), thermal treatment (0.9%), ultrasound (0.9%), chemical treatment (13.8%), electrochemical treatment (1.7%), antimicrobial drugs (0.9%), argon treatment (0.9%), and probiotics (0.9%). Table 1 presented the decontamination methods using specialized instruments and the ﬁndings of the respective studies. These decontamination methods were then divided into two types based on whether the bioﬁlm was already established (bioﬁlm-treatment) or not (bioﬁlm-prevention). Supplementary Information 2 shows the coating of the Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 11 Mechanical cleaning and laser treatment Gracey (dentin) or titanium curets (CUR), Er:YAG, photodynamic therapy (PDT) and CUR with adjunctive PDT (CUR/PDT) In Vivo Studies Bioﬁlm-Treatment (Lee et al., 2018) Bacterial grading showed low levels of bacteria with no signiﬁcant differences (p < 0.05) within any of the groups at the two time points. Also, no signiﬁcant differences were evident between the groups at either baseline or 12 months after regular preventive therapy (p > 0.05). The four treatment modalities in the present study are comparable and generally resulted in positive outcomes over the course of a year following attachment of superstructures to implants. Bioﬁlm-Treatment (Schmidt et al., 2019) Two of the tested side-to-side toothbrushes were able to reduce an in-vitro bioﬁlm by noncontact brushing. The efﬁcacy of the tested toothbrushes for noncontact bioﬁlm removal differed signiﬁcantly. Bioﬁlm-Treatment (Schmidt et al., 2014) Ti brushes were more effective than curets and Er: YAG laser in the removal of surface contaminants, whereas Er: YAG laser was more effective than curets and Ti brushes in killing the bioﬁlm bacteria. None of the methods tested in this study was able to completely eliminate Ti surface contaminants. Bioﬁlm-Treatment (Al-Hashedi et al., 2017) The use of Er:YAG laser yielded clear advantages compared to the other debridement modalities. All treatment methods decreased statistically signiﬁcantly (p < 0.001) total CFUs in bioﬁlms compared with untreated titanium surfaces. Bioﬁlm-Treatment (Eick et al., 2017) (Continued) Neither a sole CAP treatment nor a sole brushing provided a completely decontaminated surface. A combined treatment of brushing and subsequent CAP treatment led to a cleaned surface, which allowed cells to grow comparable to cells on the sterile control. Bioﬁlm-T The combination of mechanical and chemical cleansing used in the present study was ineffective in complete removal of the bioﬁlm from all four titanium discs. Bioﬁlm-T Neither a sole CAP treatment nor a sole brushing provided a completely decontaminated surface. A combined treatment of brushing and subsequent CAP treatment led to a cleaned surface, which allowed cells to grow comparable to cells on the sterile control. Bioﬁlm-Treatment (Duske et al., 2015) The combination of mechanical and chemical cleansing used in the present study was ineffective in complete removal of the bioﬁlm from all four titanium discs. Bioﬁlm-Treatment (Charalampakis et al., 2015) Treatment of titanium ﬁxture surfaces with ultrasonic metal, plastic, or carbon tip signiﬁcantly enhanced bacterial removal efﬁcacy of brushing. Thorough instrumentation that can smooth the whole exposed surface may facilitate maintenance of the implants. Curet debridement, normal saline irrigation, and placement in 0.12% chlorhexidine Titanium curets (TC), stainless steel ultrasonic tip (PS), erythritol air-polishing powder (EP), and rubber cup polishing (CON) In Vivo Studies Twenty-two studies applied oral normal ﬂora in human participants who were either healthy or diagnosed with peri- implantitis or periodontitis (n = 22, 19.0%) (Schwarz et al., 2005; Größner-Schreiber et al., 2009; Rehman et al., 2012; Diab Al- Radha et al., 2013; Cochis et al., 2013; Idlibi et al., 2013; Abdulkareem et al., 2015; Charalampakis et al., 2015; de Avila et al., 2015; Duske et al., 2015; Park et al., 2015; John et al., 2016; October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. TABLE 1 \| Decontamination methods using specialized instruments and the ﬁndings of the respective studies. No. Type of method Method of decontamination Study ﬁndings TABLE 1 \| Decontamination methods using specialized instruments and the ﬁndings of the respective studies. No. Type of method Method of decontamination Study ﬁndings Type of decontamination method (Bioﬁlm- Prevention or Bioﬁlm- Treatment) Reference Mechanical cleaning Titanium surface treatment instruments Metal curets are not recommended for smooth titanium surface debridement due to severe texture alteration. Rough surfaces treated with a metal curet and the air-powder abrasive system were less susceptible to bacterial adhesion, probably due to texture modiﬁcation and the presence of abrasive deposits. Bioﬁlm-Prevention (Duarte et al., 2009) 2 Mechanical cleaning Stainless steel (SSC), titanium curets (TC), air-polisher using glycine-based, perio (PP), soft d (SP) th it l d No signiﬁcant differences were observed in the surface characteristics (except for SSC) or bacterial colonization based on one-time instrumentation. Bioﬁlm-Prevention (Schmidt et al., 2017) Study ﬁndings decontamination method (Bioﬁlm- Prevention or Bioﬁlm- Treatment) Metal curets are not recommended for smooth titanium surface debridement due to severe texture alteration. Rough surfaces treated with a metal curet and the air-powder abrasive system were less susceptible to bacterial adhesion, probably due to texture modiﬁcation and the presence of abrasive deposits. Bioﬁlm-Prevention (Duarte et al., 2009) No signiﬁcant differences were observed in the surface characteristics (except for SSC) or bacterial colonization based on one-time instrumentation. Bioﬁlm-Prevention (Schmidt et al., 2017) Metal curets are not recommended for smooth titanium surface debridement due to severe texture alteration. Rough surfaces treated with a metal curet and the air-powder abrasive system were less susceptible to bacterial adhesion, probably due to texture modiﬁcation and the presence of abrasive deposits. The best cleaning effectiveness (less than 4% residual bioﬁlm) was observed with the sonic and ultrasonic oscillating PEEK plastic tips and the Air polishing. However, the instruments were not able to completely clean the implant surfaces. In Vivo Studies Bioﬁlm-Treatment (Park et al., 2015) The non-surgical treatment used in this study was not effective in removing LPS from titanium surfaces and increasing osteoblast adhesion. Bioﬁlm-Treatment (Lee et al., 2018) Bacterial grading showed low levels of bacteria with no signiﬁcant differences (p < 0.05) within any of the groups at the two time points. Also, no signiﬁcant differences were evident between the groups at either baseline or 12 months after regular preventive therapy (p > 0.05). The four treatment modalities in the present study are comparable and generally resulted in positive outcomes over the course of a year following attachment of superstructures to implants. Bioﬁlm-Treatment (Schmidt et al., 2019) Two of the tested side-to-side toothbrushes were able to reduce an in-vitro bioﬁlm by noncontact brushing. The efﬁcacy of the tested toothbrushes for noncontact bioﬁlm removal differed signiﬁcantly. Bioﬁlm-Treatment (Schmidt et al., 2014) Ti brushes were more effective than curets and Er: YAG laser in the removal of surface contaminants, whereas Er: YAG laser was more effective than curets and Ti brushes in killing the bioﬁlm bacteria. None of the methods tested in this study was able to completely eliminate Ti surface contaminants. Bioﬁlm-Treatment (Al-Hashedi et al., 2017) The use of Er:YAG laser yielded clear advantages compared to the other debridement modalities. All treatment methods decreased statistically signiﬁcantly (p < 0.001) total CFUs in bioﬁlms compared with untreated titanium surfaces. Bioﬁlm-Treatment (Eick et al., 2017) (Continued) Treatment of titanium ﬁxture surfaces with ultrasonic metal, plastic, or carbon tip signiﬁcantly enhanced bacterial removal efﬁcacy of brushing. Thorough instrumentation that can smooth the whole exposed surface may facilitate maintenance of the implants. Bioﬁlm-Treatment (Park et al., 2015) The non-surgical treatment used in this study was not effective in removing LPS from titanium surfaces and increasing osteoblast adhesion. Bioﬁlm-Treatment (Lee et al., 2018) Bacterial grading showed low levels of bacteria with no signiﬁcant differences (p < 0.05) within any of the groups at the two time points. Also, no signiﬁcant differences were evident between the groups at either baseline or 12 months after regular preventive therapy (p > 0.05). The four treatment modalities in the present study are comparable and generally resulted in positive outcomes over the course of a year following attachment of superstructures to implants. Bioﬁlm-Treatment (Schmidt et al., 2019) Two of the tested side-to-side toothbrushes were able to reduce an in-vitro bioﬁlm by noncontact brushing. In Vivo Studies Bioﬁlm-Prevention (Duarte et al., 2009) No signiﬁcant differences were observed in the surface characteristics (except for SSC) or bacterial colonization based on one-time instrumentation. Bioﬁlm-Prevention (Schmidt et al., 2017) 2 Mechanical cleaning Stainless steel (SSC), titanium curets (TC), air-polisher using glycine-based, perio (PP), soft powders (SP), erythritol powder (EP) and an ultrasonic device using stainless steel (PS) or plastic-coated instruments (PI) 3 Mechanical cleaning Manual plastic curet, manual carbon ﬁber-reinforced plastic (CFRP) curet, sonic-driven prophylaxis brush, rotating rubber cup with prophylaxis paste, sonic-driven polyether ether ketone (PEEK) plastic tip, ultrasonic-driven PEEK plastic tip, air polishing with amino acid (glycine) powder 4 Mechanical cleaning Treatment with a brush (BR), 1% oxygen/argon cold atmospheric plasma (CAP) (PL), or brushing combined with CAP (BR þ PL) 5 Mechanical cleaning Mechanical brushing and cleansing with four different cleaning agents: NaCl, Decapinol, Hexident, Listerine 6 Mechanical cleaning Instrumentation with ultrasonic scalers 7 Mechanical cleaning Curet debridement, normal saline irrigation, and placement in 0.12% chlorhexidine 8 Mechanical cleaning Titanium curets (TC), stainless steel ultrasonic tip (PS), erythritol air-polishing powder (EP), and rubber cup polishing (CON) The best cleaning effectiveness (less than 4% residual bioﬁlm) was observed with the sonic and ultrasonic oscillating PEEK plastic tips and the Air polishing. However, the instruments were not able to completely clean the implant surfaces. Bioﬁlm-Treatment (Schmage et al., 2014) Bioﬁlm-Treatment (Schmage et al., 2014) The best cleaning effectiveness (less than 4% residual bioﬁlm) was observed with the sonic and ultrasonic oscillating PEEK plastic tips and the Air polishing. However, the instruments were not able to completely clean the implant surfaces. Neither a sole CAP treatment nor a sole brushing provided a completely decontaminated surface. A combined treatment of brushing and subsequent CAP treatment led to a cleaned surface, which allowed cells to grow comparable to cells on the sterile control. Bioﬁlm-Treatment (Duske et al., 2015) The combination of mechanical and chemical cleansing used in the present study was ineffective in complete removal of the bioﬁlm from all four titanium discs. Bioﬁlm-Treatment (Charalampakis et al., 2015) Treatment of titanium ﬁxture surfaces with ultrasonic metal, plastic, or carbon tip signiﬁcantly enhanced bacterial removal efﬁcacy of brushing. Thorough instrumentation that can smooth the whole exposed surface may facilitate maintenance of the implants. Bioﬁlm-Treatment (Park et al., 2015) The non-surgical treatment used in this study was not effective in removing LPS from titanium surfaces and increasing osteoblast adhesion. Neither a sole CAP treatment nor a sole brushing provided a completely decontaminated surface. A combined treatment of brushing and subsequent CAP treatment led to a cleaned surface, which allowed cells to grow comparable to cells on the sterile control. The combination of mechanical and chemical cleansing used in the present study was ineffective in complete removal of the bioﬁlm from all four titanium discs. Cold atmospheric plasma (CAP), diode laser (DL), air-abrasion (AA), chlorhexidine (CHX) 14 Laser treatment In Vivo Studies The efﬁcacy of the tested toothbrushes for noncontact bioﬁlm removal differed signiﬁcantly. Bioﬁlm-Treatment (Schmidt et al., 2014) Ti brushes were more effective than curets and Er: YAG laser in the removal of surface contaminants, whereas Er: YAG laser was more effective than curets and Ti brushes in killing the bioﬁlm bacteria. None of the methods tested in this study was able to completely eliminate Ti surface contaminants. Bioﬁlm-Treatment (Al-Hashedi et al., 2017) The use of Er:YAG laser yielded clear advantages compared to the other debridement modalities. All treatment methods decreased i i ll i iﬁ l ( 0 001) l CFU i bi ﬁl d Bioﬁlm-Treatment (Eick et al., 2017) 9 Mechanical cleaning Various side-to-side toothbrushes 10 Mechanical cleaning, laser Metal curets, plastic curets, titanium brush, laser 10 Mechanical cleaning, laser Metal curets, plastic curets, titanium brush, laser The use of Er:YAG laser yielded clear advantages compared to the other debridement modalities. All treatment methods decreased statistically signiﬁcantly (p < 0.001) total CFUs in bioﬁlms compared with untreated titanium surfaces. The use of Er:YAG laser yielded clear advantages compared to the other debridement modalities. All treatment methods decreased statistically signiﬁcantly (p < 0.001) total CFUs in bioﬁlms compared with untreated titanium surfaces. Bioﬁlm-Treatment (Eick et al., 2017) (Continued) October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. TABLE 1 \| Continued No. Type of method Method of decontamination TABLE 1 \| Continued No. Type of method Method of decontamination Study ﬁndings Type of decontamination method (Bioﬁlm- Prevention or Bioﬁlm- Treatment) Reference g Ultrasonic scaling with a metal tip signiﬁcantly increased titanium surface roughness and signiﬁcantly increased the adherence of P. gingivalis compared to those of surfaces treated with polishing, laser, and chemical debridement. Moreover, the growth of bacterial bioﬁlm was the lowest for smoother surfaces, than polishing, CHX, and laser treated surfaces. Bioﬁlm-Treatment (Batsukh et al., 2017) The laser treatment signiﬁcantly reduced the bacterial adhesion to the surface as well as bioﬁlm formation as compared to a reference polished surfaces. Femtosecond laser texturing is a simple and promising method for endowing titanium implants with antibacterial properties, reducing the risk of implant-associated infections without requiring immobilized antibacterial substances, nanoparticles or coatings. In Vivo Studies Bioﬁlm-Prevention (Cunha et al., 2016) t Compared to the untreated controls Candida cells grown in mature in-vitro bioﬁlms were signiﬁcantly reduced by both wavelengths investigated. Comparison between the different methods of laser treatment additionally revealed a signiﬁcantly greater effect of the Er: YAG over the diode laser. Scanning electron microscopy ﬁndings proved that the diode laser light was effective in direct contact mode. In contrast, in the areas without direct contact, the fungal cells were left almost unchanged. The Er:YAG laser damaged the fungal cells to a great extent wherever it was applied. Bioﬁlm-Prevention (Sennhenn- Kirchner et al., 2009) Prior to treatment, CLSM and SEM detected adhering bacteria. Post- treatment FM recorded that the number of dead cells was higher using TTP compared to DL and C. Bioﬁlm-Treatment (Preissner et al., 2016) A), Treatment of oral bioﬁlms with CAP reduced the quantity of the bioﬁlms, destroyed their structure and reduced superﬁcial bacteria while complete removal was not achieved. However, according to the microbiological assay, CAP produced the best results with regard to disinfection of microorganisms from the oral bioﬁlm. Bioﬁlm-Treatment (Idlibi et al., 2013) s At 3W, both lasers decontaminated all surfaces no matter the type of bacteria tested, whereas at 2.5 W, the rough implants were partially decontaminated. The 980-nm diode and the 1064-nm Nd:YAG lasers were effective in decontamination of P. gingivalis and E. faecalis without promoting surface alteration on the implants. Bioﬁlm-Treatment (Gonçalves et al., 2010) m, Er,Cr:YSGG laser treatment of titanium disks coated with single – species bioﬁlm of P. gingivalis ablates >95% of the bioﬁlm with no physical changes noted to the implant material surface coating. Bioﬁlm-Treatment (Strever et al., 2017) No signiﬁcant adverse effect on subsequent colonization and proliferation of MG-63 cells or increased bacterial adhesion was found in comparison to untreated control surfaces. Bioﬁlm-Treatment (Hauser- Gerspach et al., 2014) o, An Er:YAG laser (100 mJ/pulse, 10 Hz) seems to be most suitable for the removal of supragingival early plaque bioﬁlms grown on SLA titanium implants, and (2) all treatment procedures failed to restore the biocompatibility of previously-contaminated SLA titanium surfaces. Bioﬁlm-Treatment (Schwarz et al., 2005) m Diode laser irradiation in both continuous and pulsed modes induced a statistically signiﬁcant reduction of viable bacteria and nitrite levels. Study ﬁndings Bioﬁlm-Treatment (Strever et al., 2017) g Compared to the untreated controls Candida cells grown in mature in-vitro bioﬁlms were signiﬁcantly reduced by both wavelengths investigated. Comparison between the different methods of laser treatment additionally revealed a signiﬁcantly greater effect of the Er: YAG over the diode laser. Scanning electron microscopy ﬁndings proved that the diode laser light was effective in direct contact mode. In contrast, in the areas without direct contact, the fungal cells were left almost unchanged. The Er:YAG laser damaged the fungal cells to a great extent wherever it was applied. Bioﬁlm-Prevention (Sennhenn- Kirchner et al., 2009) Prior to treatment, CLSM and SEM detected adhering bacteria. Post- treatment FM recorded that the number of dead cells was higher using TTP compared to DL and C. Bioﬁlm-Treatment (Preissner et al., 2016) Treatment of oral bioﬁlms with CAP reduced the quantity of the bioﬁlms, destroyed their structure and reduced superﬁcial bacteria while complete removal was not achieved. However, according to the microbiological assay, CAP produced the best results with regard to disinfection of microorganisms from the oral bioﬁlm. Bioﬁlm-Treatment (Idlibi et al., 2013) At 3W, both lasers decontaminated all surfaces no matter the type of bacteria tested, whereas at 2.5 W, the rough implants were partially decontaminated. The 980-nm diode and the 1064-nm Nd:YAG lasers were effective in decontamination of P. gingivalis and E. faecalis without promoting surface alteration on the implants. Bioﬁlm-Treatment (Gonçalves et al., 2010) Er,Cr:YSGG laser treatment of titanium disks coated with single – species bioﬁlm of P. gingivalis ablates >95% of the bioﬁlm with no physical changes noted to the implant material surface coating. Erbium:yttrium–aluminium–garnet and diode laser light Study ﬁndings 12 Mechanical cleaning and laser treatment Ultrasonic scaler, rubber polishing cup, gallium–aluminum–arsenide laser, and chlorhexidine (CHX) rinse 13 Laser treatment Femtosecond laser surface texturing using a Yb:KYW chirped-pulse-regenerative ampliﬁcation laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs 14 Laser treatment Erbium:yttrium–aluminium–garnet and diode laser light 12 Mechanical cleaning and laser treatment Ultrasonic scaler, rubber polishing cup, gallium–aluminum–arsenide laser, and chlorhexidine (CHX) rinse Ultrasonic scaling with a metal tip signiﬁcantly increased titanium surface roughness and signiﬁcantly increased the adherence of P. gingivalis compared to those of surfaces treated with polishing, laser, and chemical debridement. Moreover, the growth of bacterial bioﬁlm was the lowest for smoother surfaces, than polishing, CHX, and laser treated surfaces. g Compared to the untreated controls Candida cells grown in mature in-vitro bioﬁlms were signiﬁcantly reduced by both wavelengths investigated. Comparison between the different methods of laser treatment additionally revealed a signiﬁcantly greater effect of the Er: YAG over the diode laser. Scanning electron microscopy ﬁndings proved that the diode laser light was effective in direct contact mode. In contrast, in the areas without direct contact, the fungal cells were left almost unchanged. The Er:YAG laser damaged the fungal cells to a great extent wherever it was applied. Bioﬁlm-Prevention (Sennhenn- Kirchner et al., 2009) Prior to treatment, CLSM and SEM detected adhering bacteria. Post- treatment FM recorded that the number of dead cells was higher using TTP compared to DL and C. Bioﬁlm-Treatment (Preissner et al., 2016) Treatment of oral bioﬁlms with CAP reduced the quantity of the bioﬁlms, destroyed their structure and reduced superﬁcial bacteria while complete removal was not achieved. However, according to the microbiological assay, CAP produced the best results with regard to disinfection of microorganisms from the oral bioﬁlm. Bioﬁlm-Treatment (Idlibi et al., 2013) At 3W, both lasers decontaminated all surfaces no matter the type of bacteria tested, whereas at 2.5 W, the rough implants were partially decontaminated. The 980-nm diode and the 1064-nm Nd:YAG lasers were effective in decontamination of P. gingivalis and E. faecalis without promoting surface alteration on the implants. Bioﬁlm-Treatment (Gonçalves et al., 2010) Er,Cr:YSGG laser treatment of titanium disks coated with single – species bioﬁlm of P. gingivalis ablates >95% of the bioﬁlm with no physical changes noted to the implant material surface coating. Er:YAG laser device (KEY3, KaVo, Biberach, Germany) In Vivo Studies These results indicate that in addition to its bactericidal effect laser irradiation can also inhibit LPS-induced macrophage activation and thus blunt the inﬂammatory response Bioﬁlm-Treatment (Giannelli et al., 2016) Tissue tolerable plasma Study ﬁndings Bioﬁlm-Treatment (Strever et al., 2017) 15 Laser treatment Tissue tolerable plasma 16 Laser treatment Cold atmospheric plasma (CAP), diode laser (DL), air-abrasion (AA), chlorhexidine (CHX) 17 Laser treatment 980-nm gallium aluminum arsenide (GaAlAs) and 1064-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) lasers 18 Laser treatment Radiation from an irrigated erbium, chromium–doped yttrium, scandium, gallium, garnet (Er,Cr: YSGG laser) (Waterlase iPlus, Biolase, Irvine, CA) 19 Laser treatment Er:YAG laser 20 Laser treatment Er:YAG laser device (KEY3, KaVo, Biberach, Germany) 21 Laser treatment Diode laser emitting at l 808 nm (Dental Laser System 4 × 4™, General Project Ltd., Montespertoli, Florence, Italy) 22 Laser treatment and photodynamic therapy Photodynamic laser treatment (PDT) 15 Laser treatment 15 Laser treatment Tissue tolerable plasma 16 Laser treatment Cold atmospheric plasma (CAP), diode laser (DL), air-abrasion (AA), chlorhexidine (CHX) 17 Laser treatment 980-nm gallium aluminum arsenide (GaAlAs) and 1064-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) lasers 18 Laser treatment Radiation from an irrigated erbium, chromium–doped yttrium, scandium, gallium, garnet (Er,Cr: YSGG laser) (Waterlase iPlus, Biolase, Irvine, CA) 19 Laser treatment Er:YAG laser 20 Laser treatment Er:YAG laser device (KEY3, KaVo, Biberach, Germany) 16 Laser treatment 17 Laser treatment 18 Laser treatment 19 Laser treatment 20 Laser treatment Er:YAG laser device (KEY3, KaVo, Biberach, Germany) 21 Laser treatment Diode laser emitting at l 808 nm (Dental Laser System 4 × 4™, General Project Ltd., Montespertoli, Florence, Italy) 22 Laser treatment and photodynamic therapy Photodynamic laser treatment (PDT) 21 Laser treatment 22 Laser treatment and photodynamic therapy October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. Study ﬁndings Type of decontamination method (Bioﬁlm- Prevention or Bioﬁlm- Treatment) Reference MB-mediated PDT displayed the concentration-dependent, pH- dependent, and time-dependent efﬁcacy of eradication therapy on bioﬁlm-contaminated implant surfaces. The most effective treatment occurred with exposure to laser light in combination with the 200 mg/ mL MB photosensitizer at pH 10 for 60 s of irradiation time. Bioﬁlm-Treatment (Huang et al., 2019) Both PDT1 and PDT2 protocols showed a high efﬁcacy against a 3- day-old bacterial bioﬁlm on dental implants and were more effective compared with LAD. Bioﬁlm-Treatment (Azizi et al., 2018) A dose dependent efﬁcacy of a PDT treatment against F. nucleatum grown in bioﬁlm was signiﬁcant. Study ﬁndings Bioﬁlm-Treatment (Mang et al., 2016) The combination of methylene blue and a red color LED (Periowave®) was effective in reducing the viability of bioﬁlm attached to SLA titanium surface in-vitro. Bioﬁlm-Treatment (Kim et al., 2017) UVC irradiation reduced the attachment or bioﬁlm formation of wound pathogens, and promoted the hydrophilicity, and carbon elimination on various topographical titanium surfaces, rivaling or surpassing UVA irradiation in effect. Bioﬁlm-Prevention (Yamada et al., 2014) Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that while bacterial community proﬁles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as bioﬁlm formation progressed. Bioﬁlm-Prevention (de Avila et al., 2015) The surface characteristics and biological properties of TNS could be modulated via short-duration high-intensity UV treatment. UV-TNS effectively inhibited attachment and bioﬁlm formation of A. oris. UV treatment could enhance both the biological compatibility and the antimicrobial activity of TNS. Bioﬁlm-Treatment (Zhang et al., 2017) (a) PAD using LED is effective against periodontopathic microbial species even in the presence of serum, (b) PAD and HPAD reduce viability in single-species bioﬁlms. (C) the increase in antimicrobial activity following the use of H-PAD may bear potential relevance as an adjunctive antimicrobial treatment in periodontal and peri-implant infections thus warranting further clinical testing. Bioﬁlm-Treatment (Eick et al., 2013) 5 minutes is the optimal time of sonication in order to recover the maximum amount of most bacteria attached to Ti6Al4V discs. Bioﬁlm-Treatment (Prieto-Borja et al., 2017) The bacterial adhesion studies revealed that the TS-Ti ﬁlms formed at temperatures below 350 C were refractory to bacterial adhesion, while temperatures above 600 C showed signiﬁcantly enhanced afﬁnity towards bacterial adhesion. Bioﬁlm-Prevention (Gopal et al., 2016) This experiment demonstrates that the DCLFU device can clear S epidermidis bioﬁlms off of metallic implant material. At the moderate power levels and brief exposure time used in this study, the bacteria are converted to the planktonic state with bioﬁlm dispersion. Using HCA as the mechanical irrigant facilitates killing of the planktonic bacteria that are released. Bioﬁlm-Treatment (Granick et al., 2017) Air polishing with erythritol/chlorhexidine seems to be a viable alternative to the traditional glycine treatment for bioﬁlm removal. Bioﬁlm-Treatment (Drago et al., 2014) air-polishing treatment with glycine powder causes minimal damage to the implant surfaces and reduce bacterial recolonization in a short test period (24 h). 31 Sonication Inﬂuence of exposure time of sonication 32 Thermal treatment Inﬂuence of thermally (50–800 C) stabilized titanium (TS-Ti) nano oxide 33 Ultrasound treatment Direct-contact low-frequency ultrasound Study ﬁndings Bioﬁlm-Treatment (Cochis et al., 2013) Air-polishing with erythritol powder has a high potential to render a microbially contaminated implant surface cell conductive. The concomitant use of air polishing and plasma treatment did not enhance osteoblast spreading. Bioﬁlm-Treatment (Matthes et al., 2017) Study ﬁndings Type of decontamination method (Bioﬁlm- Prevention or Bioﬁlm- Treatment) Reference MB-mediated PDT displayed the concentration-dependent, pH- dependent, and time-dependent efﬁcacy of eradication therapy on bioﬁlm-contaminated implant surfaces. The most effective treatment occurred with exposure to laser light in combination with the 200 mg/ mL MB photosensitizer at pH 10 for 60 s of irradiation time. Bioﬁlm-Treatment (Huang et al., 2019) Both PDT1 and PDT2 protocols showed a high efﬁcacy against a 3- day-old bacterial bioﬁlm on dental implants and were more effective compared with LAD. Bioﬁlm-Treatment (Azizi et al., 2018) A dose dependent efﬁcacy of a PDT treatment against F. nucleatum grown in bioﬁlm was signiﬁcant. Bioﬁlm-Treatment (Mang et al., 2016) 23 Laser treatment and photodynamic therapy Methylene blue-mediated photodynamic therapy 24 Photodynamic therapy Photodynamic therapy and light- activated disinfection 25 Photodynamic therapy Photodynamic therapy (PDT), using a diode laser at 630 nm, 150 mW/cm2 for light doses ranging from 25–100 J/cm2 26 Photodynamic therapy Photodynamic therapy using methylene blue and red color diode laser 27 Radiation Ultraviolet-C pre-irradiation 28 Radiation UV treatment of titanium immediately prior to use (photofunctionalization) 29 Radiation UV-treated titanium with nanonetwork structures (UV-TNS) 30 Radiation Photoactivated disinfection with a light-emitting diode 31 Sonication Inﬂuence of exposure time of sonication 32 Thermal treatment Inﬂuence of thermally (50–800 C) stabilized titanium (TS-Ti) nano oxide 33 Ultrasound treatment Direct-contact low-frequency ultrasound 34 Air polishing Air polishing with erythritol/ chlorhexidine or glycine and amorphous silica 35 Air polishing Air polishing with glycine powder 36 Air polishing Optimized air polishing and a cold plasma device. Air powder contains erythritol (sugar alcohol, 14 mm), amorphous silica and 0.3 % chlorhexidine (CHX). Frontiers in Cellular and Infection Microbiology \| www.fron ) MB-mediated PDT displayed the concentration-dependent, pH- dependent, and time-dependent efﬁcacy of eradication therapy on bioﬁlm-contaminated implant surfaces. The most effective treatment occurred with exposure to laser light in combination with the 200 mg/ mL MB photosensitizer at pH 10 for 60 s of irradiation time. Bioﬁlm-Treatment (Huang et al., 2019) Both PDT1 and PDT2 protocols showed a high efﬁcacy against a 3- day-old bacterial bioﬁlm on dental implants and were more effective compared with LAD. Study ﬁndings One hundred and ﬁve studies performed in-vitro experiments (n = 105, 90.5%) (Duarte et al., 2009; Ewald and Ihde, 2009; Sennhenn-Kirchner et al., 2009; Tamai et al., 2009; Gonçalves et al., 2010; Baffone et al., 2011; Ercan et al., 2011; Fröjd et al., 2011; Mohn et al., 2011; Ntrouka et al., 2011; Bürgers et al., 2012; Cortizo et al., 2012; Lilja et al., 2012; Subramanian et al., 2012; Trujillo et al., 2012; Alcheikh et al., 2013; De Giglio et al., 2013; Diab Al-Radha et al., 2013; Eick et al., 2013; Holmberg et al., 2013; Roberts et al., 2013; Chen et al., 2014; Ciandrini et al., 2014; Drago et al., 2014; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Kaliaraj et al., 2014; Kang et al., 2014; Lv et al., 2014; Massa et al., 2014; Sahrmann et al., 2014; Schmidt et al., 2014; Schmage et al., 2014; Yamada et al., 2014; Abdulkareem et al., 2015; Cruz et al., 2015; de Avila et al., 2015; Duske et al., 2015; Janković et al., 2015; Jennings et al., 2015; Lewandowska et al., 2015; Narendrakumar et al., 2015; Park et al., 2015; Wood et al., 2015; Yucesoy et al., 2015; Zhang et al., 2015; Ayre et al., 2016; Chen et al., 2016; Ciandrini et al., 2016; Cochis et al., 2016; Cotolan et al., 2016; Cunha et al., 2016; Giannelli et al., 2016; Godoy-Gallardo et al., 2016; Gopal et al., 2016; Guan et al., 2016; Kotsakis et al., 2016; Mang et al., 2016; Preissner et al., 2016; Rodrı́guez-Contreras et al., 2016; Shi et al., 2016; Verardi et al., 2016; Batsukh et al., 2017; Canullo et al., 2017; Ciandrini et al., 2017; Cometa et al., 2017; Dostie et al., 2017; Eick et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Granick et al., 2017; Hirschfeld et al., 2017; Kim et al., 2017; Kulkarni Aranya et al., 2017; Li et al., 2017; Macpherson et al., 2017; Matthes et al., 2017; Prieto-Borja et al., 2017; Ramesh et al., 2017; Schmidt et al., 2017; Strever et al., 2017; Wang et al., 2017; Wiedmer et al., 2017; Ye et al., 2017; Zhang et al., 2017; Akhavan et al., 2018; Atefyekta et al., 2018; Azizi et al., 2018; Ferraris et al., 2018; Fukushima et al., 2018; Hidalgo-Robatto et al., 2018; Hoyos-Nogués et al., 2018; Lee et al., 2018; Montelongo- Study ﬁndings Type of method Method of decontamination Study ﬁndings Type of decontamination method (Bioﬁlm- Prevention or Bioﬁlm- Treatment) Reference 37 Air polishing Air-abrasive powder based on glycine and tricalcium phosphate Glycine and tricalcium phosphate seemed to be more effective than the control groups for bioﬁlm removal on titanium surfaces. Bioﬁlm-Treatment (John et al., 2016) 38 Anodizing treatment Effect of titanium anodization and nanotube diameter The adherence of oral streptococci can be modiﬁed by titanium anodization and nanotube diameter. Bioﬁlm-Prevention (Narendrakumar et al., 2015) 39 Anodizing treatment Anodized nanotubular titanium and the effect of electrical stimulation Decreased S. aureus bioﬁlm formation was found after 2 days of culture on 15–30 V stimulated anodized nanotubular titanium compared with non-stimulated conventionally used titanium. Bioﬁlm-Prevention (Ercan et al., 2011) 40 Anodizing treatment Electrochemically induced anatase at two different voltages: 90 and 130 V for pure titanium; 100 and 120 V for Ti6Al4V alloy The high voltage anodization treatments may contribute to preserving tissue integration and reduce bacteria colonization of titanium and titanium alloy for implant-based applications. Bioﬁlm-Prevention (Giordano et al., 2011) 41 Anodizing treatment Electrochemical treatment In-situ generated active oxidants at the anode caused higher bacterial reduction than the mere alkaline environment emerging at the cathode. Bioﬁlm-Treatment (Mohn et al., 2011) Study ﬁndings Type of decontamination method (Bioﬁlm- Prevention or Bioﬁlm- Treatment) Reference Glycine and tricalcium phosphate seemed to be more effective than the control groups for bioﬁlm removal on titanium surfaces. Bioﬁlm-Treatment (John et al., 2016) The adherence of oral streptococci can be modiﬁed by titanium anodization and nanotube diameter. Bioﬁlm-Prevention (Narendrakumar et al., 2015) Decreased S. aureus bioﬁlm formation was found after 2 days of culture on 15–30 V stimulated anodized nanotubular titanium compared with non-stimulated conventionally used titanium. Bioﬁlm-Prevention (Ercan et al., 2011) The high voltage anodization treatments may contribute to preserving tissue integration and reduce bacteria colonization of titanium and titanium alloy for implant-based applications. Bioﬁlm-Prevention (Giordano et al., 2011) In-situ generated active oxidants at the anode caused higher bacterial reduction than the mere alkaline environment emerging at the cathode. Study ﬁndings Bioﬁlm-Treatment (Mohn et al., 2011) Study ﬁndings 37 Air polishing Air-abrasive powder based on glycine and tricalcium phosphate 38 Anodizing treatment Effect of titanium anodization and nanotube diameter 39 Anodizing treatment Anodized nanotubular titanium and the effect of electrical stimulation 40 Anodizing treatment Electrochemically induced anatase at two different voltages: 90 and 130 V for pure titanium; 100 and 120 V for Ti6Al4V alloy 41 Anodizing treatment Electrochemical treatment Jauregui et al., 2018; Pantaroto et al., 2018; Pissinis et al., 2018; Santos-Coquillat et al., 2018; Schneider et al., 2018; Souza et al., 2018; Trobos et al., 2018; Vilarrasa et al., 2018; Wang et al., 2018; Zhang et al., 2018; Huang et al., 2019), 11 studies performed in- vivo experiments on humans (n = 11, 9.5%) (Schwarz et al., 2005; Größner-Schreiber et al., 2009; Sennhenn-Kirchner et al., 2009; Baffone et al., 2011; Fröjd et al., 2011; Rehman et al., 2012; Cochis et al., 2013; Idlibi et al., 2013; Charalampakis et al., 2015; Cruz et al., 2015; John et al., 2016; Al-Hashedi et al., 2017; Al- Hashedi et al., 2018; Schmidt et al., 2019), while two studies performed both in-vitro and in-vivo experiments (n = 2, 1.7%) (Giordano et al., 2011; Kuehl et al., 2016). Amongst the in-vivo experiments which recruited human volunteers, 11 studies used titanium discs (n = 11, 9.5%) (Schwarz et al., 2005; Größner- Schreiber et al., 2009; Giordano et al., 2011; Rehman et al., 2012; Cochis et al., 2013; Idlibi et al., 2013; Charalampakis et al., 2015; Kuehl et al., 2016; Al-Hashedi et al., 2017; Al-Hashedi et al., 2018), while the other two studies used titanium implants (n = 2, 1.7%) (John et al., 2016; Schmidt et al., 2019). Kotsakis et al., 2016; Verardi et al., 2016; Al-Hashedi et al., 2017; Dostie et al., 2017; Li et al., 2017; Matthes et al., 2017; Al-Hashedi et al., 2018; Wang et al., 2018; Schmidt et al., 2019). Study ﬁndings One hundred and ﬁve studies performed in-vitro experiments (n = 105, 90.5%) (Duarte et al., 2009; Ewald and Ihde, 2009; Sennhenn-Kirchner et al., 2009; Tamai et al., 2009; Gonçalves et al., 2010; Baffone et al., 2011; Ercan et al., 2011; Fröjd et al., 2011; Mohn et al., 2011; Ntrouka et al., 2011; Bürgers et al., 2012; Cortizo et al., 2012; Lilja et al., 2012; Subramanian et al., 2012; Trujillo et al., 2012; Alcheikh et al., 2013; De Giglio et al., 2013; Diab Al-Radha et al., 2013; Eick et al., 2013; Holmberg et al., 2013; Roberts et al., 2013; Chen et al., 2014; Ciandrini et al., 2014; Drago et al., 2014; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Kaliaraj et al., 2014; Kang et al., 2014; Lv et al., 2014; Massa et al., 2014; Sahrmann et al., 2014; Schmidt et al., 2014; Schmage et al., 2014; Yamada et al., 2014; Abdulkareem et al., 2015; Cruz et al., 2015; de Avila et al., 2015; Duske et al., 2015; Janković et al., 2015; Jennings et al., 2015; Lewandowska et al., 2015; Narendrakumar et al., 2015; Park et al., 2015; Wood et al., 2015; Yucesoy et al., 2015; Zhang et al., 2015; Ayre et al., 2016; Chen et al., 2016; Ciandrini et al., 2016; Cochis et al., 2016; Cotolan et al., 2016; Cunha et al., 2016; Giannelli et al., 2016; Godoy-Gallardo et al., 2016; Gopal et al., 2016; Guan et al., 2016; Kotsakis et al., 2016; Mang et al., 2016; Preissner et al., 2016; Rodrı́guez-Contreras et al., 2016; Shi et al., 2016; Verardi et al., 2016; Batsukh et al., 2017; Canullo et al., 2017; Ciandrini et al., 2017; Cometa et al., 2017; Dostie et al., 2017; Eick et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Granick et al., 2017; Hirschfeld et al., 2017; Kim et al., 2017; Kulkarni Aranya et al., 2017; Li et al., 2017; Macpherson et al., 2017; Matthes et al., 2017; Prieto-Borja et al., 2017; Ramesh et al., 2017; Schmidt et al., 2017; Strever et al., 2017; Wang et al., 2017; Wiedmer et al., 2017; Ye et al., 2017; Zhang et al., 2017; Akhavan et al., 2018; Atefyekta et al., 2018; Azizi et al., 2018; Ferraris et al., 2018; Fukushima et al., 2018; Hidalgo-Robatto et al., 2018; Hoyos-Nogués et al., 2018; Lee et al., 2018; Montelongo- Kotsakis et al., 2016; Verardi et al., 2016; Al-Hashedi et al., 2017; Dostie et al., 2017; Li et al., 2017; Matthes et al., 2017; Al-Hashedi et al., 2018; Wang et al., 2018; Schmidt et al., 2019). Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Study ﬁndings Bioﬁlm-Treatment (Azizi et al., 2018) A dose dependent efﬁcacy of a PDT treatment against F. nucleatum grown in bioﬁlm was signiﬁcant. Bioﬁlm-Treatment (Mang et al., 2016) y p compared with LAD. ) A dose dependent efﬁcacy of a PDT treatment against F. nucleatum grown in bioﬁlm was signiﬁcant. Bioﬁlm-Treatment (Mang et al., 2016) The combination of methylene blue and a red color LED (Periowave®) was effective in reducing the viability of bioﬁlm attached to SLA titanium surface in-vitro. Bioﬁlm-Treatment (Kim et al., 2017) UVC irradiation reduced the attachment or bioﬁlm formation of wound pathogens, and promoted the hydrophilicity, and carbon elimination on various topographical titanium surfaces, rivaling or surpassing UVA irradiation in effect. Bioﬁlm-Prevention (Yamada et al., 2014) Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that while bacterial community proﬁles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as bioﬁlm formation progressed. Bioﬁlm-Prevention (de Avila et al., 2015) The surface characteristics and biological properties of TNS could be modulated via short-duration high-intensity UV treatment. UV-TNS effectively inhibited attachment and bioﬁlm formation of A. oris. UV Bioﬁlm-Treatment (Zhang et al., 2017) The combination of methylene blue and a red color LED (Periowave®) was effective in reducing the viability of bioﬁlm attached to SLA titanium surface in-vitro. Bioﬁlm-Treatment (Kim et al., 2017) UVC irradiation reduced the attachment or bioﬁlm formation of wound pathogens, and promoted the hydrophilicity, and carbon elimination on various topographical titanium surfaces, rivaling or surpassing UVA irradiation in effect. Bioﬁlm-Prevention (Yamada et al., 2014) Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that while bacterial community proﬁles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as bioﬁlm formation progressed. Bioﬁlm-Prevention (de Avila et al., 2015) 34 Air polishing Air polishing with erythritol/ chlorhexidine or glycine and amorphous silica 35 Air polishing Air polishing with glycine powder 36 Air polishing Optimized air polishing and a cold plasma device. Air powder contains erythritol (sugar alcohol, 14 mm), amorphous silica and 0.3 % chlorhexidine (CHX). (Continued) (Continued) October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. TABLE 1 \| Continued No. In Vitro Studies 2017; Prieto-Borja et al., 2017; Strever et al., 2017; Zhang et al., 2017; Wang et al., 2017; Wiedmer et al., 2017; Ye et al., 2017; Akhavan et al., 2018; Atefyekta et al., 2018; Azizi et al., 2018; Ferraris et al., 2018; Fukushima et al., 2018; Hidalgo-Robatto et al., 2018; Hoyos-Nogués et al., 2018; Lee et al., 2018; Pissinis et al., 2018; Santos-Coquillat et al., 2018; Souza et al., 2018; Trobos et al., 2018; Zhang et al., 2018; Huang et al., 2019). Ten studies performed on mixed-species bioﬁlm (n = 10, 8.6%) (Fröjd et al., 2011; Cortizo et al., 2012; Subramanian et al., 2012; Sahrmann et al., 2014; Schmidt et al., 2014; Cruz et al., 2015; Cochis et al., 2016; Ramesh et al., 2017; Schneider et al., 2018; Vilarrasa et al., 2018), while eight studies compared single- species and mixed species bioﬁlm (n = 8, 6.9%) (Baffone et al., 2011; Ntrouka et al., 2011; Eick et al., 2013; Ciandrini et al., 2014; Annunziata et al., 2017; Schmidt et al., 2017; Montelongo- Jauregui et al., 2018; Pantaroto et al., 2018). Decontamination methods using antimicrobial drugs, chemical treatment, electrochemical treatment, probiotic, and the ﬁndings of the respective studies are presented in Supplementary Information 3. In experiments that used bacteria or fungi either as single species or mixed species (without the exposure to normal ﬂora), 143 experiments applied bioﬁlm aged less or equal to 24 h (n = 143, 64.7%), 72 experiments applied bioﬁlm aged more than 24 h or equal to 48 h (n = 72, 32.6%), 38 experiments applied bioﬁlm aged more than 48 h or equal to 72 h (n = 38, 17.2%), while 23 experiments applied bioﬁlm aged more than 72 h (n = 23, 10.4%) (Table 2). Table 2 shows the list of oral microbiota used for the development of bioﬁlm on the titanium materials. The included studies in this systematic review applied several methods after decontamination of the titanium material either to quantify the microbial bioﬁlm or to observe the morphological changes. In Vitro Studies Fifty-three studies used the number of colony forming unit (CFU) (n = 53, 45.7%) (Duarte et al., 2009; Ewald and Ihde, 2009; Tamai et al., 2009; Gonçalves et al., 2010; Giordano et al., 2011; Baffone et al., 2011; Mohn et al., 2011; Ntrouka et al., 2011; Cochis et al., 2013; Diab Al-Radha et al., 2013; Eick et al., 2013; Holmberg et al., 2013; Roberts et al., 2013; Chen et al., 2014; Ciandrini et al., 2014; Drago et al., 2014; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Massa et al., 2014; Sahrmann et al., 2014; Abdulkareem et al., 2015; Charalampakis et al., 2015; Janković et al., 2015; Narendrakumar et al., 2015; Wood et al., 2015; Chen et al., 2016; Cochis et al., 2016; Giannelli et al., 2016; Guan et al., 2016; Kuehl et al., 2016; Mang et al., 2016; Preissner et al., 2016; Rodrı́guez-Contreras et al., 2016; Verardi et al., 2016; Ciandrini et al., 2017; Cometa et al., 2017; Dostie et al., 2017; Eick et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Granick et al., 2017; Hirschfeld et al., 2017; Kim et al., 2017; Prieto-Borja et al., 2017; Wiedmer et al., 2017; Akhavan et al., 2018; Azizi et al., 2018; Ferraris et al., 2018; Pantaroto et al., 2018; Pissinis et al., 2018; Souza et al., 2018; Trobos et al., 2018; Vilarrasa et al., 2018). Fifty-eight studies used scanning electron microscopy (SEM) (n = 58, 50%) (Duarte et al., 2009; Sennhenn-Kirchner et al., 2009; Gonçalves et al., 2010; Ercan et al., 2011; Giordano et al., 2011; Trujillo et al., 2012; Cochis et al., 2013; Eick et al., 2013; Holmberg et al., 2013; Yamada et al., 2014; Duske et al., 2015; Godoy-Gallardo et al., 2014; In Vitro Studies Out of 116 studies, some studies did not use discs (n = 13, 11.2%) (Gonçalves et al., 2010; Mohn et al., 2011; Subramanian et al., 2012; Kaliaraj et al., 2014; Lewandowska et al., 2015; Narendrakumar et al., 2015; Park et al., 2015; John et al., 2016; Preissner et al., 2016; Cometa et al., 2017; Schmidt et al., 2017; Azizi et al., 2018; Schmidt et al., 2019). One study performed on discs and other form of titanium (n = 1, 0.9%) (Kulkarni et al., 2017), four studies did not state whether they used discs (n = 4, 3.4%) (Cunha et al., 2016; Macpherson et al., 2017; Ramesh et al., 2017; Akhavan et al., 2018), while the others used discs (n = 98, 84.5%) (Schwarz et al., 2005; Duarte et al., 2009; Ewald and Ihde, 2009; Größner-Schreiber et al., 2009; Sennhenn-Kirchner et al., 2009; Tamai et al., 2009; Baffone et al., 2011; Ercan et al., 2011; Fröjd et al., 2011; Ntrouka et al., 2011; Bürgers et al., 2012; Cortizo et al., 2012; Lilja et al., 2012; Rehman et al., 2012; Trujillo October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 7 Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. In Vitro Studies et al., 2012; Alcheikh et al., 2013; Cochis et al., 2013; De Giglio et al., 2013; Diab Al-Radha et al., 2013; Eick et al., 2013; Holmberg et al., 2013; Idlibi et al., 2013; Roberts et al., 2013Chen et al., 2014; Ciandrini et al., 2014; Drago et al., 2014; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Kang et al., 2014; Lv et al., 2014; Massa et al., 2014; Sahrmann et al., 2014; Schmidt et al., 2014; Schmage et al., 2014; Yamada et al., 2014; Abdulkareem et al., 2015; Charalampakis et al., 2015; Cruz et al., 2015; de Avila et al., 2015; Duske et al., 2015; Janković et al., 2015; Jennings et al., 2015; Wood et al., 2015; Yucesoy et al., 2015; Zhang et al., 2015; Ayre et al., 2016; Chen et al., 2016; Cochis et al., 2016; Ciandrini et al., 2016; Cotolan et al., 2016; Giannelli et al., 2016; Godoy-Gallardo et al., 2016; Gopal et al., 2016; Guan et al., 2016; Kotsakis et al., 2016; Kuehl et al., 2016; Mang et al., 2016; Rodrı́guez-Contreras et al., 2016; Shi et al., 2016; Verardi et al., 2016; Al-Hashedi et al., 2017; Batsukh et al., 2017; Canullo et al., 2017; Ciandrini et al., 2017; Dostie et al., 2017; Eick et al., 2017; Ferraris et al., 2017; Granick et al., 2017; Giannelli et al., 2017; Hirschfeld et al., 2017; Kim et al., 2017; Kulkarni Aranya et al., 2017; Li et al., 2017; Matthes et al., 2017; Prieto-Borja et al., 2017; Strever et al., 2017; Wang et al., 2017; Wiedmer et al., 2017; Ye et al., 2017; Zhang et al., 2017; Al-Hashedi et al., 2018; Atefyekta et al., 2018; Ferraris et al., 2018; Fukushima et al., 2018; Hidalgo-Robatto et al., 2018; Hoyos-Nogués et al., 2018; Huang et al., 2019; Lee et al., 2018; Montelongo-Jauregui et al., 2018; Pantaroto et al., 2018; Pissinis et al., 2018; Santos-Coquillat et al., 2018; Schneider et al., 2018; Souza et al., 2018; Trobos et al., 2018; Vilarrasa et al., 2018; Wang et al., 2018; Zhang et al., 2018). Studies With Development of Dental Bioﬁlm Microbiota Reference numbe 1 Acinetobacter baumannii DSM 30007 2 Actinomyces naeslundii Clinical patient ATCC 12104 OMZ 745 3 Actinomyces oris OMZ 745 MG-1 4 Actinomyces viscosus ATCC 15987 ATCC 19246 5 Aggregatibacter actinomycetemcomitans ATCC 33384 IDH 781 Serotype b VT 1169 Y4 6 Bacillus atrophaeus ATCC 9372 7 Bacillus megaterium n/s 8 Bacillus pseudomycoides n/s 9 Bacteroides fragilis n/s 10 Campylobacter rectus ATCC 33238 OMZ 698 11 Candida albicans Clinical patient Clinical patient n/s SC5314 ATCC 10231 ATCC 90028 12 Candida famata 30 13 Eikenella corrodens ATCC 23834 14 Enterobacter cloacae ATCC 13047 ATCC 23355 15 Enterococcus faecalis ATCC 29212 16 Escherichia coli ATCC 2592 ATCC DH5 ALPHA C43 HB 101 K12 JM 101 n/s n/s NCIM 2931 17 Eubacterium nodatum ATCC 33099 18 Filifactor alocis ATCC 35896 19 Fusarium solani ATCC 36031 20 Fusobacterium nucleatum ATCC 10953 ATCC 25586 OMZ 596 OMZ 598 21 Klebsiella pneumoniae ATCC 4352 22 Lactobacillus acidophilus ATCC 4356 TABLE 2 \| List of microbiota used for the development of bioﬁlm on the titanium materials. No. Studies With Development of Dental Bioﬁlm Microbiota Reference number Incubation time Reference 1 Acinetobacter baumannii DSM 30007 72 h (Cochis et al., 2016) 2 Actinomyces naeslundii Clinical patient 2, 14 h (Fröjd et al., 2011) ATCC 12104 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 24 h (Schmidt et al., 2017) OMZ 745 16.5 h (Pantaroto et al., 2018) 3 Actinomyces oris OMZ 745 40.5 h (Sahrmann et al., 2014) MG-1 6 h (Zhang et al., 2017) 4 Actinomyces viscosus ATCC 15987 2 d (Vilarrasa et al., 2018) ATCC 19246 14 d (Zhang et al., 2015) 5 Aggregatibacter actinomycetemcomitans ATCC 33384 72 h (Azizi et al., 2018) 72 h (Kim et al., 2017) IDH 781 24 h (Huang et al., 2019) Serotype b 1, 2, 4, 6 d (Massa et al., 2014) VT 1169 5 d (Ramesh et al., 2017) Y4 48 h (Eick et al., 2013) 72 h (Lee et al., 2018) 48, 72 h (Eick et al., 2017) 6 Bacillus atrophaeus ATCC 9372 1, 3, 7 d (Ewald and Ihde, 2009) 7 Bacillus megaterium n/s 10 d (Subramanian et al., 2012) 8 Bacillus pseudomycoides n/s 10 d (Subramanian et al., 2012) 9 Bacteroides fragilis n/s 48 h (Drago et al., 2014) 10 Campylobacter rectus ATCC 33238 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) OMZ 698 40.5 h (Sahrmann et al., 2014) 11 Candida albicans Clinical patient 5 d (Sennhenn-Kirchner et al., 2009) Clinical patient 8 d (Cruz et al., 2015) n/s 48 h (Drago et al., 2014) SC5314 24 h (Montelongo-Jauregui et al., 2018) ATCC 10231 120 mins (Bürgers et al., 2012) ATCC 90028 4, 18, 24 h (Akhavan et al., 2018) 12 Candida famata 30 24, 48, 72 h (Janković et al., 2015) 13 Eikenella corrodens ATCC 23834 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 14 Enterobacter cloacae ATCC 13047 24 h (Kuehl et al., 2016) ATCC 23355 24 h (Kuehl et al., 2016) 15 Enterococcus faecalis ATCC 29212 24 h (Chen et al., 2016) 24 h (Prieto-Borja et al., 2017) 18 h (Gonçalves et al., 2010) 16 Escherichia coli ATCC 2592 7, 24, 30 h (De Giglio et al., 2013) 24 h (Prieto-Borja et al., 2017) ATCC DH5 ALPHA 24 h (Chen et al., 2016) C43 60 h (Mohn et al., 2011) HB 101 48 h (Schneider et al., 2018) K12 JM 101 48 h (Schneider et al., 2018) n/s 2 h (Rodrı́guez-Contreras et al., 2016) n/s 24 h (Macpherson et al., 2017) NCIM 2931 6 h (Gopal et al., 2016) 17 Eubacterium nodatum ATCC 33099 48 h (Eick et al., 2013) 18 Filifactor alocis ATCC 35896 48, 72 h (Eick et al., 2017) 19 Fusarium solani ATCC 36031 1, 3, 7 d (Ewald and Ihde, 2009) 20 Fusobacterium nucleatum ATCC 10953 72 h (Schmidt et al., 2014) ATCC 25586 2 d (Vilarrasa et al., 2018) 24 h (Kang et al., 2014) 24 h (Mang et al., 2016) 48 h (Eick et al., 2013) 16.5, 40.5, 64.5 h (Ciandrini et al., 2014) 48, 72 h (Eick et al., 2017) 24 h (Schmidt et al., 2017) OMZ 596 16.5 h (Pantaroto et al., 2018) OMZ 598 40.5 h (Sahrmann et al., 2014) 21 Kl b i ll i ATCC 4352 24 h (T i t l 2009) TABLE 2 \| List of microbiota used for the development of bioﬁlm on the titanium materials. Studies With Development of Dental Bioﬁlm Forty-nine different types of microorganisms have been used for the development of dental bioﬁlm in the included studies for this systematic review (n = 49, Table 2). The bioﬁlm matrix was either in single species or mixed species. Seventy-eight studies performed on single-species bioﬁlm (n = 78, 67.2%) (Duarte et al., 2009; Ewald and Ihde, 2009; Sennhenn-Kirchner et al., 2009; Tamai et al., 2009; Gonçalves et al., 2010; Ercan et al., 2011; Giordano et al., 2011; Trujillo et al., 2012; Bürgers et al., 2012; Lilja et al., 2012; Roberts et al., 2013; Holmberg et al., 2013; De Giglio et al., 2013; Alcheikh et al., 2013; Chen et al., 2014; Drago et al., 2014; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Kaliaraj et al., 2014; Kang et al., 2014; Lv et al., 2014; Massa et al., 2014; Schmage et al., 2014; Yamada et al., 2014; Zhang et al., 2015; Jennings et al., 2015; Narendrakumar et al., 2015; Yucesoy et al., 2015; Wood et al., 2015; Janković et al., 2015; Lewandowska et al., 2015; Ayre et al., 2016; Chen et al., 2016; Ciandrini et al., 2016; Cotolan et al., 2016; Cunha et al., 2016; Giannelli et al., 2016; Godoy-Gallardo et al., 2016; Gopal et al., 2016; Guan et al., 2016; Kuehl et al., 2016; Mang et al., 2016; Preissner et al., 2016; Rodrı́guez-Contreras et al., 2016; Shi et al., 2016; Batsukh et al., 2017; Canullo et al., 2017; Ciandrini et al., 2017; Cometa et al., 2017; Eick et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Granick et al., 2017; Hirschfeld et al., 2017; Kim et al., 2017; Kulkarni Aranya et al., 2017; Macpherson et al., October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. TABLE 2 \| List of microbiota used for the development of bioﬁlm on the titani No. Studies With Development of Dental Bioﬁlm Incubation time Reference 72 h (Cochis et al., 2016) 2, 14 h (Fröjd et al., 2011) 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 24 h (Schmidt et al., 2017) 16.5 h (Pantaroto et al., 2018) 40.5 h (Sahrmann et al., 2014) 6 h (Zhang et al., 2017) 2 d (Vilarrasa et al., 2018) 14 d (Zhang et al., 2015) 72 h (Azizi et al., 2018) 72 h (Kim et al., 2017) 24 h (Huang et al., 2019) 1, 2, 4, 6 d (Massa et al., 2014) 5 d (Ramesh et al., 2017) 48 h (Eick et al., 2013) 72 h (Lee et al., 2018) 48, 72 h (Eick et al., 2017) 1, 3, 7 d (Ewald and Ihde, 2009) 10 d (Subramanian et al., 2012) 10 d (Subramanian et al., 2012) 48 h (Drago et al., 2014) 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 40.5 h (Sahrmann et al., 2014) 5 d (Sennhenn-Kirchner et al., 2009) 8 d (Cruz et al., 2015) 48 h (Drago et al., 2014) 24 h (Montelongo-Jauregui et al., 2018) 120 mins (Bürgers et al., 2012) 4, 18, 24 h (Akhavan et al., 2018) 24, 48, 72 h (Janković et al., 2015) 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 24 h (Kuehl et al., 2016) 24 h (Kuehl et al., 2016) 24 h (Chen et al., 2016) 24 h (Prieto-Borja et al., 2017) 18 h (Gonçalves et al., 2010) 7, 24, 30 h (De Giglio et al., 2013) 24 h (Prieto-Borja et al., 2017) 24 h (Chen et al., 2016) 60 h (Mohn et al., 2011) 48 h (Schneider et al., 2018) 48 h (Schneider et al., 2018) 2 h (Rodrı́guez-Contreras et al., 2016) 24 h (Macpherson et al., 2017) 6 h (Gopal et al., 2016) 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 1, 3, 7 d (Ewald and Ihde, 2009) 72 h (Schmidt et al., 2014) 2 d (Vilarrasa et al., 2018) 24 h (Kang et al., 2014) 24 h (Mang et al., 2016) 48 h (Eick et al., 2013) 16.5, 40.5, 64.5 h (Ciandrini et al., 2014) 48, 72 h (Eick et al., 2017) 24 h (Schmidt et al., 2017) 16.5 h (Pantaroto et al., 2018) 40.5 h (Sahrmann et al., 2014) 24 h (Tamai et al., 2009) 24 h (Ciandrini et al., 2017) (Continued) Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org October 2021 \| Volume 11 \| Article 736186 Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. Studies With Development of Dental Bioﬁlm Dhaliwal et al. TABLE 2 \| Continued No. (Continued) Studies With Development of Dental Bioﬁlm Microbiota Reference number Incubation time Reference DDS-1 24 h (Ciandrini et al., 2017) 23 Lactobacillus casei ATCC 15008 24 h (Ciandrini et al., 2017) 24 Lactobacillus paracasei B21060 24 h (Ciandrini et al., 2017) 25 Lactobacillus reuteri DSM 17938 24 h (Ciandrini et al., 2017) 26 Lactobacillus rhamnosus ATCC 53103 24 h (Ciandrini et al., 2017) ATCC 7469 24 h (Ciandrini et al., 2017) 27 Lactobacillus salivarius CCUG 17826 2 h (Godoy-Gallardo et al., 2014) 2, 24 h (Godoy-Gallardo et al., 2016) ATCC 11741 24 h (Ciandrini et al., 2017) 28 Lysinibacillus sphaericus n/s 24 h (Kaliaraj et al., 2014) 29 Parvimonas micra ATCC 33270 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 30 Porphyromonas gingivalis 381 48 h (Strever et al., 2017) A7436 24 h (Chen et al., 2016) 24 h (Huang et al., 2019) ATCC 33277 2 h (Hauser-Gerspach et al., 2014) 14 d (Zhang et al., 2015) 72 h (Lee et al., 2018) 2 h (Roberts et al., 2013) 72 h (Azizi et al., 2018) 2 d (Vilarrasa et al., 2018) 48 h (Eick et al., 2013) 8 d (Holmberg et al., 2013) 6, 12, 24 h (Batsukh et al., 2017) 3, 7 d (Kulkarni Aranya et al., 2017) 16.5, 40.5, 64.5 h (Ciandrini et al., 2014) 40.5 h (Sahrmann et al., 2014) 24 h (Kang et al., 2014) 24 h (Schmidt et al., 2017) 3 d (Gonçalves et al., 2010) ATCC BAA-308 24 h (Jennings et al., 2015) CCUG 25211 24 h (Giordano et al., 2011) DSM 20709 72 h (Schmidt et al., 2014) 31 Prevotella denticola ATCC 35308 24 h (Jennings et al., 2015) 32 Prevotella intermedia Clinical patient 72 h (Azizi et al., 2018) ATCC 25611 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 40.5 h (Sahrmann et al., 2014) 33 Pseudomonas aeruginosa ATCC 27853 24 h (Prieto-Borja et al., 2017) 24 h (Cotolan et al., 2016) 7, 24, 30 h (De Giglio et al., 2013) 24, 48, 72 h (Janković et al., 2015) ATCC 9027 1, 3, 7 d (Ewald and Ihde, 2009) 16.5, 40.5, 64.5 h (Baffone et al., 2011) CCUG 10778 24, 48 h (Atefyekta et al., 2018) DSM939 24, 48 h (Cometa et al., 2017) NBRC 3080 24 h (Tamai et al., 2009) PA 01 8, 24, 32, 48 h (Trujillo et al., 2012) 34 Staphylococcus aureus 38 4, 18, 24 h (Akhavan et al., 2018) 15981 24 h (Hidalgo-Robatto et al., 2018) 24 h (Prieto-Borja et al., 2017) H9(Clinical patient) 24 h (Lewandowska et al., 2015) ATCC 12598 (Cowan I strain) 1 h (Alcheikh et al., 2013) ATCC 12600 24 h (Kuehl et al., 2016) ATCC 13420(Newman) 24 h (Kuehl et al., 2016) ATCC 25923 24 h (Jennings et al., 2015) 1 hr, 4 hr, 8 hr, 1 d, 2 d (Ercan et al., 2011) 24 h (Wang et al., 2017) 2 h (Pissinis et al., 2018) 48 h (Giannelli et al., 2017) 48 h (Giannelli et al., 2016) 48 h (Cunha et al., 2016) (Continued number Incubation time Reference 24 h (Ciandrini et al., 2017) 24 h (Ciandrini et al., 2017) 24 h (Ciandrini et al., 2017) 24 h (Ciandrini et al., 2017) 24 h (Ciandrini et al., 2017) 24 h (Ciandrini et al., 2017) 2 h (Godoy-Gallardo et al., 2014) 2, 24 h (Godoy-Gallardo et al., 2016) 24 h (Ciandrini et al., 2017) 24 h (Kaliaraj et al., 2014) 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 48 h (Strever et al., 2017) 24 h (Chen et al., 2016) 24 h (Huang et al., 2019) 2 h (Hauser-Gerspach et al., 2014) 14 d (Zhang et al., 2015) 72 h (Lee et al., 2018) 2 h (Roberts et al., 2013) 72 h (Azizi et al., 2018) 2 d (Vilarrasa et al., 2018) 48 h (Eick et al., 2013) 8 d (Holmberg et al., 2013) 6, 12, 24 h (Batsukh et al., 2017) 3, 7 d (Kulkarni Aranya et al., 2017) 16.5, 40.5, 64.5 h (Ciandrini et al., 2014) 40.5 h (Sahrmann et al., 2014) 24 h (Kang et al., 2014) 24 h (Schmidt et al., 2017) 3 d (Gonçalves et al., 2010) 24 h (Jennings et al., 2015) 24 h (Giordano et al., 2011) 72 h (Schmidt et al., 2014) 24 h (Jennings et al., 2015) 72 h (Azizi et al., 2018) 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 40.5 h (Sahrmann et al., 2014) 24 h (Prieto-Borja et al., 2017) 24 h (Cotolan et al., 2016) 7, 24, 30 h (De Giglio et al., 2013) 24, 48, 72 h (Janković et al., 2015) 1, 3, 7 d (Ewald and Ihde, 2009) 16.5, 40.5, 64.5 h (Baffone et al., 2011) 24, 48 h (Atefyekta et al., 2018) 24, 48 h (Cometa et al., 2017) 24 h (Tamai et al., 2009) 8, 24, 32, 48 h (Trujillo et al., 2012) 4, 18, 24 h (Akhavan et al., 2018) 24 h (Hidalgo-Robatto et al., 2018) 24 h (Prieto-Borja et al., 2017) nt) 24 h (Lewandowska et al., 2015) 1 h (Alcheikh et al., 2013) 24 h (Kuehl et al., 2016) ewman) 24 h (Kuehl et al., 2016) 24 h (Jennings et al., 2015) 1 hr, 4 hr, 8 hr, 1 d, 2 d (Ercan et al., 2011) 24 h (Wang et al., 2017) 2 h (Pissinis et al., 2018) 48 h (Giannelli et al., 2017) 48 h (Giannelli et al., 2016) 48 h (Cunha et al., 2016) (Continued) 24 h (Kang et al., 2014) 24 h (Schmidt et al., 2017) 3 d (Gonçalves et al., 2010) ATCC BAA-308 24 h (Jennings et al., 2015) CCUG 25211 24 h (Giordano et al., 2011) DSM 20709 72 h (Schmidt et al., 2014) 31 Prevotella denticola ATCC 35308 24 h (Jennings et al., 2015) 32 Prevotella intermedia Clinical patient 72 h (Azizi et al., 2018) ATCC 25611 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 40.5 h (Sahrmann et al., 2014) 33 Pseudomonas aeruginosa ATCC 27853 24 h (Prieto-Borja et al., 2017) 24 h (Cotolan et al., 2016) 7, 24, 30 h (De Giglio et al., 2013) 24, 48, 72 h (Janković et al., 2015) ATCC 9027 1, 3, 7 d (Ewald and Ihde, 2009) 16.5, 40.5, 64.5 h (Baffone et al., 2011) CCUG 10778 24, 48 h (Atefyekta et al., 2018) DSM939 24, 48 h (Cometa et al., 2017) NBRC 3080 24 h (Tamai et al., 2009) PA 01 8, 24, 32, 48 h (Trujillo et al., 2012) 34 Staphylococcus aureus 38 4, 18, 24 h (Akhavan et al., 2018) 15981 24 h (Hidalgo-Robatto et al., 2018) 24 h (Prieto-Borja et al., 2017) H9(Clinical patient) 24 h (Lewandowska et al., 2015) ATCC 12598 (Cowan I strain) 1 h (Alcheikh et al., 2013) ATCC 12600 24 h (Kuehl et al., 2016) ATCC 13420(Newman) 24 h (Kuehl et al., 2016) ATCC 25923 24 h (Jennings et al., 2015) 1 hr, 4 hr, 8 hr, 1 d, 2 d (Ercan et al., 2011) 24 h (Wang et al., 2017) 2 h (Pissinis et al., 2018) 48 h (Giannelli et al., 2017) 48 h (Giannelli et al., 2016) 48 h (Cunha et al., 2016) (Continued) October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 10 Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. Studies With Development of Dental Bioﬁlm TABLE 2 \| Continued No. Studies With Development of Dental Bioﬁlm Microbiota Reference number Incubation time Reference ATCC 29213 24 h (Ferraris et al., 2018) 7, 24, 30 h (De Giglio et al., 2013) 24 h (Lewandowska et al., 2015) ATCC 35556(SA113) 24 h (Kuehl et al., 2016) ATCC 43387 16.5, 40.5, 64.5 h (Baffone et al., 2011) ATCC 6533 24, 48, 72 h (Janković et al., 2015) ATCC 6538 1, 3, 7 d (Ewald and Ihde, 2009) 1 min, 1 h, 4 h, 8 h (Yamada et al., 2014) CCUG 56489 24, 48 h (Atefyekta et al., 2018) DSM 799 24, 48 h (Cometa et al., 2017) n/s 1, 3, 5, 7, 14 d (Lv et al., 2014) n/s 4, 24, 5 d (Zhang et al., 2018) n/s 24 h (Santos-Coquillat et al., 2018) n/s 12 h (Shi et al., 2016) n/s 48 h (Drago et al., 2014) n/s 24, 48, 72, 96 h (Ayre et al., 2016) n/s 6, 12, 18, 24 h (Ye et al., 2017) Clinical patient 90 mins, 24 h (Ferraris et al., 2017) NBRC 12732 24 h (Tamai et al., 2009) NCIM 5021 6 h (Gopal et al., 2016) NTCC 8325-4 24 h (Giordano et al., 2011) 35 Staphylococcus aureus MRSA ATCC 43300 24 h (Kuehl et al., 2016) USA 300 JE2 24 h (Kuehl et al., 2016) 36 Staphylococcus epidermidis Xen 43 8 h (Wiedmer et al., 2017) RP62A 24 h (Granick et al., 2017) 8, 24, 32, 48 h (Trujillo et al., 2012) CCUG 18000A 14 h (Lilja et al., 2012) ATCC 35984 24 h (Giordano et al., 2011) 24, 48 h (Atefyekta et al., 2018) 24 h (Hirschfeld et al., 2017) 24 h (Prieto-Borja et al., 2017) ATCC 29886 2 h (Yucesoy et al., 2015) AF270147 120 mins (Bürgers et al., 2012) 35984 24 h (Hidalgo-Robatto et al., 2018) 1457 24 h (Kuehl et al., 2016) 37 Staphylococcus epidermidis MRSE ATCC 35984 24 h (Kuehl et al., 2016) 38 Streptococcus anginosus ATCC 9895(OMZ 871) 40.5 h (Sahrmann et al., 2014) 39 Streptococcus gordonii ATCC 10558 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 2, 24 h (Schmidt et al., 2017) 24 h (Schmidt et al., 2017) DL1 0, 8, 24, 48 h (Wood et al., 2015) DL1.1 24 h (Montelongo-Jauregui et al., 2018) ML-5 72 h (Chen et al., 2014) n/s 6, 24 h (Guan et al., 2016) 40 Streptococcus mitis n/s 2, 7 d (Cortizo et al., 2012) n/s 2 h (Canullo et al., 2017) BA7 8 h (Dorkhan et al., 2014) DSM 12643 24 h (Preissner et al., 2016) 41 Streptococcus mutans 3209 1 h (Narendrakumar et al., 2015) ATCC 25175 24 h (Ciandrini et al., 2017) 8 d (Cruz et al., 2015) 16.5, 40.5, 64.5 h (Ciandrini et al., 2014) 16.5, 40.5, 64.5 h (Baffone et al., 2011) 24 h (Ciandrini et al., 2016) 2 h (Roberts et al., 2013) 2 h (Yucesoy et al., 2015) ATCC 700610 24 h (Huang et al., 2019) C180-2(Clinical patient) 8, 24, 32 h (Ntrouka et al., 2011) CCUG 35176 24 h (Giordano et al., 2011) n/s n/s (Schmage et al., 2014) n/s 6, 12, 18, 24 h (Ye et al., 2017) NCTC 10449 30, 90 mins (Fukushima et al., 2018) (Continu October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. Studies With Development of Dental Bioﬁlm n/s, not stated; DSM/DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; ATCC, American Tissue Culture Collections; OMZ, Institut für Orale Mikrobiologie und Allgemeine Immunologie; NCIM, National Collection of Industrial Microorganisms, National Chemical Laboratory; CCUG, Culture Collection, University of Göteborg, Department of Clinical Bacteriology, Institute of Clinical Bacteriology, Immunology, and Virology, Sweden; NBRC, Biological Resource Center, National Institute of Technology and Evaluation, Japan; NTCC, Culture Collection, Microbiology and Cell Biology Laboratory, Indian Institute of Science; CECT, Colección Espagñola de Cultivos Tipo, Universitat de Valencia, Edeﬁcio de Investigación, Spain; h, hours; Mins, minutes; D, days. Kaliaraj et al., 2014; Abdulkareem et al., 2015; Charalampakis et al., 2015; Cruz et al., 2015; de Avila et al., 2015; Lewandowska et al., 2015; Park et al., 2015; Zhang et al., 2015 ; Preissner et al., 2016; Chen et al., 2016; Cochis et al., 2016; Cotolan et al., 2016; Cunha et al., 2016; Giannelli et al., 2016; John et al., 2016; Kotsakis et al., 2016; Mang et al., 2016; Shi et al., 2016; Al- Hashedi et al., 2017; Batsukh et al., 2017; Canullo et al., 2017; Dostie et al., 2017; Giannelli et al., 2017; Kim et al., 2017; Kulkarni Aranya et al., 2017; Li et al., 2017; Matthes et al., 2017; Ramesh et al., 2017; Schmidt et al., 2017; Strever et al., 2017; Wang et al., 2017; Wiedmer et al., 2017; Zhang et al., 2017; Al-Hashedi et al., 2018; Atefyekta et al., 2018; Azizi et al., 2018; Hidalgo-Robatto et al., 2018; Lee et al., 2018; Montelongo- Jauregui et al., 2018; Pantaroto et al., 2018; Souza et al., 2018; Trobos et al., 2018; Vilarrasa et al., 2018; Wang et al., 2018; Zhang et al., 2018; Huang et al., 2019). Studies With Development of Dental Bioﬁlm Thirty-two studies used confocal laser scanning microscopy (CLSM) (n = 32, 27.6%) (Fröjd et al., 2011; Eick et al., 2013; Drago et al., 2014; Lv et al., 2014; Sahrmann et al., 2014; Schmidt et al., 2014; Abdulkareem et al., 2015; de Avila et al., 2015; Zhang et al., 2015; Guan et al., 2016; Kotsakis et al., 2016; Preissner et al., 2016; Dostie et al., 2017; Giannelli et al., 2017; Kim et al., 2017; Kulkarni Aranya et al., 2017; Li et al., 2017; Matthes et al., 2017; Strever et al., 2017; Wiedmer et al., 2017; Ye et al., 2017; Zhang et al., 2017; Al-Hashedi et al., 2018; Akhavan et al., 2018; Hoyos-Nogués et al., 2018; Montelongo- Jauregui et al., 2018; Souza et al., 2018; Trobos et al., 2018; Wang et al., 2018; Vilarrasa et al., 2018; Zhang et al., 2018). A few of the studies that reported the use of SEM disclosed that their SEM protocol was operated between 1 and 30 kV accelerating voltage (46.7%) (Sennhenn-Kirchner et al., 2009; Ercan et al., 2011; Trujillo et al., 2012Cochis et al., 2013; ; Eick et al., 2013; Godoy-Gallardo et al., 2014; Cruz et al., 2015; Duske et al., 2015; Lewandowska et al., 2015; Park et al., 2015; Cotolan et al., 2016; Preissner et al., 2016; Al- Hashedi et al., 2017; Batsukh et al., 2017; Dostie et al., 2017; Kulkarni Aranya et al., 2017; Matthes et al., 2017; Ramesh et al., 2017; Al-Hashedi et al., 2018; Atefyekta et al., 2018; Lee et al., 2018; Pantaroto et al., 2018; Trobos et al., 2018; Wang et al., 2018). Li et al., 2017; Matthes et al., 2017; Strever et al., 2017; Wiedmer et al., 2017; Ye et al., 2017; Zhang et al., 2017; Al-Hashedi et al., 2018; Akhavan et al., 2018; Hoyos-Nogués et al., 2018; Montelongo- Jauregui et al., 2018; Souza et al., 2018; Trobos et al., 2018; Wang et al., 2018; Vilarrasa et al., 2018; Zhang et al., 2018). n/s, not stated; DSM/DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; ATCC, American Tissue Culture Collections; OMZ, Institut für Orale Mikrobiologie und Allgemeine Immunologie; NCIM, National Collection of Industrial Microorganisms, National Chemical Laboratory; CCUG, Culture Collection, University of Göteborg, Department of Clinical Bacteriology, Institute of Clinical Bacteriology, Immunology, and Virology, Sweden; NBRC, Biological Resource Center, National Institute of Technology and Evaluation, Japan; NTCC, Culture Collection, Microbiology and Cell Biology Laboratory, Indian Institute of Science; CECT, Colección Espagñola de Cultivos Tipo, Universitat de Valencia, Edeﬁcio de Investigación, Spain; h, hours; Mins, minutes; D, days. Studies With Development of Dental Bioﬁlm TABLE 2 \| Continued No. Microbiota Reference number Incubation time Reference UA 159 14 d (Zhang et al., 2015) 5 d (Ramesh et al., 2017) 42 Streptococcus oralis ATCC 35037(CCUG 13229 T) 2, 24, 48 h (Trobos et al., 2018) ATCC 6249 2 d (Vilarrasa et al., 2018) ATCC 9811 24 h (Ciandrini et al., 2017) 24 h (Ciandrini et al., 2016) SK248(OMZ 607) 40.5 h (Sahrmann et al., 2014) 43 Streptococcus pyogenes GTC 262 1 min, 1 h, 4 h, 8 h (Yamada et al., 2014) 44 Streptococcus salivarius n/s 2, 7 d (Cortizo et al., 2012) ATCC 13419 5 d (Ramesh et al., 2017) 45 Streptococcus sanguinis ATCC 10556 24 h (Chen et al., 2016) 16, 48 h (Duarte et al., 2009) 72 h (Kim et al., 2017) 2, 14 h (Fröjd et al., 2011) 5 d (Ramesh et al., 2017) CECT 480 4 h (Hoyos-Nogués et al., 2018) 2 h (Godoy-Gallardo et al., 2014) 2, 24 h (Godoy-Gallardo et al., 2016) DSM 20068 2 h (Bürgers et al., 2012; Hauser-Gerspach et al., 2014) 72 h (Schmidt et al., 2014) GW2 1 h (Narendrakumar et al., 2015) IAL 1832 16.5 h (Pantaroto et al., 2018) 1.5 h (Souza et al., 2018) n/s 6, 24 h (Guan et al., 2016) 46 Tannerella forsythia ATCC 43037 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 24 h (Schmidt et al., 2017) 47 Treponema denticola ATCC 35405 48 h (Eick et al., 2013) 48, 72 h (Eick et al., 2017) 48 Veillonella dispar ATCC 17748T 40.5 h (Sahrmann et al., 2014) 49 Veillonella parvula ATCC 10790 2 d (Vilarrasa et al., 2018) n/s, not stated; DSM/DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; ATCC, American Tissue Culture Collections; OMZ, Institut für Orale Mikrobiologie und Allgemeine Immunologie; NCIM, National Collection of Industrial Microorganisms, National Chemical Laboratory; CCUG, Culture Collection, University of Göteborg, Department of Clinical Bacteriology, Institute of Clinical Bacteriology, Immunology, and Virology, Sweden; NBRC, Biological Resource Center, National Institute of Technology and Evaluation, Japan; NTCC, Culture Collection, Microbiology and Cell Biology Laboratory, Indian Institute of Science; CECT, Colección Espagñola de Cultivos Tipo, Universitat de Valencia, Edeﬁcio de Investigación, Spain; h, hours; Mins, minutes; D, days. Bioﬁlm-Treatment Methods In a study using calcifying coating solutions (containing different ionic compositions of calcium, phosphate, and zinc), the degree of crystallinity was inversely proportional to antibacterial activity against Porphyromonas gingivalis in coated discs (Kulkarni Aranya et al., 2017). Lower the dissolution rate, lesser the antibacterial effect (Kulkarni Aranya et al., 2017). The addition of titanium dioxide nanoparticles boosted the antimicrobial efﬁcacy of hydrogen peroxide solutions and delayed the redevelopment on surfaces which were previously contaminated by bacteria (Wiedmer et al., 2017). The second most common bioﬁlm-treatment method of decontamination in this review is by chemical treatment. However, similar to the previous ﬁndings, their results showed that not all of the agents used for disinfection in the clinic are effective in the removal of the bioﬁlm. Some of the agents left behind live bacteria on the surface (Dostie et al., 2017). With regards to mechanical cleaning, a complete decontamination of a surface will not be possible as certain bacteria will continue to reside in “valleys and undercuts” (Schmage et al., 2014). In general, removal of greater than 96% bioﬁlm was reported as satisfactory for clinical health, and this was achieved by the oscillating instruments and air polishing (Schmage et al., 2014). One study focused on the application of a direct contact ultrasound at low frequency (Granick et al., 2017). They described that the ultrasound biocidal activity occurred due to acoustic microstreaming and bubble cavitation, releasing considerable energy and damaging bacterial cell walls. In another study, implants were used as cathodes to generate an alkaline environment and reactive oxygen species, and showed a twofold reduction of bacteria compared with the untreated controls (Mohn et al., 2011). Mechanisms are similar to chemical treatments. For example, the ability of hydrogen peroxide to cause bacterial bioﬁlm disruption is associated with oxidation of a number of cellular components and production of gas (Wiedmer et al., 2017). Cell Culture Studies In addition, 37 of the included studies (n = 37, 31.9%) also performed cell culture in their methodology to study re- osseointegration (Schwarz et al., 2005; Ewald and Ihde, 2009; Giordano et al., 2011; Cortizo et al., 2012; Alcheikh et al., 2013; De Giglio et al., 2013; Eick et al., 2013; Holmberg et al., 2013; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Duske et al., 2015; Janković et al., 2015; Lewandowska et al., 2015; Ayre et al., 2016; Cochis et al., 2016; Cotolan et al., 2016; Giannelli et al., 2016; Guan et al., 2016; Godoy-Gallardo et al., 2016; John et al., 2016; Kotsakis et al., 2016; Shi et al., 2016; Canullo et al., 2017; Cometa et al., 2017; Eick et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Matthes et al., 2017; Ramesh et al., 2017; Wang et al., 2017; Ye et al., 2017; Zhang et al., 2017; Atefyekta et al., 2018; Hidalgo-Robatto et al., 2018; Hoyos-Nogués et al., 2018; Lee et al., 2018; Santos-Coquillat et al., 2018). The investigated cell types in this systematic review were osteogenic sarcoma (57.4%), epithelial (2.1%), ﬁbroblast (17.0%), macrophages (4.3%), acute monocytic leukemia (2.1%), mesenchymal stem cells (4.3%), and stromal cells (2.1%). This review has grouped the decontamination methods into two types: (i) bioﬁlm-prevention method and (ii) bioﬁlm- treatment method. Staphylococcus aureus, Porphyromonas gingivalis, Streptococcus sanguinis, and Streptococcus mutans are the commonly used oral microbiota for the study of dental bioﬁlms reported in the included studies of this review. These bacteria are all responsible for peri-implantitis (Lavere et al., 2018). They can also exist in the oral saliva of healthy adults and later transform into opportunistic pathogens to become the main cause of the host diseases (Gao et al., 2018). S. aureus is a Gram- Studies With Development of Dental Bioﬁlm Cunha et al., 2016; Giannelli et al., 2016; Gopal et al., 2016; Kuehl et al., 2016; Shi et al., 2016; Cometa et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Prieto-Borja et al., 2017; Wang et al., 2017; Ye et al., 2017; Akhavan et al., 2018; Atefyekta et al., 2018; Ferraris et al., 2018; Hidalgo-Robatto et al., 2018; Pissinis et al., 2018; Santos-Coquillat et al., 2018; Zhang et al., 2018), Porphyromonas gingivalis (9.5%) (Gonçalves et al., 2010; Giordano et al., 2011; Holmberg et al., 2013; Eick et al., 2013; Roberts et al., 2013; Ciandrini et al., 2014; Hauser-Gerspach et al., 2014; Kang et al., 2014; Sahrmann et al., 2014; Schmidt et al., 2014; Jennings et al., 2015; Zhang et al., 2015; Chen et al., 2016; Batsukh et al., 2017; Kulkarni Aranya et al., 2017; Schmidt et al., 2017; Strever et al., 2017; Azizi et al., 2018; Lee et al., 2018; Vilarrasa et al., 2018; Huang et al., 2019), Streptococcus sanguinis (7.3%) (Duarte et al., 2009; Fröjd et al., 2011; Bürgers et al., 2012; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Schmidt et al., 2014; Narendrakumar et al., 2015; Chen et al., 2016; Godoy-Gallardo et al., 2016; Guan et al., 2016; Kim et al., 2017; Ramesh et al., 2017; Hoyos-Nogués et al., 2018; Pantaroto et al., 2018; Souza et al., 2018), and Streptococcus mutans (7.3%) (Baffone et al., 2011; Giordano et al., 2011; Ntrouka et al., 2011; Roberts et al., 2013; Schmage et al., 2014; Ciandrini et al., 2014; Cruz et al., 2015; Narendrakumar et al., 2015; Yucesoy et al., 2015; Zhang et al., 2015; Ciandrini et al., 2016; Ye et al., 2017; Ciandrini et al., 2017; Ramesh et al., 2017; Fukushima et al., 2018; Huang et al., 2019). bioﬁlm. In addition to the surface roughness, materials and surface treatment, too, have an impact on the early attachment of bacteria (Batsukh et al., 2017). Therefore, it is essential to assess the effect of various implant surfaces available on the attachment of initial and late colonizing bacteria. In bioﬁlm- treatment method, bioﬁlm development has already taken place. Most of the included studies were for bioﬁlm-treatment. The most common preventive method of decontamination used in the included studies was the application of a coating on the titanium implant or discs. The mechanism of decontamination did not completely rely on the physical separation of the titanium alloy and oral microbiota. Studies With Development of Dental Bioﬁlm Cunha et al., 2016; Giannelli et al., 2016; Gopal et al., 2016; Kuehl et al., 2016; Shi et al., 2016; Cometa et al., 2017; Ferraris et al., 2017; Giannelli et al., 2017; Prieto-Borja et al., 2017; Wang et al., 2017; Ye et al., 2017; Akhavan et al., 2018; Atefyekta et al., 2018; Ferraris et al., 2018; Hidalgo-Robatto et al., 2018; Pissinis et al., 2018; Santos-Coquillat et al., 2018; Zhang et al., 2018), Porphyromonas gingivalis (9.5%) (Gonçalves et al., 2010; Giordano et al., 2011; Holmberg et al., 2013; Eick et al., 2013; Roberts et al., 2013; Ciandrini et al., 2014; Hauser-Gerspach et al., 2014; Kang et al., 2014; Sahrmann et al., 2014; Schmidt et al., 2014; Jennings et al., 2015; Zhang et al., 2015; Chen et al., 2016; Batsukh et al., 2017; Kulkarni Aranya et al., 2017; Schmidt et al., 2017; Strever et al., 2017; Azizi et al., 2018; Lee et al., 2018; Vilarrasa et al., 2018; Huang et al., 2019), Streptococcus sanguinis (7.3%) (Duarte et al., 2009; Fröjd et al., 2011; Bürgers et al., 2012; Godoy-Gallardo et al., 2014; Hauser-Gerspach et al., 2014; Schmidt et al., 2014; Narendrakumar et al., 2015; Chen et al., 2016; Godoy-Gallardo et al., 2016; Guan et al., 2016; Kim et al., 2017; Ramesh et al., 2017; Hoyos-Nogués et al., 2018; Pantaroto et al., 2018; Souza et al., 2018), and Streptococcus mutans (7.3%) (Baffone et al., 2011; Giordano et al., 2011; Ntrouka et al., 2011; Roberts et al., 2013; Schmage et al., 2014; Ciandrini et al., 2014; Cruz et al., 2015; Narendrakumar et al., 2015; Yucesoy et al., 2015; Zhang et al., 2015; Ciandrini et al., 2016; Ye et al., 2017; Ciandrini et al., 2017; Ramesh et al., 2017; Fukushima et al., 2018; Huang et al., 2019). Studies With Development of Dental Bioﬁlm A few of the studies that reported the use of SEM disclosed that their SEM protocol was operated between 1 and 30 kV accelerating voltage (46.7%) (Sennhenn-Kirchner et al., 2009; Ercan et al., 2011; Trujillo et al., 2012Cochis et al., 2013; ; Eick et al., 2013; Godoy-Gallardo et al., 2014; Cruz et al., 2015; Duske et al., 2015; Lewandowska et al., 2015; Park et al., 2015; Cotolan et al., 2016; Preissner et al., 2016; Al- Hashedi et al., 2017; Batsukh et al., 2017; Dostie et al., 2017; Kulkarni Aranya et al., 2017; Matthes et al., 2017; Ramesh et al., 2017; Al-Hashedi et al., 2018; Atefyekta et al., 2018; Lee et al., 2018; Pantaroto et al., 2018; Trobos et al., 2018; Wang et al., 2018). In this review, the most commonly employed oral microbiota for the study of dental bioﬁlms were Staphylococcus aureus (15.9%) (Ewald and Ihde, 2009; Tamai et al., 2009; Baffone et al., 2011; Ercan et al., 2011; Giordano et al., 2011; Alcheikh et al., 2013; De Giglio et al., 2013; Drago et al., 2014; Lv et al., 2014; Yamada et al., 2014; Janković et al., 2015; Jennings et al., 2015; Lewandowska et al., 2015; Ayre et al., 2016; October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 12 Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2021. 736186/full#supplementary-material FUNDING The work was supported by the University Research Council grant under Universiti Brunei Darussalam (grant number: UBD/ RSCH/URC/RG (b)/2018/004). Allied Grant-Universiti Brunei Darusslam for Publication Cost. Re-osseointegration is also an important issue taken into account when decontamination methods are performed. About one-third of the included studies in this review investigated the cell adherence on the titanium implants with bioﬁlm contamination. Thisphenomenonisdescribedas “therace for the surface,” wherein bacterial and host cells compete in colonizing implant surface CONCLUSION In conclusion, this review found that the commonly used decontamination methods of microbial bioﬁlm on dental implant surfaces focused on the addition of surface coating and chemical treatment. Further efforts should be aimed at ﬁnding the optimal implant surface property that features antimicrobial treatment that is effective and does not compromise osseointegration. There are many other factors to consider if the decontamination methodissuccessfulornot.The ﬁrst isthe ageofthe bioﬁlm.Halfof the included studies in this review evaluated on bioﬁlm aged less or equalto24h. However, thismay also depend onthetype ofresident microbiota, maturity of the bioﬁlm, and the numberof bacterial cell clusters and micro colonies (Cao et al., 2018). This is a crucial factor especially for those treatment methods aimed at established bioﬁlms, such as in clinically diagnosed peri-implantitis. Additionally, in-vitro developed bioﬁlm is easily affected by surface conﬁguration than bioﬁlm that forms in the mouth (Bevilacqua et al., 2018). Nutrients and mixed bacterial species present in more favorable environment will encourage bioﬁlm formation (Bevilacqua et al., 2018). Quantitative differences detected in-vitro amongst surfaces with dissimilar features might notpredicttherateofin-vivocolonization.Variousstudiesanalyzed the efﬁcacy of their decontamination methods using a scanning electron microscopy (SEM). However, the energy beam by SEM on its own can be a potential decontamination strategy as the high energy (in kV) can cause bacterial cell wall destruction and internal reversible changes to genetic materials (Ghomi et al., 2005). Therefore, when a high-energy SEM captured the images of bacterial matrix for quantiﬁcation, their image analysis may introduce inaccuracies. Akhavan, B., Michl, T. D., Giles, C., Ho, K., Martin, L., Sharifahmadian, O., et al. (2018). Plasma Activated Coatings With Dual Action Against Fungi and Bacteria. Appl. Mater. Today 12, 72–84. doi: 10.1016/j.apmt.2018.04.003 Albrektsson, T., Jemt, T., Mölne, J., Tengvall, P., and Wennerberg, A. (2019). On Inﬂammation-Immunological Balance Theory-A Critical Apprehension of Disease Concepts Around Implants: Mucositis and Marginal Bone Loss may Bioﬁlm-Prevention Methods The studies focused on the modiﬁcation of titanium implant surfaces. For example, one of the included studies focused on the type of instrument used to modify the surfaces, either to smooth or to roughen them (Schmidt et al., 2017). This bioﬁlm- preventing step occurs before the development of assumed October 2021 \| Volume 11 \| Article 736186 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 13 Microbial Bioﬁlm Decontamination on Dental Implants Dhaliwal et al. positive, aerobic cocci and may cause infections ranging from minor skin infections to pneumonia, bacteremia, and infective endocarditis (Oliveira et al., 2018). P. gingivalis is a Gram- negative anaerobic bacteria and has a strong positive correlation with periodontal diseases (Raﬁei et al., 2017). S. sanguinis, previously known as S. sanguis, is a Gram-positive, non-spore-forming, facultative anaerobe and is typically found in a healthy plaque (Zhu et al., [[NoYear]]). S. mutans is a Gram- positive cocci, a facultative anaerobe responsible for the initiation of dental caries, which may lead to the formation of dental plaque and endocarditis (Daboor et al., 2015). (Hoyos-Nogués et al., 2018). Previous systematic review on re- osseointegration has reported that the application of single decontamination measure was not sufﬁcient to get a desirable treatment outcome for peri-implantitis (Madi et al., 2018). Therefore, further studies must be conducted to focus on the dual mechanism (or more) to encourage re-osseointegration after bioﬁlm decontamination. So far, there is no “gold standard” that has been speciﬁed in the decontamination of dental implants. A decontamination treatment will need to be combined with mechanical therapy for a successful outcome (Prathapachandran and Suresh, 2012). An important question for surface decontamination studies is whether the different patterns of bioﬁlm play a clinical role (Trobos etal.,2018). Bioﬁlm‐associated antimicrobial resistanceisrelated to a number of factors. Established bioﬁlms are resistant to antimicrobial agents as compared with planktonic bacteria which are attributed to bioﬁlm properties, including nature and structure, poor antibiotic penetration, bacterial cells metabolic state, nutrient and oxygen availability, and antimicrobial resistance acquired by gene transfer and mutation (Arciola et al., 2018). AUTHOR CONTRIBUTIONS Conceptualization: JD, JK, RA. Methodology: JD, LM, SD, JK, RA. Software: JD, NR, LM, SD, JK, RA. Validation: JD, NR, JK, RA. Formal analysis: JD, NR, LM, SD, JK, RA. Investigation: JD, NR, LM, SD, JK, RA. Resources: JD, NR, LM, SD, JK, RA. Data curation: JD, NR, LM, SD, JK, RA. Writing—original draft preparation: JD, NR, SD, JK. Writing—review and editing: JD, NR, LM, SD, JK, RA. Supervision: JD, JK. Project administration: JD, NR, LM, SD, JK, RA. Funding acquisition: JD, JK, RA. All authors contributed to the article and approved the submitted version. Conceptualization: JD, JK, RA. Methodology: JD, LM, SD, JK, RA. Software: JD, NR, LM, SD, JK, RA. Validation: JD, NR, JK, RA. Formal analysis: JD, NR, LM, SD, JK, RA. Investigation: JD, NR, LM, SD, JK, RA. Resources: JD, NR, LM, SD, JK, RA. Data curation: JD, NR, LM, SD, JK, RA. Writing—original draft preparation: JD, NR, SD, JK. Writing—review and editing: JD, NR, LM, SD, JK, RA. Supervision: JD, JK. Project administration: JD, NR, LM, SD, JK, RA. Funding acquisition: JD, JK, RA. All authors contributed to the article and approved the submitted version. Abdulkareem, E. H., Memarzadeh, K., Allaker, R. P., Huang, J., Pratten, J., and Spratt, D. (2015). 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https://openalex.org/W2510280558	https://europepmc.org/articles/pmc5042052?pdf=render	English	null	Recognising moulting behaviour in trilobites by examining morphology, development and preservation: Comment on Błażejowski et al. 2015	BioEssays	2,016	cc-by	9,564	Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Insights & Perspectives Insights & Perspectives Think again Keywords: y.arthropod; ecdysis; Ecdysozoa; fossil record; Trilobita; Trimerocephalus The fossil record of moulting is largely composed of exuviae, and fossils preserved during moulting are ex- tremely rare because these events take place on a relatively rapid time scale [5]. The most proliﬁc fossil record of moult- ing is observed in trilobites, an extinct group of marine arthropods that were very abundant and important members of the animal ecosystem from 521 to 250 million years ago (Fig. 2). Their anatomy consists of a mineralised, segmented body with three longitudinal lobes (a central axial lobe, and left and right pleural lobes, hence the name “trilobite”), and an anterior to posterior regionalisation consisting of a head (cephalon), thorax and tail (pygidium) Recognising moulting behaviour in trilobites by examining morphology, development and preservation: Comment on Bl - a_zejowski et al. 2015 Harriet B. Drage1)* and Allison C. Daley1)2) A 365 million year-old trilobite moult-carcass assemblage was described by Bl - a_zejowski et al. (2015) as the oldest direct evidence of moulting in the arthropod fossil record. Unfortunately, their suppositions are insufﬁciently supported by the data provided. Instead, the morphology, conﬁguration and preservational context of the highly fossiliferous locality (Kowala Quarry, Poland) suggest that the specimen consists of two overlapping, queued carcasses. The wider fossil record of moulting actually extends back 520 million years, providing an unparalleled opportunity to study behaviour, ecology and development in early animals. Taking cues from modern analogues, it is possible to quantify precise details about moulting behaviour to determine broad-scale evolutionary trends, ontogenetic sequences and morphological selection pressures. In this review, we argue that this rich source of data has been underused in evolutionary studies, though has great potential for investigating the life history and evolution of arthropods in deep time. damaged exoskeleton [5]. Owing to this multi-faceted function, the mode and method of moulting can greatly inﬂu- ence large-scale evolutionary trends within the arthropods; for example, the successful radiation of insects (Fig. 1A) was linked to the regulation of ecdysis-related hormones [6]. The evolutionary signiﬁcance of moulting in extinct animals can also be examined because the empty moults, or exuviae, are discarded after ecdysis, providing a record of previous developmental stages, and have the potential to be fossilised. As one of the few behaviours with palaeontological evidence, moult- ing provides a rare opportunity to study ecology, growth and development in extinct arthropods, as recently reviewed by Daley and Drage [5]. Introduction animals 541 million years ago [1–3]. Arthropod growth requires moulting of the exoskeleton (Fig. 1), a process known as ecdysis [4, 5], which is also observed in nematodes and other closely related phyla that make up the group Ecdysozoa. The exoskeleton of arthropods provides protection and structural support, but has the disad- vantage of needing to be moulted in order for the animal to grow. Each growth stage in arthropods involves a moulting event, which provides the opportunity not only for development towards an adult morphology (in juve- niles) and an increase in body size, but also for the regeneration and repair of a The extensive fossil record of Arthro- poda provides a fascinating insight into the evolution of the most diverse and abundant animal phylum, upon its appearance amongst the earliest www.bioessays-journal.com 1) Department of Zoology, University of Oxford, Oxford, UK 2) Oxford University Museum of Natural History, Oxford, UK DOI 10.1002/bies.201600027 1) Department of Zoology, University of Oxford, Oxford, UK 1) Department of Zoology, University of Oxford, Oxford, UK ) , 2) Oxford University Museum of Natural History, Oxford, UK Corresponding author: Harriet B. Drage E-mail: harriet.drage@zoo.ox.ac.uk Corresponding author: Harriet B. Drage E-mail: harriet.drage@zoo.ox.ac.uk 981 Insights & Perspectives..... H. B. Drage and A. C. Daley Figure 1. Exemplar photographs of modern arthropod moulting behaviours. A: Cicada moulting through the dorsal part of the abdomen (image credit: Wikimedia Commons). B: Horseshoe crab that died following exuviation through the anterior cephalic ecdysial g (image credit: Wikimedia Commons). C: Giant house spider (Eratigena atrica), extracting legs while emerging mid-moult from the dorsal abdomen (image credit: Wikimedia Commons). D: Mid-moult Scolopendra centipede, emerging through an anterior cephalic gape suture (image credit: Nicky Bay, http://sgmacro.blogspot.com). preservation of an individual having just emerged from its moulted exuvia, located directly above the carcass (Fig. 3C and F). The spectacular preser- vation of such a short-lived event would provide important information for studying the complex interplay between ontogeny, behaviour and fossil preser- vation, and as such it is critical that such evidence is identiﬁed correctly in the fossil record. Unfortunately, the proposed trilobite carcass-moult assem- blage hypothesised by Bła_zejowski et al. [9] is not well supported, and the lines of evidence on which their interpretation is based are discussed in order below. We then place our paper in the context of the excellent fossil record of moulting in general, and focus on its potential for studying the evolu- tion of developmental processes in deep time. Think again The earliest fossil record of moulting extends back to the Cambrian Figure 1. Exemplar photographs of modern arthropod moulting behaviours. A: Cicada moulting through the dorsal part of the abdomen (image credit: Wikimedia Commons). B: Horseshoe crab that died following exuviation through the anterior cephalic ecdysial gape (image credit: Wikimedia Commons). C: Giant house spider (Eratigena atrica), extracting the legs while emerging mid-moult from the dorsal abdomen (image credit: Wikimedia Commons). D: Mid-moult Scolopendra centipede, emerging through an anterior cephalic gape suture (image credit: Nicky Bay, http://sgmacro.blogspot.com). Bła_zejowski et al. [9] claimed that the three-dimensional specimen of T. cho- pini from Kowala Quarry in central Poland represents the oldest direct example of a fossilised moulting event at 365 million years old. However, in- the-act moulting is conclusively known from a number of older fossils from the Cambrian. An exquisite specimen of Marrella splendens from the middle Cambrian Burgess Shale (approximately 508 million years old) was illustrated emerging anteriorly from its exoskele- ton, and therefore represents the earli- est example of an arthropod preserved mid-moult [10]. Even older examples (from approximately 518 million years ago) are known for other ecdysozoans, including a loriciferan (microscopic marine sediment-afﬁxed animals with a protective outer layer) from the lower Cambrian Sirius Passet preserved exit- ing its exoskeleton [11], and a lobopo- dian (extinct worm-like ecdysozoan with paired limbs) with overlapping plates from the lower Cambrian of northern Greenland that suggests it was preserved during the process of moulting [12]. The early fossil record of moulting is also rich in examples of exuviae, particularly for Cambrian B: (Fig. 2A). Building on this basic body plan, trilobites had a diverse morphol- ogy and ecology, occupying numerous niches in marine communities. Trilo- bites had distinct moulting behaviours that mostly utilised the facial sutures, thus allowing the lateral and antero- dorsal parts of the cephalon to be displaced (the free cheeks, or librige- nae), providing an opening through which the organism could shed the smaller exoskeleton (Fig. 2B). In some trilobites, the facial sutures were fused and the animal moulted by disarticulat- ing the entire cephalon (Fig. 2C). With either scenario, the distinct conﬁgura- tion remaining as empty exuviae allows us to identify specimens as moults with relative ease. For other moulting arthro- pods with a known fossil record, such as decapods, scorpions, and eurypterids (sea scorpions), distinguishing between exuviae and carcasses is not as easy (Fig. 2A). Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. .....Insights & Perspectives H. B. Drage and A. C. Daley .....Insights & Perspectives .....Insights & Perspectives Figure 2. A: Labelled line drawing of Olenus sp., with the anterior and posterior directions indicated, and the cephalic, thoracic and pygidial regions also indicated on an adjacent photograph of an Olenus truncatus specimen (PMU, no number, 4 mm scale). B: Acidaspis coronata moult showing disarticulation and backwards displacement of the free cheeks (librigenae) (OUMNH C.17494, 5 mm scale). C: Ogygiocarella debuchii moult showing disarticulation of the cephalon and dislocation along the thorax (OUMNH B.263, 20 mm scale). moulted trilobite exoskeletons are very often recognisable in the fossil record, and can provide a great deal of behavioural and ecological information. They display several distinct moult arrangements (such as those described in Ref. [16]) with clear exuvial gapes. Most frequently, trilobites created an exuvial gape by disarticulating the free cheeks (Fig. 2B), or entire cephalon (Fig. 2C), followed by the animal extracting itself from the remaining articulated sclerites by moving anteri- orly [5]. Unfortunately, Bła_zejowski et al. [9] do not factor several crucial considerations into their description of a putative moult-carcass trilobite as- semblage. In particular, the presumed exuvial gape, relative positioning of their specimen, and the context (other specimens from the locality, and pres- ervational conditions) do not support their hypotheses. Think again T. chopini ﬁgured by Bła_zejowski et al. [9] (Fig. 3) shows two overlapping individuals, a situation that has been interpreted as a ‘moulted exoskeleton’ positioned dorsal and slightly posterior to the ‘carcass’. The putative moult is exposed, displaying a cephalon with slight dorsal angling, while the putative carcass was revealed in three-dimensions by CT-scanning. Bła_zejowski et al. [9] argued that this specimen shows the moult arrangement described for Paci- phacops preserved ‘immediately follow- ing moulting’ (p. 2). They (and we) reject the idea that this assemblage represents a specimen preserved during or immedi- ately before moulting (with the ‘carcass’ still enclosed in the soon-to-be moulted exoskeleton) because the two individu- als are ‘separated by a layer of sediment’ (Fig. 3F) and not ‘pancaked’ (Ref. [9], p. 2), as would be expected if they were still articulated at the time of preserva- tion. .....Insights & Perspectives Paciphacops is thought to utilise infaunal moulting (following burrowing for protection from predators), which occurs through an anterior gape suture created by ventral disarticulation of the cephalon and thorax, causing the ceph- alon to hinge dorsally at an angle of nearly 90˚ (Ref. [17], Fig. 2; our Fig. 3D). Other phacopid trilobite specimens also show possible infaunal moulting with their cephala angled at more than 90˚ from the sediment [18]. However, the specimen of T. chopini (Ref. [9], Fig. 2) shows a much lower angle of cephalic Figure 2. A: Labelled line drawing of Olenus sp., with the anterior and posterior directions indicated, and the cephalic, thoracic and pygidial regions also indicated on an adjacent photograph of an Olenus truncatus specimen (PMU, no number, 4 mm scale). B: Acidaspis coronata moult showing disarticulation and backwards displacement of the free cheeks (librigenae) (OUMNH C.17494, 5 mm scale). C: Ogygiocarella debuchii moult showing disarticulation of the cephalon and dislocation along the thorax (OUMNH B.263, 20 mm scale). which are found preserved in the fossil record due to their reinforced cuticle. These moult assemblages can usually be recognised by a number of criteria, described by Henningsmoen [16] and expanded upon in Daley and Drage [5]. For example, opened gape sutures (created by the opening of suture lines or articulation points) in the exoskele- ton and repeated conﬁgurations of disarticulated sclerites (individual units of the exoskeleton) are most commonly used to designate moults. Contextual information from the fossil locality (associated material, palaeoenviron- ment, preservational processes), and the distortion of sclerites in consider- ation with local preservational pro- cesses (ﬂattening, superimposition of elements, telescoping), are important for differentiating between preserved arthropod moults and carcasses, and for distinguishing abiotic or microbial exoskeleton disarticulation from true moult assemblages. Using these criteria, and Ordovician strongly biomineralised arthropods such as trilobites [5]. Several specimens from this time period show clusters of exuviae preserved in close proximity, indicating synchronous mass moulting events [13, 14]. This rich fossil record of moulting has allowed for quantitative analyses investigating moulting behaviour trends, and their impact on trilobite evolution [5, 15]. Therefore, the Devonian specimen of T. chopini described by Bła_zejowski et al. [9] cannot be considered ‘the oldest direct evidence for a moulting episode’ (p. 1). The earliest fossil record of moulting extends back to the Cambrian Building on this basic body plan, trilobites had a diverse morphol- ogy and ecology, occupying numerous niches in marine communities. Trilo- bites had distinct moulting behaviours that mostly utilised the facial sutures, thus allowing the lateral and antero- dorsal parts of the cephalon to be displaced (the free cheeks, or librige- nae), providing an opening through which the organism could shed the smaller exoskeleton (Fig. 2B). In some trilobites, the facial sutures were fused and the animal moulted by disarticulat- ing the entire cephalon (Fig. 2C). With either scenario, the distinct conﬁgura- tion remaining as empty exuviae allows us to identify specimens as moults with relative ease. For other moulting arthro- pods with a known fossil record, such as decapods, scorpions, and eurypterids (sea scorpions), distinguishing between exuviae and carcasses is not as easy because ecdysial suture lines are less distinct and the moult assemblages are not as recognisable [5]. Trilobites, therefore, represent one of the best model taxa for studying the evolution of moulting, growth and development in extinct arthropods. Trilobites have a rich fossil record of carcasses and exuviae, which has provided a wealth of information on developmental stages, behaviour and interactions with the environment (e.g. Refs. [5, 7, 8]). Trilobite specimens preserved in the short period immedi- ately post-moulting are extremely rare. However, a recent paper by Bła_zejowski et al. [9] described an Upper Devonian fossil, approximately 365 million years old from the Kowala Quarry in the Holy Cross Mountains of central Poland, that consists of two stacked exoskel- etons of the trilobite Trimerocephalus chopini, which they interpret as in situ Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. 982 Specimen position, preservation and context suggest queueing trilobite carcasses Many arthropods have distinct exoskel- etal arrangements upon moulting, 983 Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. Insights & Perspectives..... H. B. Drage and A. C. Daley Figure 3. Trimerocephalus specimens from the Early Famennian (Late Devonian), Holy Cross Mountains, central Poland. A: line drawing produced from the CT scan of the Trimerocephalus chopini ‘moult’ ﬁgured by Bła_zejowski et al. [9]. B: Line drawing from the CT scan of the T. chopini ‘carcass’. Numbering in A and B indicates 10 thoracic segments in each, particularly visible when using the appended CT scan video provided by Bła_zejowski et al. [9]. C: Original lateral-view CT scan of the T. chopini specimen from Bła_zejowski et al. (Ref. [9], Fig. 2B, cc-by licensed), illustrating the relative positioning of their putative ‘carcass’ and ‘moult’. Measures of the lateral and dorsal lengths of the cephala, and of the individual thoracic segment lengths show no appreciable difference in size between the two individuals. Scale bar 2 cm, as given in Bła_zejowski et al. [9]. D: In comparison to C, the positioning of the labelled ‘carcass’ and ‘moul suggested for a true moult-carcass assemblage [17]. E: One of several Trimerocephalus ‘queues’ of overlapping trilobite carcasses from the Holy Cross Mountains, adapted from Plate 2, Fig. 4 of Radwanski et al. [19]. The white box shows the contextual positioning of G. No scale bar provided in Radwanski et al. [19]. F: Dorsal-view photograph of the T. chopini suggested moult-carcass assemblage from Bła_zejowski et al. (Ref. [9], Fig. 2A, cc-by licensed) Scale bar 2 cm, as given in Bła_zejowski et al. [9]. G: Dorsal-view close-up of two overlapping Trimerocephalus carcasses, adapted from Radwanski et al. (Ref. [19], Plate 2, Fig. 4A and B) and shown in situ in E. Shown free of context in comparison to F, the two specimens appear extremely similar. No scale bar provided in Radwanski et al. [19]. moulting infaunally, because sediment would immediately inﬁll the empty exoskeleton after moulting [18]. Even if the specimen were moulting above- ground, a larger displacement of the cephalon would still be expected, as is regularly seen in taxa that moult in this manner (Fig. 2C) [5, 16]. Specimen position, preservation and context suggest queueing trilobite carcasses Regardless of whether Trimerocephalus was moulting infaunally or not, similar minor dis- placements of the cephala have fre- quently been observed in other trilobites from the Kowala Quarry, and interpreted as ‘resulting from a diage- netic event of compaction’ (Ref. [19], p. 461), rather than moulting. This further suggests that the dorsal individ- ual ﬁgured in Bła_zejowski et al. [9] is not a moult. Think again The position of the ‘carcass’ relative to the ‘moult’ raises further issues with the moult-carcass assemblage interpre- tation. The ‘carcass’ component is positioned very much beneath, and only slightly forward, of the ‘moult’ (Ref. [9], Fig. 2). As mentioned earlier, this cannot represent preservation be- fore or during moulting, because the layer of sediment separating the dorsal and ventral individuals indicates that the supposed ‘carcass’ was not enclosed in the soon-to-be moulted ‘exoskeleton’. To result in this position in a specimen preserved immediately after moulting, the egressing individual would have had either to detach the dorsal exoskel- etal shield as a single unit and move downwards vertically, or to have exited anteriorly through a ventral gape suture under the cephalon and burrowed backwards into the sediment. Trilobites do not have sutures in the thorax, meaning that the pleural and pygidial doublure (the parts of the thorax and pygidium that extend laterally onto the ventral side) would inhibit the ﬁrst of these scenarios. Some trilobites did move anteroventrally through the cephalon while exiting the old exoskeleton [16], but this would have required disarticula- tion of ventral sclerites (rostral plate, hypostome), and no convincing event involves backward movements within the sediment post-moulting. The direc- tion of exuvial movement in most trilo- bites is towards the anterior (Ref. [17], Fig. 3; Ref. [20], Figs. 2–6), and therefore a more convincing moult-carcass as- semblage would have the carcass pre- served entirely in front of the moult on a Figure 3. Trimerocephalus specimens from the Early Famennian (Late Devonian), Holy Cross Mountains, central Poland. A: line drawing produced from the CT scan of the Trimerocephalus chopini ‘moult’ ﬁgured by Bła_zejowski et al. [9]. B: Line drawing from the CT scan of the T. chopini ‘carcass’. Numbering in A and B indicates 10 thoracic segments in each, particularly visible when using the appended CT scan video provided by Bła_zejowski et al. [9]. C: Original lateral-view CT scan of the T. chopini specimen from Bła_zejowski et al. (Ref. [9], Fig. Specimen position, preservation and context suggest queueing trilobite carcasses 2B, cc-by licensed), illustrating the relative positioning of their putative ‘carcass’ and ‘moult’. Measures of the lateral and dorsal lengths of the cephala, and of the individual thoracic segment lengths show no appreciable difference in size between the two individuals. Scale bar 2 cm, as given in Bła_zejowski et al. [9]. D: In comparison to C, the positioning of the labelled ‘carcass’ and ‘moult’ suggested for a true moult-carcass assemblage [17]. E: One of several Trimerocephalus ‘queues’ of overlapping trilobite carcasses from the Holy Cross Mountains, adapted from Plate 2, Fig. 4 of Radwanski et al. [19]. The white box shows the contextual positioning of G. No scale bar provided in Radwanski et al. [19]. F: Dorsal-view photograph of the T. chopini suggested moult-carcass assemblage from Bła_zejowski et al. (Ref. [9], Fig. 2A, cc-by licensed). Scale bar 2 cm, as given in Bła_zejowski et al. [9]. G: Dorsal-view close-up of two overlapping Trimerocephalus carcasses, adapted from Radwanski et al. (Ref. [19], Plate 2, Fig. 4A and B) and shown in situ in E. Shown free of context in comparison to F, the two specimens appear extremely similar. No scale bar provided in Radwanski et al. [19]. Figure 3. Trimerocephalus specimens from the Early Famennian (Late Devonian), Holy Cross Mountains, central Poland. A: line drawing produced from the CT scan of the Trimerocephalus chopini ‘moult’ ﬁgured by Bła_zejowski et al. [9]. B: Line drawing from the CT scan of the T. chopini ‘carcass’. Numbering in A and B indicates 10 thoracic segments in each, particularly visible when using the appended CT scan video provided by Bła_zejowski et al. [9]. C: Original lateral-view CT scan of the T. chopini specimen from Bła_zejowski et al. (Ref. [9], Fig. 2B, cc-by licensed), illustrating the relative positioning of their putative ‘carcass’ and ‘moult’. Measures of the lateral and dorsal lengths of the cephala, and of the individual thoracic segment lengths show no appreciable difference in size between the two individuals. Scale bar 2 cm, as given in Bła_zejowski et al. [9]. D: In comparison to C, the positioning of the labelled ‘carcass’ and ‘moult’ suggested for a true moult-carcass assemblage [17]. E: One of several Trimerocephalus ‘queues’ of overlapping trilobite carcasses from the Holy Cross Mountains, adapted from Plate 2, Fig. 4 of Radwanski et al. [19]. The white box shows the contextual positioning of G. No scale bar provided in Radwanski et al. [19]. Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. Understanding moulting allows for the study of growth and development in trilobites Think again During development, juvenile arthro- pods grow in a stepwise pattern, with distinct instars separated by moulting events and accompanied by morpholog- ical change such as thoracic segment addition (anamorphic growth). Adult trilobites may have grown indetermi- nately, meaning they increased in size with each adult moult until death while maintaining the same morphology [22, 23]. Continued moulting during adulthood is suspected because of the presence of healed injuries in adult exoskeletons, a process that only takes place during moult growth events [24]. Examining moults therefore allows direct interpretation of growth and developmental patterns from the fossil record, and to date there have been numerous studies detailing variation in trilobite ontogeny (e.g. Refs. [25–28]). Bła_zejowski et al. [9] interpreted their putative moult-carcass assemblage as a growing juvenile, based on a suggested increase in thoracic segment number. However, we ﬁnd no clear evidence for the increased number of thoracic seg- ments, and suggest that the lack of overall increase in body size argues against this being a moult assemblage. Figure 4. A and B: Specimen of Olenoides serratus described as freshly moulted [33] (USNM 57685, part and counterpart). C: Freshly moulted specimen of Phacops rana (USNM 403875, described in Refs. [31, 32]). Both show wrinkling, ﬂattening, and low relief of features; these are considered characteristic of freshly moulted trilobites and led to the original publications describing them as such. C is in contrast to an adjacent, smaller specimen considered a normal intermoult trilobite because it is darker in colour, and shows no ﬂattening, wrinkling, or evidence of compression. Scale bars all 1 cm. Bła_zejowski et al. [9] argued that the ‘carcass’ part of their specimen displays one additional thoracic segment as compared to the supposed empty moult, and use this to support the idea of a moult-carcass assemblage. This sug- gested increase in segment number may actually reﬂect complications in preservation of the dorsal specimen because posterior segments are obscured by pygidium enrolment. Bła_zejowski et al. [9] did not publish their segment number observations, but we can distin- guish 10 thoracic segments in the CT- scan reconstructions of both individuals (Fig. 3A and B). This argues against the juvenile moult assemblage interpreta- tion. Furthermore, pygidium enrollment results from muscle contraction on death [19], and is a classic indicator of a pre- served carcass rather than a moult, becausecomplexmusculatureisrequired. nine trilobites do not show any clear evidence of moulting (e.g. Specimen position, preservation and context suggest queueing trilobite carcasses F: Dorsal-view photograph of the T. chopini suggested moult-carcass assemblage from Bła_zejowski et al. (Ref. [9], Fig. 2A, cc-by licensed). Scale bar 2 cm, as given in Bła_zejowski et al. [9]. G: Dorsal-view close-up of two overlapping Trimerocephalus carcasses, adapted from Radwanski et al. (Ref. [19], Plate 2, Fig. 4A and B) and shown in situ in E. Shown free of context in comparison to F, the two specimens appear extremely similar. No scale bar provided in Radwanski et al. [19]. hinging and displacement (Fig. 3C) than these deﬁnitive moult specimens, sug- gesting either very minor disruption during moulting or subsequent closure of the gape suture. Both scenarios would be unlikely if the animal had been Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. 984 H. B. Drage and A. C. Daley .....Insights & Perspectives Figure 4. A and B: Specimen of Olenoides serratus described as freshly moulted [33] (USNM 57685, part and counterpart). C: Freshly moulted specimen of Phacops rana (US 403875, described in Refs. [31, 32]). Both show wrinkling, ﬂattening, and low relief of features; these are considered characteristic of freshly moulted trilobites and led to the original publications describing them as such. C is in contrast to an adjacent, smaller specimen considered a normal intermoult trilobite because it is darker in colour, and sho no ﬂattening, wrinkling, or evidence of compression. Scale bars all 1 cm. Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. Understanding moulting allows for the study of growth and development in trilobites While there may have been a very brief period of time immediately after moulting when inﬂation of body size had not yet occurred to an observable extent, the exoskeleton would necessarily be rela- tively soft during this freshly moulted stage, but there is little evidence of this in the Bła_zejowski et al. [9] specimen, as discussed in detail in the next section. A freshly moulted Phacops trilobite specimen found immediately adjacent to its moulted exoskeleton (Ref. [30], Plate 1, Fig. 5) clearly has a larger body size and is in the softer, post-moult state, as evidenced by the ﬂattened and wrinkled preservation. This specimen suggests that an increase in body size occurs almost immediately after exiting the exoskeleton, and should be ob- served if the specimen in Bła_zejowski et al. [9] really were a moult assemblage. the individual sclerites of the ventral specimen are not signiﬁcantly larger than the dorsal specimen. Trilobites grew at each moulting event [22], par- ticularly when considered juvenile [29], as seen in the H. primitivus specimen ﬁgured by Dean (Ref. [21], Plate 5, Fig. 9) and suggested by Henningsmoen [16] to represent a larger carcass to the anterior of its moulted exoskeleton. While there may have been a very brief period of time immediately after moulting when inﬂation of body size had not yet occurred to an observable extent, the exoskeleton would necessarily be rela- tively soft during this freshly moulted stage, but there is little evidence of this in the Bła_zejowski et al. [9] specimen, as discussed in detail in the next section. A freshly moulted Phacops trilobite specimen found immediately adjacent to its moulted exoskeleton (Ref. [30], Plate 1, Fig. 5) clearly has a larger body size and is in the softer, post-moult state, as evidenced by the ﬂattened and wrinkled preservation. This specimen suggests that an increase in body size Understanding moulting allows for the study of growth and development in trilobites disarticulated cephala) or the presence of disarticu- lated exuvial debris. The specimens described by Bła_zejowski et al. [9] more likely represent two individual aligned carcasses preserved on top of each other and separated by sediment deposition, in the queued arrangement typical of this locality [19]. While this does not necessarily exclude the specimen of Bła_zejowski et al. [9] from also being a moult assemblage, it demands a clearer explanation of how this particular speci- men differs from the other completely preserved body clusters found there. In isolation, several queued carcasses from Radwanski et al. (Ref. [19], Plate 2) are indistinguishable (see our Fig. 3E and G) from the specimen described by Bła_zejowski et al. [9]. similar depositional plane (Fig. 3D). For example, Henningsmoen [16] identiﬁed a Hanchungolithus primitivus specimen (Ref. [21], Plate 5, Fig. 9) possibly preserved shortly after moulting in this speciﬁc conﬁguration (Fig. 3D) ‘again [indicating] forwards movement of the exuviating trilobite, since the larger specimen lies in front of the smaller’ (Ref. [16], p.188). The broader preserva- tional context of the locality must also be considered. Kowala Quarry preserves aligned (‘queued’) carcasses of Trimer- ocephalus (Ref. [19], Plate 1, Fig. 5C–D, Plate 2, Figs. 3A and B, 4A and B, 6C and D) overlapping in the same way as the specimen illustrated by Bła_zejowski et al. [9]. Radwanski et al. [19] described these very similar specimens as ‘live trilobites entombed while migrating’ (p. 460), and these queues of up to Our measurements also indicate that the overall body size and size of Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. 985 Insights & Perspectives..... H. B. Drage and A. C. Daley compression’ (Ref. [31], Fig. 3D, p. 91; reiterated in Ref. [32], Fig. 7E, p. 214). A single Olenoides serratus (Fig. 4A and B), in contrast to other individuals of the i h l li f i Figure 5. Percentage of sampled trilobite species from seven orders that show each of four different moulting behaviours (disarticulation of: free cheeks; cephalon; free cheeks þ remain- ing cephalon; thoracic segments). Data obtained from the descriptive literature (see [5] for methodology, data normalised to 100%). Figure 5. Percentage of sampled trilobite species from seven orders that show each of four different moulting behaviours (disarticulation of: free cheeks; cephalon; free cheeks þ remain- ing cephalon; thoracic segments). Understanding moulting allows for the study of growth and development in trilobites Data obtained from the descriptive literature (see [5] for methodology, data normalised to 100%). compression’ (Ref. [31], Fig. 3D, p. 91; reiterated in Ref. [32], Fig. 7E, p. 214). A single Olenoides serratus (Fig. 4A and B), in contrast to other individuals of the Figure 5. Percentage of sampled trilobite species from seven orders that show each of four different moulting behaviours (disarticulation of: free cheeks; cephalon; free cheeks þ remain- ing cephalon; thoracic segments). Data obtained from the descriptive literature (see [5] for methodology, data normalised to 100%). Think again Figure 5. Percentage of sampled trilobite species from seven orders that show each of four different moulting behaviours (disarticulation of: free cheeks; cephalon; free cheeks þ remain- ing cephalon; thoracic segments). Data obtained from the descriptive literature (see [5] for methodology, data normalised to 100%). compression’ (Ref. [31], Fig. 3D, p. 91; reiterated in Ref. [32], Fig. 7E, p. 214). A single Olenoides serratus (Fig. 4A and B), in contrast to other individuals of the species, has low relief, conspicuous wrinkling, poorly deﬁned structures, and soft-part preservation of appen- dages and the entire alimentary canal (including midgut), the latter indicating that it is a carcass [33]. Phacops rana milleri trilobites from the Middle Devo- nian Silica Shale of Ohio show the stages of post-ecdysial cuticle harden- ing, freshly moulted individuals being ‘pale, thin and wrinkled’ (Ref. [30], p. 33). Cuticle thickness was quantita- tively measured in thin section, and freshly moulted individuals had a thinner cuticle (25–135 mm) than inter- moult specimens (300–500 mm) [30]. All of these specimens show a charac- teristic wrinkling, ﬂattening and thin- ness in the exoskeletons of freshly moulted trilobites. Wrinkles may have resulted from incomplete decompres- sion of the new larger exoskeleton after exiting the old exoskeleton, and/or from deformation and compaction during burial in sediment, the latter of which also caused ﬂattening of the soft exoskeleton [30]. Because other preser- vational processes unrelated to moult- ing (e.g. tectonic deformation) could the individual sclerites of the ventral specimen are not signiﬁcantly larger than the dorsal specimen. Trilobites grew at each moulting event [22], par- ticularly when considered juvenile [29], as seen in the H. primitivus specimen ﬁgured by Dean (Ref. [21], Plate 5, Fig. 9) and suggested by Henningsmoen [16] to represent a larger carcass to the anterior of its moulted exoskeleton. Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. Preservation of freshly moulted trilobites is characterised by wrinkling and ﬂattening This is because in centipedes the cuticle undergoes extensive reab- sorption prior to moulting and the remaining moulted exuvia is almost always consumed by the individual, which greatly reduced the chance of exuviae being preserved in the fossil record [48]. Myriapods are appropriate modern analogues for studying general moulting mode, but because they are terrestrial rather than aquatic, they make poor analogues for understanding trilobite exuvial behaviour and moult preservational processes. The density difference between air and water pre- sumably leads to divergent exuvial procedures, requiring distinct move- ments for terrestrial compared to aquatic moulting. Marine isopod crus- taceans may be a more useful compara- tive group. These are extant arthropods that have a unique biphasic moulting method, in which the posterior part of the body is moulted ﬁrst, followed by the anterior sclerites a few days later [49–51]. Separate moulting of the body and head is reminiscent of moulting in trilobites, although in trilobites the cephalic sclerites are moulted ﬁrst and the remainder of the exoskeleton is shed after. Biphasic moulting in marine isopods inﬂuences preservation of these arthropods in the palaeontological re- cord, the majority of fossils being interpreted as moulted exuviae because they consist of posterior body regions only, without the head [50, 51]. Studying how marine isopod moulting affects their fossil preservation could therefore Think again Błazejowski et al. [9] used horseshoe crabs as a comparison for understand- ing the preservation and behaviour of moulting trilobites. The horseshoe crab (Fig. 1B) is one of the most renowned ‘living fossils’, generally considered to have a morphology that has changed little in over 400 million years. In reality, horseshoe crabs (xiphosurans) have undergone striking changes to their morphology during their long evolutionary history [38]. Horseshoe crabs may not be the most appropriate modern analogue for interpreting the trilobite fossil record of moulting. While trilobites and horseshoe crabs are both marine arthropods and some recent phylogenetic analyses ally them (and other Artiopoda, the extinct group containing trilobites) with the Chelicerata group to which horseshoe crabs belong [39], other analyses place trilobites with the Mandibulata, rather than the Cheli- cerata [1, 2, 40]. Further, xiphosurans possess an anterior exuvial gape that closes after egress, making it difﬁcult to distinguish moults from carcasses in the fossil record [5]. Preservation of freshly moulted trilobites is characterised by wrinkling and ﬂattening Unlike most trilobites, horseshoe crabs have an exuvial suture that splits open along the anterolateral margin of the head, through which the animal exits, but during which no element of the head is removed [41]. The resulting uncertain moult fossil record makes it difﬁcult to study evolu- tionary aspects of xiphosuran moulting behaviour. Horseshoe crabs also differ from trilobites in having determinate growth, meaning that they stop growing and moulting after reaching adulthood, whereas trilobites are suggested to have continued to moult and increase in size as adults [4, 22, 23, 42, 43]. Wrinkles in the anterior cephalon of the T. chopini ‘carcass’ specimen are taken by Bła_zejowski et al. (Ref. [9], p. 3) as evidence that the cuticle was soft as a result of recent moulting. However, these wrinkles are difﬁcult to discern in specimen images (Ref. [9], Fig. 2E), and could alternatively be cracks in the rock, particularly given that numerous cracks are visible elsewhere in the ‘carcass’ and ‘moult’ (Ref. [9], Fig. 2C– F), and in other trilobites from this locality [19, 35], because trilobite exo- skeletons are known to fracture during compression [30, 36]. Wrinkles alone would not be enough to identify a freshly moulted trilobite because all other documented examples (Fig. 4) show pronounced ﬂattening in addition to wrinkles (Ref. [30], Plate 1, Fig. 5; Ref. [31], Fig. 3D; Ref. [33], Plate 17, Fig. 3, Plate 19), owing to the greater compressibility of the softer, newly moulted exoskeleton. In contrast, the Bła_zejowski et al. [9] specimen shows very little difference in the height of the ‘carcass’ as compared to the ‘moult’ (Fig. 3A and B), and this lack of ﬂattening does not support the interpretation of a freshly moulted individual. Preservation of freshly moulted trilobites is characterised by wrinkling and ﬂattening Several trilobite carcasses (Fig. 4) have been described as preserved very shortly after a moulting event in a soft-shelled state before the new exoskeleton min- eralised and hardened [30–33]. In this duration of time, arthropods are vulnerable to predation. Individuals of Phacops rana from the same beds as trilobite exuviae clusters at the Penn Dixie shale pit, New York (Fig. 4C), ‘exhibit a distinctly thinner, pale brown to grey cuticle, which may be ﬂattened and slightly wrinkled’ (Ref. [31], p. 96) and are found directly associated with specimens showing ‘little or no 986 Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. .....Insights & Perspectives H. B. Drage and A. C. Daley allowed for the analysis of brain mor- phology in a 518 million year-old arthropod, Fuxianhuia protensa from the Chengjiang Biota, by comparison with the neuroanatomy of modern hermit crabs [37]. Such approaches are particularly important when studying extinct animals with no direct modern relatives, as long as the modern ana- logue is appropriate in afﬁnity and/or mode of life. have caused wrinkling and/or ﬂattening in whole assemblages of trilobite exo- skeletons [34], trilobites with ﬂattened and wrinkled exoskeletons need also to be found in close proximity to trilobites with unwrinkled, high-relief, fully min- eralised exoskeletons before they can be soundly interpreted as freshly moulted individuals. Size frequency distribu- tions of the assemblages should also be examined to determine if differences in relief could be the result of varying preservation mechanics of differently sized trilobites. Crustacea (crabs, lobsters, shrimp, etc.) and Hexapoda (insects). Many myriapods and crustaceans show con- tinuous growth and moulting during adulthood [4, 44], as suggested for trilobites [22, 23, 42, 43]. In myriapods (Fig. 1D), moulting often proceeds by a transverse dorsal suture opening in the head to create the exuvial gape, through which the animal egresses anteri- orly [45, 46]. Other myriapods moult by disarticulating the entire head from the remainder of the body. The moulting behaviour and similar repeated nature of the trunk segments is therefore comparable to that of trilobites. The myriapod fossil record largely consists of millipedes, which have a robust cuticle reinforced with calcium carbon- ate, although most fossils are inter- preted as carcasses rather than moults [47, 48]. Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. Why study moulting in trilobites? Think again Bła_zejowski et al. [9] compared their proposed trilobite moult assemblage to the fossil horseshoe crab Crenatoli- mulus from the Late Jurassic Owadow- Brzezinki Quarry in Central Poland, to examine both preservation and behav- iour. The proposed wrinkles on the exoskeletons of both the horseshoe crab and the trilobite are taken as evidence for these being freshly moulted individ- uals that have not yet undergone post- moulting ‘decompression’ (Ref. [9], p. 3). This is in contrast to the well- established interpretation of wrinkles in otherwise complete horseshoe crab fossils as taphonomic artefacts resulting from sediment compaction and ﬂatten- ing of high relief structures [34, 41, 52]. Bła_zejowski et al. [9] imply that the horseshoe crab specimens, and by extension the T. chopini trilobite speci- men, are too three-dimensional for the wrinkling seen on their exoskeletons to be from taphonomic processes. How- ever, it has been shown that even small amounts of compression can cause taphonomic wrinkles in horseshoe crab specimens that retain high relief [34, 52]. The absence of pronounced ﬂatten- ing in these specimens suggests that these specimens had fully hardened exoskeletons at the time of preserva- tion, and does not support the claim of Bła_zejowski et al. [9] that they were freshly moulted individuals. The enormous wealth of trilobite moult fossils provides a unique opportunity to explore the development and behaviour of an ecologically important group during the early history of animal evolution. Each individual study on trilobite moulting feeds into a larger aggregation of occurrences and behav- ioural descriptions that can be analysed to interpret evolutionary trends for the group. This is why specimens such as that described by Bła_zejowski et al. [9] must be examined with a critical eye, in order to determine how appropriate they are for inclusion in future studies that examine broader trends in trilobite moulting. While most published occur- rencesoftrilobitemoultingaredescriptive in nature, rare quantitative studies have given us a glimpse of the huge potential residing in this growing database of behavioural information. Brandt [15] contrasted trilobite moulting – which is actually highly variable even within a single species (e.g. Ref. [66]) and lacks reabsorption of the mineralised exoskel- eton before moulting – with the canal- ised habit and efﬁcient reabsorption seen in modern arthropods. Why study moulting in trilobites? Preliminary survivorship analyses suggested that the ultimate extinction of trilobites may have been related to the cumulative effects of a variable and metabolically expensive moulting behaviour, reveal- ing the evolutionary importance of ecdysialefﬁciency fortaxonomiclongev- ity [15], though the extent of this inﬂuence has been debated (see Ref. [67]). Our own recent analyses of published occurrences quantitatively showed that trilobites exhibit a high levelof variabilityin moulting behaviour in all orders and throughout their evolutionary history (Fig. 5, Ref. [5]). These proof-of-concept studies show that a huge breadth of information on early arthropod development remains to be extracted and interpreted from the moult fossil record. Gregarious behaviour in trilobites has been documented in many assemb- lages [19, 60–63] for protection [14], moulting [31], burrowing [60], feed- ing [64] and reproduction [65]. Unlike in modern arthropods, where behaviour can be observed directly, trilobite fossil aggregations must be considered in context to try to assess whether they are the result of gregarious behaviour or abiotic accumulations. Currents, sedi- ment reworking, gravity and other transportation mechanisms could act to accumulate numerous individuals in one area after death, leading to a time- averaging effect where individuals that were separated in space and time during life are combined and preserved to- gether after death. Detailed analyses of the taphonomy, sedimentology and palaeoenvironment of the locality and size-frequency distribution of the popu- lation have been successful in separat- ing abiotic trilobite accumulations from actual examples of gregarious behav- iour [31, 62, 65]. Bła_zejowski et al. [9] described their Devonian T. chopini trilobite assemblage as preserved in close association with many juvenile individuals, and suggest that they were ‘growing in a nursery ground’ (Ref. [9], p. 4). Prior descriptions of synchronised moulting-mating behaviour in trilobites and migration of the offspring to nursery grounds were based on ﬁrmly documented evidence of moulting, in combination with quantitative analyses of the environmental context and pop- ulation structure [31, 65]. These data were not described for the Trimeroce- phalus queues at the Kowala Quarry locality [9], but if documented, could allow further inferences about trilobite gregarious behaviour to be made. Modern analogues can be used to understand trilobite moulting One of the better ways to understand the anatomy, behaviour and development of extinct animals is to compare them to modern animals with similar afﬁnity, mode of life or environmental prefer- ences. The use of modern analogues has shed light on a diverse set of enigmatic palaeontological evidence. For exam- ple, a comparative anatomy approach Given that trilobites have been considered closely related to Mandibu- lata, modern analogues may be more appropriate if drawn from within this clade, which is comprised of the Myr- iapoda (centipedes and millipedes), 987 Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. Insights & Perspectives..... Insights & Perspectives..... H. B. Drage and A. C. Daley H. B. Drage and A. C. Daley visual communication, as seen in krill [13, 57]. Mass moulting reduces preda- tion pressure on individuals during this vulnerable stage of their development [58], but the beneﬁt is balanced by higher disease transmission rates within the population [13, 59]. Synchronised moulting events are well established in modern arthropods, but identifying this behaviour in the fossil record is more difﬁcult. Trilobites are one of the only fossil arthropod groups found in large enough numbers that we can access group behavioural information [19, 60–65] and examine synchronised moulting events in deep time. provide insight into the taphonomy and behaviour of moulting in the extinct trilobites. Mass moulting occurs in modern arthropods and trilobites One of the most signiﬁcant social behaviours linked with moulting is the occurrence of synchronised moulting events, or ‘mass moulting’, involving a large number of individuals [31, 53–57]. In modern arthropods, mass moulting events are sometimes triggered by external abiotic cues, as is seen in the lunar-rhythmic moulting of the crayﬁsh Astacus [53]. Direct communication between individuals can also trigger synchronised moulting, either through pheromones, as seen in some colonial spiders and Hexapoda such as beetles and springtails [54–56], or through References 21. Dean TW. 1966. 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Quanti- tative studies on moulting in the fossil record should be further expanded, based upon their demonstrated poten- tial to reveal information on the life histories and behaviour of extinct animals. Understanding what the fossil record can tell us about moulting and growth provides a unique deep-time insight into the evolution and develop- ment of Arthropoda and other members of Ecdysozoa. 6. Cheong SP, Huang J, Bendena WG, Tobe SS, et al. 2015. Evolution of ecdysis and metamorphosis in arthropods: the rise of regulation of juvenile hormone. Integr Comp Biol 55: 878–90. 26. Conclusions and outlook Bła_zejowski et al. [9] described a speci- men of T. chopini, from the Upper Devonian (365 million years ago) 988 Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. .....Insights & Perspectives H. B. Drage and A. C. Daley H. B. Drage and A. C. 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Bioessays 38: 981–990, 2016 The Authors. Bioessays published by WILEY Periodicals, Inc. Acknowledgements New York: McGraw-Hill. p 486. 54. Baker JE. 1977. Synchronized pupation in starved and fed larvae of the black carpet beetle. Ann Entomol Soc Am 70: 299–302. 64. Fortey RA, Owens RM. 1999. Feeding habits in trilobites. Palaeontol 42: 429–65. 43. Bright RC. 1959. A paleoecologic and biomet- ric study of the middle Cambrian trilobite Elrathia kingii (Meek). J Paleontol 33: 83–98. 55. Leinaas HP. 1983. Synchronized moulting controlled by communication in group-living Collembola. Science 291: 193–5. 65. Paterson JR, Jago JB, Brock GA, Gehling JG. 2007. Taphonomy and palaeoecology of the emuellid trilobite Balcoracania dailyi (early Cambrian, South Australia). Palaeogeogr Palaeocl 249: 302–21. 44. Enghoff H, Dohle W, Blower JG. 1993. Anamorphosis in millipedes (Diplopoda) the present state of knowledge with some developmental and phylogenetic consider- ations. 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https://openalex.org/W3049036569	https://pubs.rsc.org/en/content/articlepdf/2020/ra/d0ra05701k	English	null	Preparation and photophysical properties of quinazoline-based fluorophores	RSC advances	2,020	cc-by	5,003	Department of Chemistry, Zhejiang University, Hangzhou 310027, P. R. China. E-mail: pinglu@zju.edu.cn; orgwyg@zju.edu.cn † Electronic supplementary information (ESI) available: Structural characterization data, TGA, crystallographic data, photophysical spectra, DFT, CV. CCDC 1961660, 1961661, 1961663 and 1961672. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0ra05701k RSC Advances View Article Online View Journal \| View Issue Open Access Article. Published on 17 August 2020. Downloaded on 10/24/2024 6:16:21 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 17 August 2020. Downloaded on 10/24/2024 6: This article is licensed under a Creative Commons Attribution 3.0 Un The donor–acceptor design is a classic method of synthesizing new ﬂuorescent molecules. In this study, a series of new ﬂuorescent compounds (1–10) were synthesized based on 2-(3,5-bis(triﬂuoromethyl) phenyl)-quinazoline acceptor and various amino donors. The ﬂuorescent emissions of 1–10 cover the spectrum from 414 nm to 597 nm in cyclohexane solutions with various amino donors on 4- or 7- positions of quinazoline. Ultimately, compounds 1 and 2 presented the highest photoluminescence quantum yield (QY) over 80%, while compound 10 provided the largest Stokes shift (161 nm) in cyclohexane. Most of them have strong emissions in aggregated states such as in nanoparticles, in powders, in crystals and in ﬁlms. Mechanochromic properties were observed for compounds 1, 2, 4 and 7. Furthermore, blue OLEDs were fabricated by using compound 2 or 7 as the active layer. Received 30th June 2020 Accepted 7th August 2020 DOI: 10.1039/d0ra05701k rsc.li/rsc-advances Received 30th June 2020 Accepted 7th August 2020 Received 30th June 2020 Accepted 7th August 2020 relationship (FSPR)18,19 studies which are instructive in the development of new uorophores with a unique application. Preparation and photophysical properties of quinazoline-based ﬂuorophores† Zhichao Wang, Hanjie Li, Zhixing Peng, Zaibin Wang, Yanguang Wang * * Introduction Quinazoline is one of the most attractive heterocycles in alka- loids. Their derivatives are abundant in natural products and show a broad spectrum of bioactivities in living organisms.1–5 For instance, some quinazoline-based compounds exhibited eﬀective and selective CYP1A2 inhibition and might function in cancer chemoprevention.6 CID 9998128 (2,4-disubstituted qui- nazoline) has been revealed as a good candidate for Alzheimer's disease (AD) treatment.7 Besides the unique attributes of qui- nazolines in medicinal chemistry, a series of uorescent qui- nazolines have also been designed and synthesized for optoelectronic devices8–10 and uorescent chemosensors.11–14 Recently, quinazoline was successfully functioned as an anchor to prevent the stacking between uorophores and turned aggregation-caused quenching (ACQ) to aggregation-enhanced emission (AEE).15 Covalently bonded with coumarin, the coumarin–quinazoline dyad displayed solvatochromic and ACQ properties. Taking advantage of this disaggregation-induced emission (DIE), a turn-on uorescent probe was designed and was capable of probing parallel G4 DNA topologies over other G4 DNA structures.16 However, in contrast to frequently used electron-decient benzothiadiazole (BTD),17 the uorescent compounds based on quinazolines largely remained immature. The same goes for the uorescent structure–property The multicomponent reaction provided a new avenue in the construction of quinazolines with various substituents in a single step and made the investigation and establishment of FSPR practical and possible.20 For instance, assisted by CuCl2, 2,4-diarylquinazolines could be eﬀectively prepared from 2- methylquinoline, 2-aminobenzophenome, ammonia acetate and oxygen in a single step.21 2,3-Dihydroquinazolin-4(1H)-ones could be eﬃciently prepared by three component reaction from isatoic anhydride, aniline and arylaldehyde in the presence of Al2(SO4)3 with a variety of functional groups.22 In the presence of Cu(OTf)2, a [2 + 2 + 2] cascade annulation of diaryliodonium salts with two nitriles produced 2,4-disubstituted quinazolines in excellent yields.23 Mediated by 4-hydroxy-TEMPO radical24 or iodine,25 2-aryl-quinazolines could be obtained from arylme- thanamines, 2-aminobenzoketones and oxidant. Here, we report the preparation procedure for 2,4-diarylquinazolines and 2,4,7-triarylquinazolines and their uorescent properties as well as their applications in OLED. Open Access Article. Published on 17 August 2020. Downloaded on 10/24/2024 6:16:21 This article is licensed under a Creative Commons Attribution 3.0 Unported Compounds 1–10 are highly uorescent in dilute solutions. Compound 1 emits light at 414 nm with Stokes shiof 45 nm (2982 cm1) and quantum yield of 84.67%. As the amino group Scheme 1 Key intermediates A, B and compounds 1–10. Fig. 1 (a) Absorptions and normalized emissions of 1–5 in cyclohex- anes; (b) absorptions and normalized emissions of 6–10 in cyclohexanes. or B with various secondary aromatic amines aﬀorded target molecules 1–10 in yields varying from 84% to 99%. Structures of these synthesized compounds (A, B, 1–10) were conrmed by 1H NMR, 13C NMR and 19F NMR (Fig. S2–S37†). Further conr- mation was made by HRMS and IR as well as single crystal analysis of compounds 1–4 (Table S1†). These compounds are thermally stable (Fig. S38† and Table 1). The decomposition temperatures were determined to be 369 C, 380 C, 382 C, 363 C, 383 C, 377 C, 342 C, 379 C, 351 C and 393 C for compounds 1–10, respectively. Absorption and emission of 1–10 in cyclohexanes All the absorption and emission spectra were taken in a concentration of 2 105 M in order to avoid the intermo- lecular interactions between solutes (Fig. S39–S40 and Table S2†). Compound 1 absorbs lights at 338 nm and 369 nm with molar absorptivities of 9200 and 9550 L mol1 cm1 in Fig. 1 (a) Absorptions and normalized emissions of 1–5 in cyclohex- anes; (b) absorptions and normalized emissions of 6–10 in cyclohexanes. Table 1 Photophysical property of 1–10 in cyclohexanes and thermal stability of 1–10 Compounds Absorptiona Emission Td f (C) lmax (nm) 3 (L mol1 cm1) lmax a,b (nm) Stokes (nm cm1) Life timec (ns) QYd (%) kr e (107 s1) knr e (107 s1) 1 338, 369 9200, 9550 414 45/2982 3.37 84.67 25.1 4.5 369 2 403 18 050 450 47/2592 3.96 87.59 22.1 3.1 380 3 415 13 900 500 85/4079 6.03 22.75 3.8 12.8 382 4 420 1150 514 94/4354 1.32(40.4%) 21.94 2.9 10.2 363 9.71(59.6%) 5 444 7500 575 131/5152 4.1 7.35 1.8 22.6 383 6 378, 397 5200, 5550 412 15/930 3.29 18.80 5.7 24.5 377 7 400 6550 458 58/3153 5.81 43.32 7.5 9.8 342 8 411 6100 519 108/5063 8.36 19.30 2.3 9.7 379 9 456 750 533 77/3168 1.18(52.9%) 16.26 2.7 14 351 9.06(47.1%) 10 436 4550 597 161/6185 4.31 17.57 4.1 19.1 393 a Measured in cyclohexane at 2 105 M. b Excited at 320 nm (4 and 9), 445 nm (5) and 365 nm (1–3, 6–8, and 10). c According to uorescence decay traces of compounds in cyclohexane (5, 10) and toluene (1–4 and 6–9) at 2 105 M. d Quantum yields were obtained from an integrating sphere. e Calculated by QY ¼ skr ¼ kr/(kr + knr). f Td was determined at 5% weight loss. Table 1 Photophysical property of 1–10 in cyclohexanes and thermal stability of 1–10 a Measured in cyclohexane at 2 105 M. b Excited at 320 nm (4 and 9), 445 nm (5) and 365 nm (1–3, 6–8, and 10). c According to uorescence decay traces of compounds in cyclohexane (5, 10) and toluene (1–4 and 6–9) at 2 105 M. d Quantum yields were obtained from an integrating sphere. e Calculated by QY ¼ skr ¼ kr/(kr + knr). f Td was determined at 5% weight loss. a Measured in cyclohexane at 2 105 M. b Excited at 320 nm (4 and 9), 445 nm (5) and 365 nm (1–3, 6–8, and 10). c According to uorescence decay traces of compounds in cyclohexane (5, 10) and toluene (1–4 and 6–9) at 2 105 M. d Quantum yields were obtained from an integrating sphere. e Calculated by QY ¼ skr ¼ kr/(kr + knr). f Td was determined at 5% weight loss. Synthesis of compounds 1–10 The preparatory routes leading to compounds 1–10 were shown in Fig. S1† and structures of intermediates A and B as well as the structures of compounds 1–10 were presented in Scheme 1. Key intermediates A and B were synthesized according to the liter- ature reporting palladium-catalyzed three components reaction starting from arylboronic acids, cyanoanilines and arylalde- hydes.26 In this way, A and B were prepared in yields of 60% and 70%, respectively. Subsequent Buchwald–Hartwig coupling of A RSC Adv., 2020, 10, 30297–30303 \| 30297 This journal is © The Royal Society of Chemistry 2020 View Article Online View Article Online RSC Advances RSC Advances Paper Scheme 1 Key intermediates A, B and compounds 1–10. RSC Advances cyclohexane (Table 1). Red-shied absorption was observed when carbazole (CZ) was changed into diphenylamino (PA), di(4-methoxyphenyl)amino (MPA), phenoxazinyl (POZ) and di(4-(N,N-dimethylamino)phenyl)amino (DMPA). Thus, compounds 2, 3, 4 and 5 absorb lights in dilute cyclohexanes at 403, 415, 420 and 444 nm with molar absorptivities of 18 050, 13 900, 1150 and 7500 L mol1 cm1, respectively (Fig. 1a). Similar results were found for the series of compounds 6–10 when these amino groups occupied on the 7-position of qui- nazoline (Fig. 1b). It indicates that DMPA is the strongest electron-donating group among these secondary aromatic amines. Emission of 1–10 in aggregates Nanoparticles were prepared by quickly pouring water into their dilute THF solutions and their emission spectra were recorded accordingly (Fig. S42 and S43†). The shape and size were pho- tographed by TEM (Fig. S44†) and recorded by DLS as well. Emissions of aggregates could be classied into two categories in general. For those compounds with strong uorescence in dilute solutions, such as compounds 1, 2, 6 and 7, aggregation- causing quenching (ACQ) was rstly observed and followed by aggregation-induced emission enhancement (AIEE) as the water fraction increases. As the water fraction increase to 60%, the emission of compound 2 red-shied to 564 nm and the emis- sion intensity decreased to 5.1% of its original value (Fig. 3a). ACQ was observed in this stage. Further increment of water fraction to 95%, a steady blue-shiemission as well as the increment of the emission intensity was observed. Nano- particles formed in ball shape with the diameter about 100 nm, determined by DLS (Fig. 3b) and conrmed by TEM (Fig. 3c). A similar situation was observed for compounds 1, 6 and 7. To those compounds with weak or non-uorescent in dilute THF solutions (3–5, 8–10), increment of water fraction induced emission enhancement signicantly. Compound 3 presented the typical aggregation-induced emission (AIE).30 As the water fraction increased to 60%, the uorescence of compound 3 was turned on. Beyond this point, a steady increment of emission intensity was recorded. As the water fraction reached 90%, the emission intensity was magnied 21.6 times of its initial. For compound 9, lifetimes of 1.18 ns (52.9%) and 9.06 ns (47.1%) were recorded and the average lifetime was calculated to be 5.98 ns. That both 4 and 9 exhibited emissions with bi- components might be the respective decays from S1 and T1 due to the phenoxazine-caused steric repulsion between hydrogens at donor–acceptor linkage and the resulted large dihedral angle between donor and acceptor.28 Tuning the donor on the 4-position of quinazoline aﬀected the absorption spectra to a greater extent, while the larger Stokes shis were observed for those compounds with the donor on 7-position except for compounds 6 and 9. Finally, compound 10 provided the largest Stokes shiwith a value of 161 nm in cyclohexane. Absorption and emission of 1–10 in various solvents These compounds are soluble in normal organic solvents, such as cyclohexane (CH), toluene (TOL), dioxane (DIO), tetrahydro- furan (THF), dichloromethane (DCM) and acetonitrile (MeCN). Blue-shied absorption and red-shied emission of these D–A compounds were observed as the solvent polarity increased from cyclohexane to toluene, dioxane, tetrahydrofuran, dichloro- methane and acetonitrile (Fig. S39, S40 and Table S2†). Mean- while, rainbow emissions were recorded and could be clearly photographed under UV light as the solvent polarity changes. As a typical example, compound 1 absorbs light at 338/369, 340/363, 339/358, 339/354, 340/354 and 339 nm (Fig. 2a), while emits light at 414, 453, 461, 495, 501 and 548 nm in CH, TOL, DIO, THF, DCM and MeCN, respectively (Fig. 2b). The absorption at the lower energy gap of 1 disappeared as the solvent changed to MeCN. Finally, the largest Stokes shi(209 nm) was found for 1 in MeCN.29 As the solvent polarity increases, the HOMO/LUMO levels of these D–A compounds decrease through the salvation of compounds both in ground states and in excited states. However, in a more polar solvent, a larger energy gap was found for D–A compounds in ground state because of the more stabi- lization of the HOMO level, while a smaller energy gap for D–A compounds in excited states was observed because of the more stabilization of the LUMO level. Thus, blue-shied absorption Absorption and emission of 1–10 in cyclohexanes 30298 \| RSC Adv., 2020, 10, 30297–30303 This journal is © The Royal Society of Chemistry 2020 Paper Fig. 2 (a) Absorptions and (b) rainbow emissions of 1 in various solvents. changed to better electron-donating groups, compounds 2, 3, 4 and 5 emit lights at 450, 500, 514 and 575 nm with Stokes shis of 47 (2592), 85 (4079), 94 (4354) and 131 nm (5152 cm1), respectively (Fig. 1a). The highest emission quantum yield (87.59%) was found for compound 2 with MPA occupied on the 4-position of quinazoline. Similar results were observed for compounds 6–10. These compounds emit lights at 412, 458, 519, 533 and 597 nm with Stokes shis of 15 (930), 58 (3153), 108 (5063), 77 (3168) and 161 nm (6185 cm1), respectively (Fig. 1b). Among this series, compound 7 presented the highest emission quantum yield (43.32%). To both series, emission spectra t the single exponential function with uorescence lifetime varying from 3.29 to 8.36 ns except for compounds 4 and 9 with POZ substituted (Fig. S41†). The decay trace is bi- exponential with lifetimes of 1.32 ns (40.4%) and 9.71 ns (59.6%) for the emission band at 514 nm of compound 4. The average lifetime was calculated to be 7.62 ns. The average life is calculated using the literature method.27 Open Access Article. Published on 17 August 2020. Downloaded on 10/24/2024 6:16:21 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 (a) Absorptions and (b) rainbow emissions of 1 in various solvents. and red-shied emission were presented for these D–A compounds as the solvent polarity increases. This journal is © The Royal Society of Chemistry 2020 Emission of 1–10 in powders Published on 17 August 2020. Downloaded on 10/24/2024 6:16:21 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. position of quinazoline changes from CZ to PA, MPA, and POZ, the maximum emission wavelengths in powders were deter- mined to be 450, 495, 558 and 539 nm, respectively (Fig. 4a). When DMPA occupied on the 4-position of quinazoline, the maximum emission wavelength of 5 in powder was determined to be 467 nm with a shoulder peak (620 nm) at a larger energy gap. A colourful solid emission was photographed under UV light, altering the colour from bright blue to green, orange, yellow and nally to red, as the secondary aromatic amine changed on the 4-position of quinazoline (Fig. 4c). Emission quantum yields were determined to be 56.55%, 49.91%, 28.03%, 27.51% and 3.08% with the lifetimes of 4.34, 2.86, 5.44, 15.04 and 2.45 ns for compounds 1–5, respectively. For the series of electron-donating compounds on the 7-position of quinazoline, compounds 6–9 exhibited similar emissions with the colour changed from bright blue to red, but with relatively lower quantum yields, accordingly (Fig. 4b and c). As for compound 10, the emission presented weak and cannot be detected. Emission of 1–10 in lm The transparent lms of these compounds were fabricated by dipping their dilute methylene chloride solutions onto quartz plates and let it evaporate to dry aerwards. For the series of amines occupied on the 4-position of quinazoline, emissions of compounds 1–5 in lms were red-shied in contrast to emis- sions of these compounds in powders, while quantum yields decreased accordingly too (Table 2). Film emission of compound 4 was bi-exponential with the combination of 0.48 ns (60.8%) and 3.94 ns (39.2%). The average lifetime was calcu- lated to be 2.62 ns. Film of compound 5 presented weak uo- rescence and the quantum yield was not detectable. For the series of the substituent on 7-position of quinazoline, lms of compounds 6 and 7 exhibited larger quantum yields with respect to those in powders. Film emission of compound 9 also showed bi-exponential with the combination of 0.65 ns (6.5%) and 5.28 ns (93.5%). The average lifetime was calculated to be 4.50 ns. Emission of 1–10 in powders These compounds are also strongly uorescent in powders (Fig. 4 and Table 2). As the electron-donating group on 4- Fig. 3 (a) 3D emissions of 2, (b) DLS (fw ¼ 90%, 95%) of 2 and (c) TEM images of 2 (95%). Fig. 3 (a) 3D emissions of 2, (b) DLS (fw ¼ 90%, 95%) of 2 and (c) TEM images of 2 (95%). RSC Adv., 2020, 10, 30297–30303 \| 30299 This journal is © The Royal Society of Chemistry 2020 View Article Online RSC Advances Fig. 4 (a) Powder emission spectra of 1–5; (b) powder emission spectra of 6–9; (c) colorful powders of 1–10 under UV light. Compounds Emission in powder Absorption in lm lmax e (nm) Emission in lm Emission in crystalline lmax f (nm) lmax a (nm) Life timeb (ns) QYc (%) kr d (107 s1) knr d (107 s1) lmax f (nm) Life timeb (ns) QYc (%) kr d (107 s1) knr d (107 s1) 1 450 4.34 56.55 13.0 10.0 364 467 5.04 31.55 6.3 13.6 468 2 495 2.86 49.91 17.5 17.5 410 509 3.00 26.06 8.7 24.6 491 3 559 5.44 28.03 5.2 13.2 429 555 4.84 25.99 5.4 15.3 539 4 540 15.04 27.51 1.8 4.8 412 506 0.48(60.8%) 20.86 8.0 30.2 532 3.94(39.2%) 5 617 2.45 3.08 1.3 39.6 413 —g —g —g — g —g —h 6 460 2.34 15.21 6.5 36.2 388 467 1.31 32.84 25.1 51.3 —h 7 488 4.88 31.70 6.5 14.0 416 536 3.34 35.70 10.7 19.3 —h 8 558 7.61 17.92 2.4 10.8 430 543 7.4 11.35 1.5 12.0 —h 9 568 9.58 11.33 1.2 9.3 455 540 0.65(6.5%) 17.96 4.0 18.2 —h 5.28(93.5%) 10 —g —g —g —g —g —g —g —g —g —g —g —h a Excited at 380 nm (9), 400 nm (5) and 365 nm (1–4, 6–8 and 10). b According to uorescence decay traces. c Quantum yields were obtained from an integrating sphere. d Calculated by QY ¼ skr ¼ kr/(kr + knr). e Films were prepared by spin-coating a dilute DCM solution on a quartz plate. f Excited at 365 nm. g Emission is too weak to be detected. h No single crystal was obtained. This journal is © The Royal Society of Chemistr Fig. 4 (a) Powder emission spectra of 1–5; (b) powder emission spectra of 6–9; (c) colorful powders of 1–10 under UV light. Open Access Article. Mechanochromic properties of 2 and 4 Some of them showed mechanochromic property, such as compounds 1, 2, 4 and 7 (Fig. 6 and S45†). While grinding the crystalline of compound 2, the maximum emission wavelength changed from 494 nm to 505 nm and emission colour under UV light changed from blue to green (Fig. 6a). Aer methylene chloride fumigation, maximum emission wavelength changed back to 492 nm while the colour was turning back to blue (Fig. 6a). The cycle of blue and green was reproducible without the loss of the emission intensity (Fig. 6b). SAXS analysis indi- cated that the phase alternation existed during the grinding and fumigation process (Fig. S46†). Grinding crystalline of compound 4 altered the maximum emission wavelength from Fig. 5 Single crystal structures of 1–4: (a) monomeric structure; (b) dimeric structures; (c) tetrameric structures and (d) CLSM images under 405 nm laser of 1–4. As shown in Fig. 5, the dimers of 1, 4 are stacked together in an anti-parallel manner that might be the nature of the planarity of carbazole in 1 and phenoxazine in 4. Intermolecular p–p stacking between carbazole and quinazoline in molecule 1 was observed at a distance of 3.690 A. A shorter distance (3.545 A) in 4 was measured between phenoxazine and quinazoline which might be the dislocated alignment arising from the larger dihedral angle (72.71) between phenylene and phenoxazine in comparison with that in 1 between phenylene and carbazole (40.84). Finally, multiple C–H/p and C–H/F interactions hold molecules together. The stacking of molecule 4 was addi- tionally assisted by the methylene chloride solvent in which C– H/Cl interactions were seen. Single crystal 1 emits light at 468 nm which is similar to its lm emission, while single crystal Fig. 7 (a) HOMO/LUMO orbitals calculated by Gaussian (DFT using the B3LYP functional and the 6-31G(d) basis set). (b) Energy gaps (HOMO/ LUMO) from theoretical calculation, UV observation and CV versus compounds 1–10. Fig. 6 Mechanochromic property of compounds 2 and 4; (a) normalized emission spectra; (b) emission cycles. Fig. 7 (a) HOMO/LUMO orbitals calculated by Gaussian (DFT using the B3LYP functional and the 6-31G(d) basis set). (b) Energy gaps (HOMO/ LUMO) from theoretical calculation, UV observation and CV versus compounds 1–10. Fig. 7 (a) HOMO/LUMO orbitals calculated by Gaussian (DFT using the B3LYP functional and the 6-31G(d) basis set). (b) Energy gaps (HOMO/ LUMO) from theoretical calculation, UV observation and CV versus compounds 1–10. Fig. Emission of 1–4 in crystalline Four single crystals (1–4) were obtained by slow evaporation of the dilute solutions of dichloromethane and hexane. The single crystal data were shown in Table S1.† Four single crystals are all granular, of which compound 1, 3 and 4 are clusters of small particles, and compound 2 are large regular crystals. 30300 \| RSC Adv., 2020, 10, 30297–30303 RSC Advances View Article Online RSC Advances View Article Online Paper 4 emits light at 532 nm which is red-shied in comparison with its lm emission in 506 nm. Fig. 5 Single crystal structures of 1–4: (a) monomeric structure; (b) dimeric structures; (c) tetrameric structures and (d) CLSM images under 405 nm laser of 1–4. Because of the propeller structure of triarylamine, intermo- lecular p–p stacking between donor and acceptor were not seen in the cases of 2 and 3. The main interactions are intermolec- ular C–H/p and C–H/F interactions. Thus, both of them showed blue-shied emissions in comparison to their lm emissions. Single crystal 2 emits light at 491 nm while single crystal 3 emits light at 539 nm (Table 2). Open Access Article. Published on 17 August 2020. Downloaded on 10/24/2024 6:16:21 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Acknowledgements The authors gratefully acknowledge the nancial support from the National Natural Science Foundation of China (21871229). RSC Advances 533 nm to 564 nm with the colour alternation from yellow to brown (Fig. 6a). The colour could be tuned back to yellow by fuming the sample with DCM vapour (Fig. 6b). Open Access Article. Published on 17 August 2020. Downloaded on 10/24/2024 6:16:2 This article is licensed under a Creative Commons Attribution 3.0 Unpor 3 C. Wattanapiromsakul, P. I. Forster and P. G. Waterman, Phytochemistry, 2003, 64, 609–615. 4 V. Alagarsamy, K. Chitra, G. Saravanan, V. R. Solomon, M. T. Sulthana and B. 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The devices were structured of ITO/PEDOT: PSS (40 nm)/ compound (65 nm)/TBPI (40 nm)/LiF (1.5 nm)/Al (50 nm) and the device performances were listed in Table S4.† When the pure compound 2 was sandwiched in a thickness of 65 nm, device A emitted light at 509 nm with the EQE of 0.47% and the turn-on voltage of 4.5 V. By doping compound 2 in PVK (2 : PVK ¼ 1 : 4) as emitter, device B emitted light at 513 nm. Increased EQE (1.09%) and decreased turn-on voltage (4.2 V) were recorded. As the ratio of 2 : PVK changed from 1 : 4 to 1 : 8 (device C), the EQE value was further increased to 1.35%. Device D, fabricated with the emitter of 7 in PVK (7 : PVK ¼ 1 : 4) emitted light at 510 nm with the EQE of 0.83% and the turn-on voltage of 4.8 V. 7 N. Q. Thai, Z. Bednarikova, M. Gancar, H. Q. Linh, C. K. Hu, M. S. Li and Z. Gazova, ACS Chem. Neurosci., 2018, 9, 2588– 2598. 8 G. N. Lipunova, E. V. Nosova, V. N. Charushin and O. N. Chupakhin, Curr. Org. Synth., 2018, 15, 793–814. 9 D. Liu, Z. 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Moreover, these quinazolines-based D–A compounds emit bright light in diﬀerent states with excellent quantum yields. The emission colour could be nely tuned by the intrinsic substituent, the extrinsic solvent polarity and grinding. More- over, some of them might be used as the emissive material in the fabrication of OLEDs. 16 M. Deiana, K. Chand, J. Jamroskovic, I. Obi, E. Chorell and N. Sabouri, Angew. Chem., Int. Ed. Engl., 2020, 59, 896–902. 17 Z. Peng, K. Zhang, Z. Huang, Z. Wang, S. Duttwyler, Y. Wang and P. Lu, J. Mater. Chem. C, 2019, 7, 2430–2435. 18 J. Yuan, Y. Yuan, X. Tian, Y. Liu and J. Sun, J. Phys. Chem. C, 2017, 121, 8091–8108. 19 S. Achelle, J. Rodr´ıguez-L´opez and F. Robin-le Guen, J. Org. Chem., 2014, 79, 7564–7571. 20 R. Afshari and A. Shaabani, ACS Comb. Sci., 2018, 20, 499– 528. 21 E. Liang, Y. Wu, J. Chen, W. Xiong, J. Zhao, X. Yao and X. Tang, Tetrahedron, 2019, 75. 22 A. Barmak, K. Niknam and G. Mohebbi, ACS Omega, 2019, 4, 18087–18099. Mechanochromic properties of 2 and 4 6 Mechanochromic property of compounds 2 and 4; (a) normalized emission spectra; (b) emission cycles. This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 30297–30303 \| 30301 Paper View Article Online Paper View Article Online View Article Online View Article Online RSC Advances Conﬂicts of interest 23 X. Su, C. Chen, Y. Wang, J. Chen, Z. Lou and M. Li, Chem. Commun., 2013, 49, 6752–6754. Notes and references Three methods were used to evaluate the HOMO/LUMO orbitals, including UV (Fig. S39 and S40†) and CV measure- ments (Fig. S47†) as well as the theoretical calculation (Fig. 7a) for comparison. Results were listed in Table S3.† Based on the calculation, donor and acceptor are clearly separated by HOMO and LUMO orbital distributions. Energy gaps obtained from CV and UV measurements are in good accordance with the results from calculation (Fig. 7b). Gradually decreased energy gaps from 1 to 5 as well as from 6 to 10 explained the full spectrum emissions of these compounds in solutions (Fig. 1) and in solids (Fig. 4c). Open Access Article. Published on 17 August 2020. Downloaded on 10/24/2024 6:16:21 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 1 Z.-Z. Ma, Y. Hano, T. Nomura and Y.-J. Chen, Heterocycles, 1997, 541–546. 2 S. Yoshida, T. Aoyagi, S. Harada, N. Matsuda, T. Ikeda, H. Naganawa, M. Hamada and T. Takeuchi, J. Antibiot., 1991, 44, 111–112. RSC Adv., 2020, 10, 30297–30303 \| 30303 This journal is © The Royal Society of Chemistry 2020 The authors declare no conict of interest. The authors declare no conict of interest. 30302 \| RSC Adv., 2020, 10, 30297–30303 30302 \| RSC Adv., 2020, 10, 30297–30303 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Paper RSC Advances 24 B. Han, C. Wang, R. F. Han, W. Yu, X. Y. Duan, R. Fang and X. L. Yang, Chem. 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https://openalex.org/W3177776407	https://cris.brighton.ac.uk/ws/files/25763967/387149_Article_Text_564074_2_10_20210705.pdf	English	null	Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia performance in the age of global pandemic	Artnodes	2,021	cc-by	9,645	Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia performance in the age of global pandemic Ada Xiaoyu Hao University of Brighton Date of submission: May 2021 Accepted in: May 2021 Published in: July 2021 Recommended citation Hao, Ada Xiaoyu. 2021. «Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia performance in the age of global pandemic». In: González Díaz, Paloma; García Méndez, Andrea (coord.) «In the limits of what is possible: art, science and technology». Artnodes, no. 28: 1-13. UOC. [Accessed: dd/mm/yy]. http://doi.org/10.7238/a.v0i28.387149 The texts published in this journal are – unless otherwise indicated – covered by the Creative Commons Spain Attribution 4.0 International license. The full text of the license can be consulted here: http://creativecommons.org/licenses/by/4.0/ E-JOURNAL ON ART, SCIENCE AND TECHNOLOGY https://artnodes.uoc.edu NODE «IN THE LIMITS OF WHAT IS POSSIBLE: ART, SCIENCE AND TECHNOLOGY» Resumen A raíz de la pandemia de la COVID-19, nos hemos ido adaptando como fugitivos de esta lucha contra el virus «into cable» a la vez que se vuelve un escenario virtual compartido, donde nuestros cuerpos prolijos se encuentran dentro, incrustados y teletrabajando. Nuestra creciente dependencia de la interacción en línea y de las videoconferencias durante la pandemia no solo facilitan la conexión social, sino que también nos hace considerar lo siguiente: ¿Cómo prolongar nuestra sensibilidad somática y hacernos eco de la experiencia corporal de tocar a través de la conectividad virtual incorpórea? En este ensayo me centro en la naturaleza encarnada de la interpretación de la telesinestesia, su efecto potencial de forjar una conexión rítmica de una modalidad sensorial a otra, y la aparición de fallos técnicos y la latencia de Internet como indicios no verbales de la comunicación a través Internet. En referencia a la teoría de la «visualidad háptica» de Laura Mark, en que la visión desencadena una experiencia táctil en el cuerpo; al concepto «tacto virtual» de Naomi Bennett, con el que se puede provocar una respuesta sensorial afectiva del tacto a través de sentidos no táctiles; a las producciones artísticas Telematic Quarantine (2020) y Pandemic Encounter (2020), de Paul Sermon, que telepresentan las historias del aislamiento; y en relación con el concepto «heterotopia», de Michel Foucalt en el contexto del comportamiento en Internet, exploro el trabajo de interpretación en el que he trabajado durante el confinamiento desde marzo de 2020, The Best Facial (2021), con la ayuda del Centro de Investigación de Culturas de Medios Digitales y de la investigación de posgrado de la Escuela de Arte de la Universidad de Brighton. Se trata de una serie de sesiones de 25 minutos de representaciones participativas de telesinestesia de persona a persona en Zoom, en las que me convierto en esteticista virtual y hago uso de la telepresencia para llevar a cabo estrategias faciales virtuales contemplativas, y hacer improvisaciones de telecontacto en la piel del rostro para despertar experiencias táctiles a través de la visualidad háptica, el tacto virtual y la fantasía auditiva. Abstract In the wake of the COVID-19 pandemic, we have been adapting as fugitives of this accidental encounter with an “untouchable” virus, while being rendered into the shared virtual arena, where our discursive bodies are situated within, embedded in and tele-commuted to. Our increasing dependence on online interaction and video conferencing during the pandemic is not only facilitating social connectedness, but also contemplating the question: How to elongate our somatosensation and echo the embodied experience of touching through the incorporeal virtual connectivity? This essay focuses on the embodied nature of tele-synaesthesia performance, A UOC scientific e-journal A UOC scientific e-journal Artnodes, no. 28 (2021) I ISSN 1695-5951 Universitat Oberta de Catalunya artnodes Making touch visible with the suture of fantasy with virtual aesthetician in chy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in https://artnodes.uoc.edu Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic a g ouc s b e e su u e o a asy ua aes e c a “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic its potential effect of forging a rhythmic connection from one sensuous modality to another, and the concurrent emergences of glitch and internet latency as non-verbal cues of internet-situated communication. In reference to Laura Mark’s theory of haptic visuality, where vision triggers a tactile experience in the body; Naomi Bennett’s concept of virtual touch, in which an affective sensory response of touch can be elicited through non-tactile senses, Paul Sermon’s artistic production of Telematic Quarantine (2020) and Pandemic Encounter (2020), that telepresents the stories of self (isolation), and in relation to Michel Foucault’s concept Heterotopia in the context of internet-situated performance, I examine the performance work I have been developing during lockdown since March 2020: The Best Facial (2021), supported by Centre for Digital Media Cultures Research and School of Art Postgraduate Research at University of Brighton, a number of sessions of 25-minute 1-to-1 participatory tele-synaesthesia performances that take place on Zoom, where I become a virtual aesthetician and use telepresence to perform meditative virtual facial tactics, and to make tele-contact improvisation upon the surface of the participant’s face to trigger tactile experiences through haptic visuality, virtual touching, and auditory fantasization. Keywords Keywords Participatory performance, Haptic visuality, Heterotopia, Telecommunication, Synaesthesia, Glitch Hacer «visible» el tacto mediante la unión de la fantasía con la esteticista virtual en The Best Facial Clinic: el índice de fallos técnicos en la telesinestesia en época de pandemia mundial Introduction Using the Michel Foucault’s concept of heterotopia2 (Foucault 1986), I propose to use heterotopia as a conceptual backdrop to frame internet-situated tele-synaesthesia performance space as a virtual-real heterotopia: a deviant space and a virtual-real instantiation of a temporary utopia ideal that carries the potential of maintaining the embodied expe riences of distanced bodies and their network of relations during the time of lockdown. This essay considers embodied nature of tele-synaesthesia per formance as a potential for forging connections between distanced bodies and isolated sensory modalities, and focuses on the situated technological possibilities, such as glitch and internet latency, that mediate non-verbal communication. In reference to Laura Mark’s theory of haptic visuality, where vi sion triggers a tactile experience in the body; Naomi Bennett’s concept of virtual touch, in which an affective sensory response of touch can be elicited through non-tactile senses, Paul Sermon’s artistic production of Telematic Quarantine (2020) and Pandemic Encounter (2020), that tele-presents the stories of self isolation, and in relation to Michel Foucault’s concept Heterotopia in the context of internet-situated performance, I examine a performance work I developed during lockdown since March 2020: The Best Facial 1 (2021): a 25-minute 1-to-1 participatory tele-synaesthesia performance that took place on Zoom, where the participants were invited to receive virtual “facial treatment” performed by a fictional character – a virtual aesthetician. This project uses fiction as a method to create phantasmagorical fantasy of a virtual facial clinic in order to test the threshold of embo died experience in a heterotopic space where the body’s the haptic visuality (Marks 2002) and sensuous virtual touching (Bennett 2020) are performed in relation to the distanced participants. Additionally, I propose to identify internet latency and glitch in online communi cation as open contacts, like surfaces in contact improvisation, that expands the intensity of the embodied experience in internet-situated performances. In this essay, I will first use the “suture of fantasy” as a metaphor to illustrate how I embrace virtuality and transpose visual tactics in tele-synaesthesia performance. Then, I will trace my reflective practice-based research, along with the presentation, dissemination, documentation, and research feedback from participants, to identify the performative technological anxiety - glitch- as open contact, that triggers non-verbal cues for participation and interaction in internet-situated performances. Introduction In the end, I will conclude with my research findings to reflect the embodied nature of internet-situated performance, and to provide a positive affirmation for the symbiotic relationship between the mutability of technological anxiety and the creativity for art practice and research. Introduction translation of […] the embodied experiences […] from one sense modality to another’ (Marks 2002, ix), and occurs when ‘stimulation of one sensory modality automatically triggers a perception in a second modality’ (R. E. Cytowic 2002, 2), with the absence of the direct contact or stimulation with the second modality. In this essay, paraphrasing the definition of synaesthesia given by Richard Cytowic in his book Synaesthesia: A Union of Senses (1989), and following the hypothesis of tele-synaesthesia proposed by Dr. Hugo Heyrman in his lecture, ‘Tele-Synaesthesia: the Telematic Future of the Senses’ (Heyrman 1997), I propose to evaluate the question: how to create a virtual heterotopia to reflect the embodied experience of online communication using the techno-aesthetics of tele-synaesthesia. Coming in and out of… ‘When so much more can be achieved and saved by reframing our approach to face-to-face coexistence through being creative with our videoconferencing. Making it memorable now could make a difference in the future.’ (Sermon, Be creative with your videocon ferencing, make it memorable, it makes a difference 2020, n.pag.) As we are adapting to the effusive after-effects of living through the sudden irregularities teased out by the pandemic, how to extend the face-to-face communication and the shared consciousness at a distance? Our increasing dependence on online interaction during the pandemic is not only facilitating social connectedness, but also contemplating the question: How to elongate our somatosensation along the fiber-optic interface and echo the embodied experience of touching through the incorporeal virtual connectivity? As we are adapting to the effusive after-effects of living through the sudden irregularities teased out by the pandemic, how to extend the face-to-face communication and the shared consciousness at a distance? Our increasing dependence on online interaction during the pandemic is not only facilitating social connectedness, but also contemplating the question: How to elongate our somatosensation along the fiber-optic interface and echo the embodied experience of touching through the incorporeal virtual connectivity? According to Foucault, ‘places like rest homes, clinics, psychiatric hospitals, and prisons’ are the ‘heterotopias of deviance’, in which ‘people whose actions deviate from the norms in some way, and hence need to be spatially isolated’ (Foucault 1986). artnodes artnodes Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu https://artnodes.uoc.edu 1. The selected documentation of the project can be viewed here: https://vimeo.com/563633992 2. According to Foucault, heterotopias refer to “counter-sites” that juxtapose with the unreality of utopias to provide a ‘curious property of being…to suspect, neutralize, or invert the set of relations’ that the reality ‘happen to designate, mirror, or reflect’. (Foucault 1986, 3). Palabras clave Actuación participativa, visualidad háptica, heterotopia, telecomunicación, sinestesia, fallo técnico Actuación participativa, visualidad háptica, heterotopia, telecomunicación, sinestesia, fallo técnico A UOC scientific e-journal A UOC scientific e-journal Artnodes, no. 28 (2021) I ISSN 1695-5951 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao Universitat Oberta de Catalunya 3. ‘Suture’, Merriam-Webster.com Dictionary, Merriam-Webster, https://www.merriam-webster.com/dictionary/suture. Accessed 27 Mar. 2021. 4. ‘Suture’ is a concept introduced by Jacques-Alain Miller in his essay Suture: Elements of a Logic of the Signifier (Miller 1977). 5. Here, the Covid-19 pandemic has made “the first cut”, which cuts face-to-face communication and tactile interaction away from the everyday life. 6. Even though we have already been living in a permanent state of changes before the pandemic, it is still difficult to adapt to the sudden change without being nostalgic about the memories of the past or betting on certain possible future that might take place after the change. Instead of obsessively looking for the differences that are generated by the ‘cut’ or affirming the changes that have already become overwhelmingly present in our everyday life, as artists, it is urgent now to cope with these challenges of adaptation with the great capacity of the human creativity. 7. The posture of the virtual aesthetician in the advertising poster, along with the slogan at the bottom, is an ironic parody of the society’s mainstream notions of care that are commercialized with a high frequency of irresponsible promises and idealized fantasy of beauty. The Suture of Fantasy: The First Cut ‘Suture names the relation of the subject to the chain of its discourse; we shall see that it figures there as the element, which is lacking, in the form of a stand-in. For, while there lacking, it is not purely and simply absent.’ (Miller 1977) During the Covid-19 pandemic, the thought of suturing fantasy and reality allow me to adapt my art practice to blur the virtual embodied experience with its counter-real part.5 By cutting off the multifaceted supply of habitual physical contact, there is a higher demand for virtual communication and nostalgia of the past.6 The Best Facial was born in such suture. It triggers new sensorimotor gestural channels and perceptual pathways through the spatiotemporal online encounters enabled by videoconferencing tool - Zoom, and virtual corporeal touch envisioned by the use of live audio-visual performance software Resolume Avenue. Suture, as a verb, means to unite, close or secure with sutures3. In surgery, a suture is a basic speedy sewing technique that holds together the edges of a cut, a wound, or an incision. A suture performs a viscous action to the cut, while simultaneously immersing what was foreign into the subject itself surreptitiously during the operation. As the wound heals, the suture will eventually become the skin as a part of the body. Cutting across the spectrum of internet-situated performance, live performance, and digital performance, this project draws two parallel sutures: one connects haptic vision with tactile sensation of touching with virtual-real synaesthesia experiences, the other one draws the link between the tele-synaesthesia performance space and heterotopia. This project addresses the unpresentable void of communication by presenting the conceptual suture that blur our physical and digital presences. In this essay, ‘suture’4 is considered as a jump-off point to weave together fantasy and reality in flux: a temporal space for the tele- synaesthesia performance to take place, as illustrated in the Figure 1 below. Rather than looking at the ‘suture’ as a concealer of differences or a closure for the relationships between subjects in the chain of structured discourses, I would like to ask the reader to think of act of suturing as a poetic gesture, that not only reveals the absence of elements that has been lacking as a form of a stand-in, but also creates spaces to situate the tractions and relationships that are generated between different subjects in process of suturing. artnodes Making touch visible with the suture of fantasy with virtual aesthetician in glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic with the aim of transposing the distanced bodies through a nonunitary vision of the digital communication platforms. Tele-synaesthesia performance in virtual-real heterotopia Tele-synaesthesia performance links tele and synaesthesia to each other. It generates the multiple possibilities of creating synaesthetic effect with people at a distance and people of the future through the transmission of data. Synaesthesia, which means “jointed sensation”, which is derived from the Greek word: ‘syn’, which means together; and ‘aisthèsis’, which means perception. Synaesthesia means ‘the A UOC scientific e-journal Artnodes, no. 28 (2021) I ISSN 1695-5951 A UOC scientific e-journal 3 2021 f thi diti b C 2021, Ada Xiaoyu Hao Universitat Oberta de Catalunya artnodes 8. In the virtual environment, it appears to be difficult to translate all of the information from one physical end to another. As a result, such absence of embodied experience, such as being touched or touch, could be trapped to be revealed through gesture of touch in tele-synaesthesia performance. 9. In relation to what Puig de la Bellacasa writes, ‘attention to what it means to touch and to be touched deepens awareness of the embodied character of perception, affect, and thinking’ (Puig de la Bellacasa 2017, 96), the contact made by human eyes could be understand as touch that intensifies the sensuous relationship between the screen and the flesh. 10. Here, my proposition for distant touching echoes with Cytowic’s conceptualisation of synaesthesia: ‘while sensing emotions concretely as particular colours, shapes, or tastes can lead to intensely felt experiences, it can also facilitate identifying those feelings and resolving conflicts within oneself and with others.’ (R. E. Cytowic 2018) Making touch visible – inhale from the webcam and exhale into the internet as an umbilical cord When our bodies are confined to a finite space, we should imagine the counter-actualization of the limitation we face by ‘transforming negative into positive relations, encounters, and passions’ (Braidotti 2011, 31). Echoing Braidotti’s nomadic theory that transposes the subjectivity with multiple ecologies of becoming, I created this project Inspired by Paul Sermon’s Pandemic Encounter and Telematic Qua rantine (Paul Sermon 2020) (Sermon, Pandemic Encounters 2020), I created an open call to the public to invite participants to join me for a 25-minute 1-to-1 participatory tele-synaesthesia performance that takes place on Zoom, where I become a virtual aesthetician in The Best Facial Clinic. I propose to use The Best Facial project to explore a speculative mode of connectivity and care with a creative use of the videoconferencing tool, while embracing the ambivalence of online communication as a tentative ground. Figure 1. The Suture of Fantasy and Reality, Courtesy of the artist. Building from Maria Puig de la Bellascasa’s speculative ethics of care, that is to ‘propose modes to contribute to [the] re-articulation, re-conception, and “re-enactment”’ (Puig de la Bellacasa 2017, 11) of care, the poster of the open call advertisement (fig. 2.) for participation says: ‘The Best Facial: Say Goodbye to Hopelessness’.7 Figure 1. The Suture of Fantasy and Reality, Courtesy of the artist. A UOC scientific e-journal Artnodes, no. 28 (2021) I ISSN 1695-5951 A UOC scientific e-journal 4 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao Universitat Oberta de Catalunya artnodes https://artnodes.uoc.edu Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Figure 2. Advertisement Poster for The Best Facial, Courtesy of the artist/writer Figure 4. Performance still from The Best Facial, Courtesy of the artist/writer Figure 3. Performance still from The Best Facial, Courtesy of the artist/writer Figure 3. 11. To start the session (fig. 5), I would guide the participant to start a short breathing exercise, by asking them to breath in from their nose, and breath out through the mouth. Followed by a synaesthesia game to imagine the web camera as a breath chamber that provides air, that is passed through the umbilical cord of the fibreoptic internet cable, which allows me to start the virtual connection with them. 12. The pre-session assessment can be viewed through this link: https://docs.google.com/forms/d/1nbnFOY6IIPgr968QyFr_kYsc1u2s8VxHfDZKRxo3PsA/ viewform?edit_requested=true 13. As the soundtrack is playing, I use Resolume Avenue to playfully apply different transformations using the built-in function of the software, such as scale of the participant’s face (fig.8), the closeness between the face and the frame of the screen, the position of the face on the screen (fig.9), and the level of transparency of the participant face. The action of these playful transformations is synced live with the soundtrack, as I drag the computer mouse adjust the level of intensity for each transformation. 14. The full version of the soundtrack can be read here: https://drive.google.com/file/d/1QP7LAxh0Q_im3iWYlveqnBTkT0c4261q/view?usp=sharing; The full version of the soundtrack can be listened to here: https://drive.google.com/file/d/1-5PV_kcdXzpNiE6wIW6ovwZ91yqn9Kc8/view?usp=sharing 15. The participant is encountered with the contextual emplacement first with the sentence, ‘you are always in contact’, which is repeated in the beginning of the first act of the soundtrack in four segments. artnodes artnodes Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible – inhale from the webcam and exhale into the internet as an umbilical cord Performance still from The Best Facial, Courtesy of the artist/writer Figure 2. Advertisement Poster for The Best Facial, Courtesy of the artist/writer Figure 3. Performance still from The Best Facial, Courtesy of the artist/writer Figure 3. Performance still from The Best Facial, Courtesy of the artist/writer Figure 4. Performance still from The Best Facial, Courtesy of the artist/writer Figure 2. Advertisement Poster for The Best Facial, Courtesy of the artist/writer Figure 4. Performance still from The Best Facial, Courtesy of the artist/writer the computer screen. It could appear to the participant, that they are superimposed into a phantasmagoria when a hand starts to crawl outside of their eyes, mouths and the pores on their faces (fig. 3 & 4). When participant is witnessing their face being touching, their eyes also initiates an optical contact as a corporeal device. Rather than using videoconferencing tool as an alternative of the face-to-face communication, I use fiction as the method to play a role in this virtual fantasy that highlights the sliding relationship between haptic and optical. How to provoke the experience of being touched by witnessing the gestural touching? How to awake the memory of our tactile sen sitivity through synaesthesia, the translation of information from one sensational modality to another?8 In the performance, I repeatedly draw connections between touch and surface, touch and contact, contact and screen, the contact with skin and the contract with skin we each have. The contact made by human eyes could be understand as touch that intensifies the sensuous relationship between the screen and the flesh.9 The gesture of virtual touching is a tele-synaesthesia tactic that fulfils the eye’s corporeal cavity with the link drawn between desire and fantasy also connects the physical sensation of being touched and the virtual touching that is witnessed by the participant from One could propose that we have being in contact with or without the physical sensation of being in contact.10 In the performance, Artnodes, no. 28 (2021) I ISSN 1695-5951 A UOC scientific e-journal A UOC scientific e-journal 5 2021 f thi diti b C 2021, Ada Xiaoyu Hao 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao Universitat Oberta de Catalunya https://artnodes.uoc.edu https://artnodes.uoc.edu I blur relationship between optic and haptic by engaging with the distant vision that provides alternative ways for the participants to feel being touched.11 Figure 6. Performance still from The Best Facial, Courtesy of the artist/writer Figure 5. Performance still from The Best Facial, Courtesy of the artist/writer Figure 6. Performance still from The Best Facial, Courtesy of the artist/writer Figure 7. Performance still from The Best Facial, Courtesy of the artist/writer Figure 5. Performance still from The Best Facial, Courtesy of the artist/writer Prior to the virtual facial session, the participants are asked to fill an online assessment form12 to resemble the facial service that is provided by commercial beauty parlour and build up the sense of suspense and expectation for the participant. This pre-session assessment form also allows me to make the first “contact point” with the participant without any knowledge about who they are, but with enough information to design a “treatment plan” for the virtual facial session with the participant. Figure 7. Performance still from The Best Facial, Courtesy of the artist/writer The “treatment plan” is a composed of choreographed audio soundtrack and improvised actions. During the session, I virtually superimpose the participant’s face into the virtual background of The Best Facial Clinic, in which is presented with animated purple waves against a black background and an ongoing subtitle that says, ‘The Best Facial’ by the bottom of the screen, using virtual camera output, Camtwist and live VJ software Resolume Avenue, as shown in fig.6. & 7. Then, I start to play the pre-recorded narration about touch with a meditative soundtrack that is composed digitally, so to cultivate an atmospherically vacant and peaceful environment, where the participant could be teleported into a different space than their own home. The soundtrack13 is an essential element in this tele-synaesthesia performance.14 It functions as the score for the performance and guides the participant to use the auditory sensation to further trigger the possible haptic sensation with the distant vision. 15 Artnodes, no. 16. During this segment, I leave the participant in “The Best Facial Clinic”, so that the participant could continue with the beginning breathing exercise and to be comfortable with the spectacle of their own face superimposed in the virtual background, shown in fig 10. 17. To activate the participants’ self- consciousness and self-awareness of the physical body, I annunciate the less-attended parts of the body, such as the roof of the mouth, fingertips, scalp, teeth, togue, lips, hair, nails; then I bring the relational awareness of the body in reference to the other bodies in the everyday life, such as neighbour, strangers encountered on the street, mailman, lover, friends and people who extend the sensible dimension of the self; and then I construct speculative contacts that could provoke memory of the body in a domestic environment, such as household appliances, old clothes, abandoned things, things one have touched once before, and things forgotten and left in the corner of the room. 18. In the performance, the scale of the participant’s face was enlarged to increase the surface area of experience. https://artnodes.uoc.edu Figure 10. Performance still from The Best Facial, Courtesy of the artist/writer Figure 8. Performance still from The Best Facial, Courtesy of the artist/writer Figure 9. Performance still from The Best Facial, Courtesy of the artist/writer Figure 10. Performance still from The Best Facial, Courtesy of the artist/writer Figure 9. Performance still from The Best Facial, Courtesy of the artist/writer Figure 11. Performance still from The Best Facial, Courtesy of the artist/writer The first segment of act 1 in the performance is focused on the peripheral vision and subjective perspective of the participants in relation to the surfaces that their bodies are in contact with.16 In segment two to four, of approximately 5 minutes, I focus on the visceral awareness of the fragmented body parts. 17 During these three segments, I start to play with the scale and position of the participant’s face, in order to accentuate on the relationship between the participant’s physical spatial awareness of their face and the visual spectacle of their face. At the same time, I use the chroma-key function in Resolume Avenue and substitute the participant’s facial surface with found films, pre-recorded moving image and recordings of the natural landscape, as shown in fig.11 & 12. p g You are the contact, you are the surface, you are the interface, you are becoming the contact with yourself by becoming yourself. You have become the contact that reveal what, who, where and how you are in contact with. Figure 11. Performance still from The Best Facial, Courtesy of the artist/writer You are the contact, you are the surface, you are the interface, you are becoming the contact with yourself by becoming yourself. You have become the contact that reveal what, who, where and how you are in contact with. Figure 12. Performance still from The Best Facial, Courtesy of the artist/writer In the second act of the performance, I then leave the participant wait in the clinic for the second time, in order to allow time for the participant to perceive what has happened in the last 5 minutes. 18 Where is your skin? Isn’t it amazing that you are held in contract with your skin without the need to balance the tension between each of Figure 12. Performance still from The Best Facial, Courtesy of the artist/writer Figure 12. Performance still from The Best Facial, Courtesy of the artist/writer 16. https://artnodes.uoc.edu 28 (2021) I ISSN 1695-5951 A UOC scientific e-journal A UOC scientific e-journal 6 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao C 2021, Ada Xiaoyu Hao Universitat Oberta de Catalunya artnodes Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu https://artnodes.uoc.edu Figure 13. Studio installation of The Best Facial performance set-up, Courtesy of the artist/ writer I will now introduce a friend, a slug. It will move across your face to take away the dead skin cells that have become alien to your face. It’s leaving a wet trail as it’s moving slowing around your face, with a path left behind. You face is now all moisturized, all fresh, all flesh, all anew, like a new born. In the last act of the performance, I use the web camera that is installed on the ceiling of the studio in order to emplace my entire body into the participant’s face to perform the last set of virtual facial treatment.21 Figure 15. Performance still from The Best Facial, Courtesy of the artist/writer Figure 13. Studio installation of The Best Facial performance set-up, Courtesy of the artist/ writer the organs in your body? Isn’t it amazing that you are able to feel what’s outside without ripping your skin open? Isn’t it amazing that you can see what is outside while being inside of the skin? (Selected text from Act 3 of the soundtrack) Figure 14. Performance still from The Best Facial, Courtesy of the artist/writer Figure 15. Performance still from The Best Facial, Courtesy of the artist/writer Toward the ending of the performance, I would use my own body as a “ruler” to align the participant’s face in relation to my physical body lying on top of the greenscreen on the studio floor, as shown in fig.16.22 Figure 14. Performance still from The Best Facial, Courtesy of the artist/writer Figure 16. Performance still from The Best Facial, Courtesy of the artist/writer the organs in your body? Isn’t it amazing that you are able to feel what’s outside without ripping your skin open? Isn’t it amazing that you can see what is outside while being inside of the skin? (Selected text from Act 3 of the soundtrack) the organs in your body? Isn’t it amazing that you are able to feel what’s outside without ripping your skin open? Isn’t it amazing that you can see what is outside while being inside of the skin? 19. Practically, I use two web cameras, with one installed on the ceiling of the studio, and another one positioned in front of the green screen. As shown in fig.13, the web camera installed on the tripod by the lower right corner of the photograph is the front camera. The web camera that is taped to the ceiling by the top of the photograph is the other image capture device I use. 20. For example, I slowly stroke the eyebrows of the participant when the soundtrack says, ‘Can you feel your eyebrow? Try to move your eyebrow to scale the size your face’. Or, I would gradually stick my arms outside of the participant’s mouth, when the soundtrack says, ‘take another deep breath you’re your mouth open wider. When the breath is out, let your breath penetrate what you see’, as shown in fig.14. 21. For example, I would wrinkle a transparent blue plastic sheet in front of the web camera, when the soundtrack says, ‘I will now introduce a friend, a slug. It will move across your face to take away the dead skin cells that have become alien to your face’, as shown in fig.15. 22. This is what I refer to as virtual contact improvisation. Because I was touching my own body, while it appears to the participant that I was touching their face. According to the participant’s feedback, this section invokes the most effective sensation of touch, because of the accuracy of the virtual touch. https://artnodes.uoc.edu During this segment, I leave the participant in “The Best Facial Clinic”, so that the participant could continue with the beginning breathing exercise and to be comfortable with the spectacle of their own face superimposed in the virtual background, shown in fig 10. 17. To activate the participants’ self- consciousness and self-awareness of the physical body, I annunciate the less-attended parts of the body, such as the roof of the mouth, fingertips, scalp, teeth, togue, lips, hair, nails; then I bring the relational awareness of the body in reference to the other bodies in the everyday life, such as neighbour, strangers encountered on the street, mailman, lover, friends and people who extend the sensible dimension of the self; and then I construct speculative contacts that could provoke memory of the body in a domestic environment, such as household appliances, old clothes, abandoned things, things one have touched once before, and things forgotten and left in the corner of the room. 18. In the performance, the scale of the participant’s face was enlarged to increase the surface area of experience. A UOC scientific e-journal A UOC scientific e-journal Artnodes, no. 28 (2021) I ISSN 1695-5951 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao 2021 f hi di i b C 2021, Ada Xiaoyu Hao Universitat Oberta de Catalunya artnodes artnodes Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Glitch as a non-performative refusal – Embracing the change in the suture of fantasy In the beginning of the lockdown in March, 2020, I was overwhel med by the over-saturation of the glorification of using internet as a medium to disseminate artworks.23 However, it has occurred to me that, it is more urgent than ever to stop comparing the differences between face-to-face and internet-situated communication, but to create symbiotically with technology as more than a mediator or a one-way output-orientated machine. Hence, I use digital improvisation in The Best Facial project to create more possible “glitches” in addition to the existing glitches and the ephemeral changes, as shown in fig. 17. During the performance, the participants and the technological beings I work with both undergo the process of ‘having technical ensemble and becoming reabsorbed in a new technical [and virtual] ensemble, performing a similar or different function there’ (Halyes 2012, 103) I would ask questions, such as, did you feel me touching you during the performance, and which action give you the most effective feeling in relation to touch and being touched. Instead of working on ways to overcome the technical imper fection of internet-situated communication, I started to pay more attention to the messiness and failures activated by the use of internet and internet-based software.24 Going along with the feedback loops between the artists and audiences and between the artists’ creation and individual’s need to self-identify and self-actualize, the increasing demand for internet-situated performances and artworks, especially during the time of the pandemic. Figure 17. Performance still from The Best Facial, Courtesy of the artist/writer Such demand also creates an archive for the contemporary art and performance practice, while cultivating a historical background for the future development and innovation of internet-situated per formance. Hence, it is crucial to diversify the practice of internet- situated performance. In order to do that, I would like to focus on the understanding of glitch, as a correlated feature and an often forgotten characteristic of internet-situated performance. Figure 17. Performance still from The Best Facial, Courtesy of the artist/writer A glitch is an error. It is invisible until it is visible. A glitch can be accidentally triggered, yet the appearance of glitch cannot be predicted. The power of a glitch could cut the continuous happening that connect our perception of time and space. 23. Such as using different social media platform to publish and share documentation of live performances, virtual gallery tour, representation of live theatrical production; or online Zoom panel discussions that ends with how to get over “Zoom fatigue” that leads to tiredness and physical exhaustion. 24. However, I do not want to dismiss the increasing quantity of performances that use the internet as a medium to disseminate work. As a survival mechanism, digital media is imbued with ‘fantasy today is not a retro act of mythologizing’ (Russell 2020) 25. I also embrace the limit performance capacity of my performance hardware to allow a continuous feed of glitches, such as face or body freezes, stuttered or unclear communication, low frequency buzzing in the soundtrack’s background, and sudden disappearances of imagery or disconnection during the course of online performance platform. https://artnodes.uoc.edu (Selected text from Act 3 of the soundtrack) In the third act of the performance, I then start to virtually superimpose my hands, arms and my body inside of the virtual clinic.19 During this act, I first use the front camera that captures the greenscreen hanging up. I start to play with the visual tropes of superposition and surrealistic imagery using my hands and arm in order to trigger tactile sensation.20 According to the feedback of the participants, some of them could actually feel a tingling sensation when my hands were tapping their forehead and eyebrow. This is one of the most effective synaesthesia experiences that the participant could be able to describe and locate. Figure 16. Performance still from The Best Facial, Courtesy of the artist/writer The proprioceptive choreography of body movements, facial ex pressions and hand gestures are key components to any conversation, often used unconsciously, but by simply combing these views within the same image we become kinaesthetically conscious and in control of our combined coexistence, escaping our individual isolation. Revista científica electrónica impulsada por la UOC Artnodes, no. 28 (2021) I ISSN 1695-5951 Revista científica electrónica impulsada por la UOC Revista científica electrónica impulsada por la UOC 8 2021, Ada Xiaoyu Hao 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao C Universitat Oberta de Catalunya artnodes Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu Glitch as a non-performative refusal – Embracing the change in the suture of fantasy this motion makes an error difficult to see but its interference ever present’. (Russell 2020) In order to identify the glitches, I identify glitches not as errors, but as open points of contacts that creates spaces that fore-ground potential interaction with the participant. In the performance, I re-membered glitch into the process of crea ting internet-situated tele-synaesthesia performance, by addressing glitch as a digital mutation, in parallel to the organic mutality of Coronavirus – that simultaneously performs the “unsanitary error”: the technological anxiety that is independent from the human will, and calls out for a ‘non-performative’ action. I purposefully chose to use the low-resolution output to increase the duration and frequency of glitch.25 Using the nostalgic affect of low-resolution related to early days of internet, the glitches triggered by internet latency or CPU overload during the performance would not only introduce new movement into the ‘static space’, but also make the interference of glitches into organic ephemeral changes that are organic to tele-synaesthesia performance. These ephemeral changes are the metaphorical contact points, for which become a virtual interface for performative interactions, actions and reactions. Hence, I use digital improvisation in The Best Facial project to create more possible “glitches” in addition to the existing glitches and the ephemeral changes, as shown in fig. 17. During the performance, the participants and the technological beings I work with both undergo the process of ‘having technical ensemble and becoming reabsorbed in a new technical [and virtual] ensemble, performing a similar or different function there’ (Halyes 2012, 103) I would ask questions, such as, did you feel me touching you during the performance, and which action give you the most effective feeling in relation to touch and being touched. I purposefully chose to use the low-resolution output to increase the duration and frequency of glitch.25 Using the nostalgic affect of low-resolution related to early days of internet, the glitches triggered by internet latency or CPU overload during the performance would not only introduce new movement into the ‘static space’, but also make the interference of glitches into organic ephemeral changes that are organic to tele-synaesthesia performance. These ephemeral changes are the metaphorical contact points, for which become a virtual interface for performative interactions, actions and reactions. https://artnodes.uoc.edu to moving my head as a way of interesting with the imagery and Ada’s ‘touch’ - whilst that didn’t lead to any tactile sensation it did make my experience feel more generally embodied and engaged. (Selected response from participant’s post-performance feedback) My intension of creating this internet-situated performance is to substitute the practice of live performances we witness in person. My attempt of discovering creative use of online communication tool is to perform, reflect and archive my share of the collective experiences of living through the global crisis in the age of the tactical new media. Rather than thinking of create an artwork that is only responding to the temporal changes, as Boris Groys proposed: ‘how can artists resist the destructiveness of progress? How can one make art that would escape permanent change—art that would be atemporal, transhistorical?’ (Groys 2010) 26 When I ask participants what their faces feel like, 66.7% of the participants felt that their face has become a playground, with an equal percentage of the rest of participants saying that their faces has become a void or a new speculative that they can’t name. This data shows that tele-synaesthesia performance is such playground, which creates potential embodied experience that sutures the fantasy together with reality for participants. In relation to Foucault’s proposi tion of heterotopia as deviant spaces in the reality, internet-situated performance creates such ‘heterotopias of deviance’, that provides a virtual playground that invites spatially isolated bodies to perform durational deviancy. Within these virtual-real heterotopias, the virtual embodied experience of the body could be remembered, displaced and com posed differently despite not being altered, changed or moved in its physical presence. If we think virtual-real heterotopias as if they are experiments in scientific lab, where the body would appear to be transformed for the viewer’s eye and mind, like phenomenon scientists could observe in a practical experiment in the lab, the reaction or metamorphosis of the body would only happen with the visual spectacle, instead of the actual body. Is that so? I disagree. Like the phantom limb pain, tele-synaesthesia performance could activate such virtual-real heterotopias, where one would embody the vision, feel the image, and even become tempted to react to such invisible sources of sensorimotor activation. 26. I agree that change should be the status quo, and ‘permanent change is our only reality… to change the status quo would be to change the change—to escape the change… every utopia is nothing other than an escape from this change’ (Groys 2010), and hence every virtual-real heterotopia could be a multiple temporary traps to reveal and archive what has or has not escaped from such change. https://artnodes.uoc.edu Figure 18, Participants’ feedback form generated by the questionare using Google Form, Courtesy of the art-ist/writer Tele-synaesthesia performance does not necessarily offer a solution to cope with limitation imposed by the physical distance for human-technology and human-human communication, but to propose a question that artists could ask themselves: What is the creative use of the technological uncertainty and the em bodiment of the precariousness in the society? How to become present in relation to the advancement virtual spaces and digital developments? Why do we use work with technology and internet in the first place? Glitch as a non-performative refusal – Embracing the change in the suture of fantasy When we experience glitch in online communication, words become fragmented, and images start to embody an involuntary instability that disrupts the integrity of our perception and re-contextualize our vision in a different time and space. Aesthetically, the “clumsiness” of the tech worked really well with the absurdity of the interaction (even if the initial glitching was quite maddening). I was very curious to see how Ada would re-imagine haptic interaction across the virtual divide and was gratified to expe rience her wonderfully barmy and playful approach to this challenge. (Selected response from participant’s post-performance feedback) …When Ada moved her ‘hands’ along the hairline on my forehead in longer movements I could ‘feel’ that - days later I still can. As the session progressed I went from trying to keep my head still Each glitch is unique, as described in Glitch Feminism: A Manifes to, by Legacy Russel: ‘errors bring new movement into static space; Revista científica electrónica impulsada por la UOC Revista científica electrónica impulsada por la UOC Artnodes, no. 28 (2021) I ISSN 1695-5951 2021, Ada Xiaoyu Hao 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao f hi di i b C Universitat Oberta de Catalunya artnodes artnodes Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in e glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu Conclusion Figure 18, Participants’ feedback form generated by the questionare using Google Form, Courtesy of the art-ist/writer Tele-synaesthesia performance could be a novel performance art practice that transposes differences between individuals into open- contacts that make the most use of online communication. Instead of only using online communication as a platform for presentation, this essay stress the awareness of the mutability of glitch in internet- situated performance and its potential to activate new sensorimotor through the techno-aesthetics that re-cartograph the embodied experience in a globalised network culture, without ‘upholding the unity of space and time as its governing principle’. (Terranova 2004) In addition to Foucault’s conception of heterotopia, heterotopia also refers to the displacement of a part of the body from its normal position in the context of medicine. The playful connections and intersections between body and space the internet-situated perfor mances that I discussed in this essay , like Stelarc’s implantation of a third ear on his own arm, have enabled the performing bodies to virtually be in contact with each other in different precarious Revista científica electrónica impulsada por la UOC Artnodes, no. 28 (2021) I ISSN 1695-5951 10 2021, Ada Xiaoyu Hao 2021 f hi di i b C Universitat Oberta de Catalunya artnodes Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu spaces of time, while bearing the spatial existence and movement of each other virtually. relationship between techno-aesthetic and embodied experience of participation, but also creates a spatio-temporal virtual healing scenario to allow individuals to experience the frictions of adapting to change and to transpose the feeling of lonliness and hopeless into individual’s structural ability to actualize oneself as a process of transformation. Even though it is urgent to think of online com munication not only as a medium to disseminate the network of relations or a technology for human-computer interaction, but also a material-based practice that posits a code of conduct, which could be disrupted, interrogated and dismantled for the purpose of making the most complete use of its potential for personal expression. It is more urgent that ever to create a sustainable practice of care that initiates interconnections among individuals that are not bound by the consumptive nature of online communication and the nostalgic desire for “normal” socialization that resorts into human-human interaction only. Conclusion When the physical reciprocal contact is absent, the eye become an organ that takes on a tactile functionality. Is touch visible? Could you feel me touching you with your eyes? When the continuity and normality of the common everyday places are disrupted, how do we recontextualize our relationships with each other? Based on Marks’s theory of haptic visuality, that uses visual sensation to create tactile response in the body (Marks 2002), and Bennett’s virtual touch, that embodies a visceral experience with the feelings of touch to be elicited through the visual senses, ‘transcending the presence of physical contact’ (Bennett 2020), these internet-situated performances have constructed a model of creating virtual-real-heterotopic spaces, where virtual touching could become the reciprocal sensor recep tors that feed into the playful interaction between the performer, the action-performers, and the participants. The Best Facial operates as a ‘heterotopia of deviance’, that gestures toward the ‘fissure within which new possibilities of being and becoming manifest’ (Russell 2020, 11)27, and emplaces the de layed feedback without inserting a compulsory etiquette that asks for immediate reaction or interaction from the participant. The embodied nature of internet-situated performance, as dis cussed in the essay, should be placed at the very heart of online communication to “suture” together the imaginable and the possible. The very reason that I consider internet-situated performance hete rotopic is a reflection of the absence of deviant spaces in our society. If the pandemic is a spatial-temporal phenomenon that demands a remapping of our social interactivity, we should consider this change of pace as a catalyst to find new coordinates and scores for online communication, such as glitch and internet latency, to sustain the symbiotic relationship between the mutability of technological anxiety and the creativity in art practice and research. This new preceptive displacement of the way we interact with each other has shifted from the pre-Covid-19 times and emplaced our perception toward change with a new perspective. The virtual displacement of our body could potentially change our habitual way of interacting with each other post-Covid-19 pandemic. Because the focus of this change is not necessarily lying at the core of what has changed, but invoking questions such as: How do we adapt to the change? How to access our vulnerability and incapability during the time of change? Conclusion How to use the mutability of technology as a contact point to extend the embodied experience in the temporarily encapsulated space and time? Could there be more virtual-real heterotopias out there? 27. It also resembles the pandemic and delivers a message that we could walk out of them with hope for the unforeseen future. It is important to know that we could live through the pandemic and continue living without retreating to the sheltered position. References (SAGE), Scientific Advisory Group for Emergencies. 2020. “SPI-B: Well- being and Household Connection: the behavioural considerations of ‘Bubbles’.” GOV.UK. June 12. Accessed March 21, 2021. https:// assets.publishing.service.gov.uk/government/uploads/system/ uploads/attachment_data/file/892048/S0356_SPI-B_Well- being_and_Household_Connection.pdf. (SAGE), Scientific Advisory Group for Emergencies. 2020. “SPI-B: Well- being and Household Connection: the behavioural considerations of ‘Bubbles’.” GOV.UK. June 12. Accessed March 21, 2021. https:// assets.publishing.service.gov.uk/government/uploads/system/ uploads/attachment_data/file/892048/S0356_SPI-B_Well- being_and_Household_Connection.pdf. These questions about change function as guiding navigational tools to allow us to tell the difference between different flow of change and mutation, in particular the reactive changes made out of fear and the affirmative empowerment of alternative embodied experiences we could create and develop in the process of changing. Bennett, Naomi Petrea. 2020. “Virtual Touch: Embodied Experiences of (dis)Embodied Intimacy in Mediatized Performance.” LSU Digital Commons. May. Accessed March 22, 2021. https://digitalcommons. lsu.edu/gradschool_dissertations/5225. Like getting a facial treatment in beauty clinics, this performance is a creative resource of imagination that constantly renews the sense-making process for the subject self. The slogan ‘Say Goodbye to Hopelessness’ ironically creates a false hope, which negates the assumption that we could forget about what happened in the past and get back to “normal” way of living, especially to a life post- pandemic. The Best Facial not only exposes a new virtual territory for performative interventions that attempt to negotiate a reciprocal Braidotti, Rosi. 2011. Nomadic Theory: The Portable Rosi Braidotti. New York: Columbia University Press. Cytowic, Richard E. 2018. Synesthesia / Richard E. Cytowic, M.D. Cambridge, MA: MIT Press. DOI: https://doi.org/10.7551/mitpress/10568.001.0001 Revista científica electrónica impulsada por la UOC Artnodes, no. 28 (2021) I ISSN 1695-5951 11 Universitat Oberta de Catalunya artnodes s://artnodes.uoc.edu Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic Making touch visible with the suture of fantasy with virtual aesthetician in glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic artnodes artnodes Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic https://artnodes.uoc.edu Miller, Jacques-Alain. 1977. “Dossier Suture: Suture (elements of the logic of the signifier).” Screen 18 (4): 24-34. DOI: https://doi. org/10.1093/screen/18.4.24 Cytowic, Richard E. 2002. Synaesthesia: A Union of the Senses. Cambridge, Massachusetts: The MIT Press. DOI: https://doi. org/10.7551/mitpress/6590.001.0001 Foucault, Michel. 1986. “Des Espace Autres (Of Other Spaces: Utopias and Heterotopias).” October: 1-9. Paul Sermon, Randall Packer, Gregory Kuhn. 2020. Pandemic Encou nters: BE-ING[TOGETHER] IN THE DEEP THIRD SPACE. May 23. Accessed March 28, 2021. https://research.brighton.ac.uk/en/ publications/pandemic-encounters-beingtogether-in-the-deep- third-space. Groys, Boris. 2010. “The Weak Universalism.” e-flux (15). Halyes, N. Katherine. 2012. How we think: digital media and contemporary technogenesis. Chicago and London: The Uni versity of Chicago Press. DOI: https://doi.org/10.7208/chica go/9780226321370.001.0001 Puig de la Bellacasa, María. 2017. Matters of care: speculative ethics in more than human worlds. Minneapolis, Minnesota: University of Minnesota Press. Heyrman, Dr. Hugo. 1997. “doctorhugo.org.” July. Accessed March 22, 2021. http://www.doctorhugo.org/synaesthesia/e-tsyn.htm#:~:t ext=Tele%2Dsynaesthesia%3A%20virtual%20interactions%20 between,constraints%20of%20the%20human%20senses. Russell, Legacy. 2020. Glitch Feminism: A Manifesto. London, NY: Verso. Sermon, Paul. 2020. Be creative with your videoconferencing, make it memorable, it makes a difference. 21 April. Accessed March 20, 2021. https://blogs.brighton.ac.uk/creative/2020/04/21/ be-creative-with-your-videoconferencing-make-it-memorable- it-makes-a-difference/. Marks, Laura U. 2002. Touch: sensuous theory and multisensory media. Minnesota, MN: University of Minnesota press. Mental Health Foundation. 2021. “One year of covid: loss and gain.” mentalhealth.org.uk. February 25. Accessed March 21, 2021. https://www.mentalhealth.org.uk/coronavirus/one-year-covid- loss-and-gain. Sermon, Paul. 2020. Pandemic Encounters. May 23. Accessed March 27, 2021. http://paulsermon.org/pandemic-encounters/. Terranova, Tiziana. 2004. Network culture: politics for the information age. London: Pluto Press. 12 Revista científica electrónica impulsada por la UOC 2021, Ada Xiaoyu Hao Artnodes, no. 28 (2021) I ISSN 1695-5951 2021, of this edition by FUOC CC CC Artnodes, no. 28 (2021) I ISSN 1695-5951 Revista científica electrónica impulsada por la UOC 2021, Ada Xiaoyu Hao 2021, of this edition by FUOC CC CC https://artnodes.uoc.edu artnodes Universitat Oberta de Catalunya Making touch visible with the suture of fantasy with virtual aesthetician in “The Best Facial Clinic” – The glitchy-score of tele-synaesthesia perfor-mance in the age of global pandemic CV Universitat Oberta de Catalunya CV CV Ada Xiaoyu Hao University of Brighton oahada@gmail.com Current PhD Projects: https://aproductionofmultiplebecomingsofs jectivities.cargo.site/Desktop Artist website: https://www.adahao.org/ Performance Artist Researcher Born in China. Currently live and work in London, UK Current PhD Projects: https://aproductionofmultiplebecomingsofsub jectivities.cargo.site/Desktop Artist website: https://www.adahao.org/ Performance Artist Researcher Born in China. Currently live and work in London, UK Current PhD Projects: https://aproductionofmultiplebecomingsofsub jectivities.cargo.site/Desktop Artist website: https://www.adahao.org/ Performance Artist Researcher Born in China. Currently live and work in London, UK I’m an artist-researcher, born in China and currently doing a practice- based Ph.D. in the Doctoral School of Art & Communication, at University of Brighton. My practice is situated within performance practice, using role-playing and fiction as methods to trigger and explore the emer gences of spatial-temporal ‘heterotopias’ (spatial otherness), in relation to the futurity of human beings under the veil of “becoming multiple”. I’m interested in exploring ways to renew individual subjectivity and to embody a speculative envisaging of what the human body could be or become. During such diffractive process of becoming, the performan ce space becomes a virtual site where social attitudes and cultural identities are negotiable. My recent research output includes a digital peer-reviewed visual essay for The International Journal of Creative Media Research’s Digital Ecologies: Fiction Machines (Issue 5), digital collaboration with British Art Studies’ Issue 13, and a peer-reviewed article for the Journal of Contemporary Chinese Art 2021’s Special Issue: ‘The World, Two Meters Away’. 13 A UOC scientific e-journal 2021, of this edition by FUOC CC CC Artnodes, no. 28 (2021) I ISSN 1695-5951 2021, Ada Xiaoyu Hao A UOC scientific e-journal A UOC scientific e-journal Artnodes, no. 28 (2021) I ISSN 1695-5951 Artnodes, no. 28 (2021) I ISSN 1695-5951 2021, of this edition by FUOC CC CC 2021, Ada Xiaoyu Hao
https://openalex.org/W2906015197	https://qmj.qu.edu.iq/index.php/QMJ/article/download/652/542	Arabic	null	Phylogenetic Analysis of MERSCoV in Human and Camels in Iraq	Al-Qadisiah medical journal	2,018	cc-by	5,189	الخالصة ا ان فايروس متالزمة الشرق االوسط التنفسيه MERS-CoV يصيب االنسان يقع ضمن التصنيف الوراثي لفايروسMERS-CoV المعزول من الجمال ممايؤكد االهميه المشتركة والدور الذي تلعبة الجمال ف ي نقل االصابه الى االنسان.صممت الدراسة الحالية للتحري عن المرض ألول مرة في العراق ودرا سة بعض المظاهر الوبائية له اضافة الى التوصيف الجزيئي وتحديد العالقه الفيلوجينيه لهذا الفايرو س ولل فترة من ت شرين األول2015 الى شباط2016 .عن طريق جمع100 نموذج 94 من ال مسحات االنفية و و9 من الم س حات الفمية البلعومية في الجمال من مختلف االعمار وفي مناطق مختلفه,اذ تم التحري عن المستض دات الفيروس بأستخدام اختبار الترحيل المناعي السريعImmunochromatographic assay (ICA) اظهرت النتائج ان28 من العينا ت المفحوصة اعطت نتائج موجبة في حين لم تسجل فروق%م عوية ( بين الجنسين و سجلت الفئة العمرية من5-10) سنه اعلى نسبة اصابة36.84%)) مع وجود فروق م عنويه تحت مستوى احتماليهp<0.05 كما اعطت كال النوعيين من العينات نتائج موجبة مع عدم وجود فروق . معنوية احصائيا بين المسحة االنفية والفموية البلعومية ا كما اظهرت النتائج طبقا الى المناطق الدراسة المختلفة ان الشنافية كانت اعلى النسب (50%) مع وجود فروقات معنوية بين المناطق الدراسة تحت مستوى احتماليةp<0.05 ,في حين سجل شهر ت شرين االول ( اعلى نسبة اصابة (40%بالمقارنة مع اشهر الدراسة مع وجود فرق معنو ية تحت مستوى احتمال يةp<0.05 . تم في هذه الدراسة استخدام التقنية الجزيئية للتحقق من النتائج وذلك عن طريق استخالص الحامض النووي الرايبيRNA لفيروسMERS-CoV وقياس تركيزه ونقاوته بواسطة جهازNanodrop بعد ذلك تم تحويلRNA الىcDNA عن طريق استخدام االستنسا خ العكسيReverse transcriptase ليتسنى التحري عن قطعة الجين المشفرة لبروتين الغالف النوويNucleocapsid gene وذلك بأستخدام بادئات primers , ومجس خاصTaq man pobe ( وتطبيقها تقنية تفاعل سلسة البلمرة في الوقت الحقيقيRT- qPCR ) . كانت النسبة الكلية لألصابة 15% في حين تراوحت دورات العتبةThreshold cycles ( (CT ( من12-22 ) دورة. على اساس الجنس لم تكن هناك فروقات معنوية بين الجنسين بينما سجلت الفئة العمرية اكثر (من عشرة سنوات اعلى نسبة اصابة(25% مقارنة بالفئات العمرية االخرى مع وجو د فروق معنويه تحت مستوى احتماليةp<0.05 ( .وقد سجلت شهر تشرين االول اعلى النسب%40 ) مقارنة بأشهر الدراسة االخرى مع وجود فروق المعنوية تحت مستوى احتمالp<0.05 . عp كما اظهرت النتائج تفاعل سلسلة البلمرة في الوقت الحقيقي لعينات الجمال في المناطق المختلفة ان اعلى النسب ( تم تسجيلها في المنطقة الشنافية35 % ) وهي مطابقة لما تم تسجيلها لهذة العينات عن طريق الفحص السريع (ICA ) مع وجود فروقات معنويه تحت مستوى احتماليهp<0.05 . Phylogenetic Analysis of MERSCoV in Human and Camels in Iraq q Saba F. Al salihi,Zoonotic diseases unit, Vet. Med. Collage, Al-Qadisiyah University. Mohsen A. Alrodhan,collage of pharmacy, Al-Qadisiyah University.ا الخالصة ان نتائج بناء الشجرة الوراثية والتحليل الفيلوجيني اظهرت ان جميع العتر العراقية و قعت ف ي ()العرق(بB Clade )الذي يضم العتر السعودية ويشمل عتر الجمال واالنسان وفي نفس الفرع عل ى الشجرة الفيلوجينية بينما كانتMERS_IQ.2Huh برقم تسلسل KX150501.1 تعود الى عتر اال نسان في هذ ة ( الدراسة ضمن العرق أ ) ) Clade A ) ) والذي وقعت ضمن نفس الفرع المنفصل للعتر االردن ية والمصريه وEMC , بينما كانت عتر الخفافيش والسارس خارج مجموعةMERS-CoV ومن ضمن عائلة الكورونا خارج المجوعة تماما وبشكل منفصل في جذر خاص( Out group ) يمكن ان نستنتج ان فيروساتMERS-CoV منتشرة وبشكل واسع في الجمال وخصوصا في المناطق الغربية القريبة من الحدود السعودية في حين سجل عدد من الحاالت الموجبة في الحجا ج الع ائدين من % الديار المقدسة في موسم الحج والعمرة وان االنواع الجينية السائدة كانت100 مطابقة ا لى ا لعتر المنتشرة في . الجزيرة العربية ABSTRACT ABSTRACT The present study was conducted to evaluate the genetic relationship among Middle East respiratory syndrome coronavirus (MERSCoV) of human and camels origin at the period from October 2015 to February 2016.One hundred samples were collected from camel and 100 from human. Nighty four from nasal swabs and six from oropharyngeal swabs Camel samples secerned by immunochromatographic assay (ICA) for detection of viral antigen. The total percentage of ICA positivity was 28%. Human and camel samples subjected to Revers transcription real time- PCR and carried out by RNA extraction by using specific primers and Taq- Man-Probe for detection of nucleocapsid gene 113 bp. The total positive result in camels were 15% ,there was no significant difference between sex and type of samples, in relation to the age group the results showed that age group more than ten years of camel was the heights percent. With significant difference at P<0.05. According to the months of the year October recorded the highest infection rate with significant difference at p<0.05. the result of RT-qPCR according to the regions of study showed that Al-shinafyah in western borders of Iraq-Saudi was the highest infection rate 35% .On the other hand ,100 human 81 nasal swabs and 19 bronchial lavage samples were collected from pilgrims and non-pilgrims. The total positive result was 5%. The pilgrims recorded the highest infection rate. The results of conventional PCR by using specific primers for detection of Nucleocapsid gene (217 bp) of MERSCoV. The results were confirmative. Three human and 11 camel positive samples were used in further sequencing and phylogenetic analysis by extraction and purification of the PCR products. Our clones sequence submitted in GenBank-NCBI for accession number. The phylogenetic tree construction and analysis results showed that most of Iraqi variants of camel and human were located in clade-B in which Saudi Arabia strains were clustered. One of our clones (MERS-Iq.2Huh) of accession number KX150500.1 was located in clade-A in the same branch of Jordanian strain while bat corona virus, SARS d i i t l t d i t d The phylogenetic tree construction and analysis results showed that most of Iraqi variants of camel and human were located in clade-B in which Saudi Arabia strains were clustered. ABSTRACT One of our clones (MERS-Iq.2Huh) of accession number KX150500.1 was located in clade-A in the same branch of Jordanian strain while bat corona virus, SARS corona and neoromica corona virus was out group clustered in separated branch. Keywords: MERSCoV, Real Time PCR, Phylogenetic analysis, camel Introduction Middle East Respiratory Syndrome Corona Virus (MERSCoV) is widly spread in Arabian Peninsula and many other Middle East countries surrounded of Iraq [1, 2,3,4,5] . It has become one of the most important emerging human health threating virus [6]. الخالصة بين مناطق الدراسة المختلفة لقد تم في هذه الدراسة جمع100 عينة من الحجاج وغير الحجاج من المسحات االنفية والغ سيل القصبي ( (Nasal swabs &Broncho alveolar lavage sample وفحصهاRT-qPCR للتحري عن قطعة ( الجين المشفر للبروتين الغالف النووي(Nucleocapsid gene لفايروس MERS-CoV اذ كانت النسب % الكلية لألصابة5 وقد تراوحت دورات العتبةCT ( 13 - 17 ) Cycle ولم تسج ل فروقات معنويه ب ين الجنسين كما سجلت الفئة العمرية اكثر من50 ( سنة اعلى نسبة اصابة%10.81) مع وجود فروق معنوية ت حت مستوى احتماليةp<0.05 بين المجاميع العمرية المختلفة . كما اظهرت نتائج الدراسة الحالية ان ج ميع العينا ت الموجبة كانت تعود الى المسحات االنفية ( % 6.71 (ولم يسجل الغسيل القصبي اي نتائج موجبة, كما سجل ش هر تشرين االول %( اعلى نسبة اصابة(13.33 مع وجود فروق معنويه بين اشهر الدراسة تحت مستوى احتم الية p<0.05 . p ( ان النتائج الموجبة بين الحجاج كانت هي%13.33 ) مقارنة مع غير الحجاج مع وجود فرق معنوي تحت مستوى احتمالp<0.05 .وسجلت نتائج تفاعل سلسلة البلمرة التقليديPCR Conventional لل cDNA ( للتحري عن قطعة جين الغالف النوويNucleocapsid gene ) 217b في جل االكاروز ال مصبوغ بروميد االثيديومEthidium bromide . وبأستخدام بادئات متخصصة وكانت احدى عشر عينة من عينات الجمال موجبة لهذا االختبار ,في حين ثالثة فقط عينات االنسان كانت موجبة وقد تم استخالص وتنقية ( قطعة الجين ألستخدامها في قراءة تعاقب النيكلوتيداتGene sequence) وا لتحليل الفيلوجيني ( Phylogenetic Analysis ( ) وبعد الحصول على اربعة عشر نسيلةClones ) قدمت في بنك الجينات في 150 AL-Qadisiyah Medical Journal Vol.13 No.23 2017 ( المركز الوطني لمعلومات التقنات االحيائية في الواليات المتحدة االمريكيةGenBank /NCBI ) لغرض التسجيل والحصول على رقم االنضمام وقد اظهرت النتائج الرسال ة االلكترونية لبنك الجينات أن اربعة عشر نسيلة قد سجلت وأعطيت رقم االنضمام الخاص في االنسان كما تم تسجيلها ايضا في ارشيف النيكلوتيدات ( االوروبي(ENA و بنك الجينات الياباني(DDBJ) . Introduction Middle East Respiratory Syndrome Corona Virus (MERSCoV) is widly spread in Arabian Peninsula and 151 Vol.13 No.23 2017 No.23 AL-Qadisiyah Medical Journal 2017 MERSCoV is beta corona virus within coronaviridae family which are enveloped, positive sense RNA genome with nucleocapsid of helical symmetry infect human and variety of animal species [7]. The ability of high recombination, unique viral replication and low fidelity of corona virus polymerases allows for unexpected viral evolution to infect other host.[8,9 ] immunochromatographic assay (ICA)for detection of MERSCoV antigen by using Rapid ( ICA ) MERS-COV Camel Strip kit Bionote Korea. [14] Viral RNA has been extracted by Total RNA Extraction KitAccuZolTM kit bioneer Korea. The extracted RNA has been measured for concentration and purity by Nanodrop. Reverse transcription conducted by AccuPower® Rocket ScriptTM RT PreMix 96 plate kit bioneer Korea and Real Time PCR applied by AccuPower ® Dual star TM qPCR PreMix 96 plate kit bioneer Korea using specific primers (F- TGCAAGCTTTTGGTCTTCGC) (R- AGCAAGCTCAGCAATTTGGG) and Taq- Man-Probe (FAM- TCGGCACTGAGGACCCACGT- BHQ1) for detection of N gene fragment 113 bp [15,16 ]. Phylogenetic analysis of African bat virus belonging to the same species of MERSCoV and indicate that the evolution of the virus in camels precede that in human suggesting the possible spreading from bats to camels took place in Africa and involved exchange of genetic materials among ancestral virus strains[10,11] Nucleocapsid gene (N gene) is common target for cloning phylogenetic analysis and generation recombinants portions. N protein is highly immunogenic phosphoprotein and modulation of cell signaling method. The N gene have been used for corona virus genotyping and phylogenetic analysis which helped our knowledge of virus temporal geographic origins and evolution[12,13].This study was conducted to evaluate the genetic relationship among local circulating MERSCoV variants in human and camel at the first time in Iraq. Conventional end point PCR for detection of N gene fragment 217 bp, The PCR products of positive samples were extracted and purified and sequenced by dye-terminator based sequenced illumina. The genomic sequences were assembled and submitted in GenBank- NCBI then multiple sequence alignment was done by clustal Omega for phylogenetic tree construction and phylogenetic analysis [12, 17]. Results This study was carried out by collection of 100 nasal and oropharyngeal swap samples from camels and 100 nasal swap and bronchial lavage samples from human at the period from October 2015 to February 2016 from both sexes and different ages in many locations of Middle Euphrates/Iraq .Camel samples were subjected to rapid Results The total positive results of MERSCoV antigen detection in camel by rapid test (ICA) was 28% .Statistically, there was no significant difference between sexes and type of samples Table (1). While there were significant difference at P<0.05 among age groups, study locations and month of study Table (2). Table (1) Infection rate according to the sexes and type of sample in camel by using by (ICA) The total positive results of MERSCoV antigen detection in camel by rapid test (ICA) was 28% .Statistically, there was no significant difference between sexes and type of samples Table (1). While there were significant difference at P<0.05 among age groups, study locations and month of study Table (2). e sexes and type of sample in camel by Sex No. of examined samples No. of positive samples infection Percentage (%) Females 83 22 (26.5%)A 152 AL-Qadisiyah Medical Journal Vol.13 No.23 2017 Males 17 6 (35.29%)A Type of sample collection Nasal swabs 94 27 (28.72%)A Oropharyngeal swabs 6 1 (16.66%)A  Similar letters refers to the non-significant differences Table (2) Iinfection rate according to the age, region and month in camel by using ICA. Age No. of examined samples No.  Difference letterers refers to the significant differences at P<0.05  Similar letters refers to the non-significant differences While there were significant difference at P<0.05 between type of sample, age group, months of the study and type of patients (Table 6). Results of positive samples infection Percentage (%) 1month-1 year 9 0 (0%)A 1-5year 41 11 (26.82%)B 5-10 38 14 (36.84%)B >10 12 3 (25%)B Region Al-Diwanyah/Al- shnifyah 20 10 (50 ) A Al-Diwanyah/Al- Shafayah 20 8 (40%)A Al-Diwanyah/ Afak 15 3 (20%)B Al-Diwanyah/ Sumer 10 1 (10%)B Babel/Hamza 5 1 (20%)B Al-Muthana/ Al- Rumetha 10 1 ( 10%)B Slaughterhouse 20 4 (20%)B Month of the year October 20 8 (40%) A November 24 6 (25%)A December 32 8 (25%)A January 8 0 (0%)B February 16 6 (37.5%)A  Difference letterers refers to the significant differences at P<0.05 AL-Qadisiyah Medical Journal Vol.13 No.23 2017 Males 17 6 (35.29%)A Type of sample collection Nasal swabs 94 27 (28.72%)A Oropharyngeal swabs 6 1 (16.66%)A  Difference letterers refers to the significant differences at P<0.05 The positive result of MERSCoV infection in camel by RT-RT-PCR in camel was 15% with different cycles of threshold (CT) ranging from 12-22. These result again statistically showed no significant difference in relation to the sex and type of samples Table (3). The positive result of MERSCoV infection in camel by RT-RT-PCR in camel was 15% with different cycles of threshold (CT) ranging from 12-22. These result again statistically showed no significant difference in relation to the sex and type of samples Table (3). ( ) Table (3) Infection rate according to the sex and type of sample in camel by using RT-qPCR Sex No. Sample No. of positive sample Infection percentage Female 83 12 (14.45%)A Male 17 3 (17.64%)A Type of sample Nasal swab 94 14 (14.89%)A Table (3) Infection rate according to the sex and type of sample in camel by using RT PCR 153 AL-Qadisiyah Medical Journal Vol.13 No.23 2017 Oropharyngeal swab 6 1 (16.66%)A  Similar letters refers to the non-significant differences While there were significant difference at P<0.05 among age group, geographical location and months of the study Table (4). Table (4) The results of infection rate in relation to age groups, region and month in camel by using RT-qPCR technique: AGE groups NO. Samples No.  Similar letters refers to the non-significant differences g While there were significant difference at P<0.05 between type of sample, age group months of the study and type of patients (Table 6).  Difference letterers refers to the significant differences at P<0.05 Results of positive sample Infection Percentage (%) 1month-1 year 9 0 (0%)B 1-5year 41 6 (14.63%)A 5-10 38 6 (15.78%)A >10 12 3 (25%)A Region Aldiwanyah/Al shnifyah 20 7 (35%)A Aldiwanyah/Al shafayah 20 5 (25%)A Aldiwanyah/ Afak 15 1 (6.66%)B Aldiwanyah /Sumer 10 0 (0%)C Babel/Hamza 5 0 (0%)C A-lRumetha/Al sumawa 10 0 (0%)C Slaughterhouse 20 2 (10%)B Month of the year No. Sample No. of positive sample Infection \ Percentage(%) October 20 8 (40%)A November 24 3 (12.5%)B December 32 1 (3.12%)C January 8 0 (0%)D February 16 3 (18.75%)B  Difference letterers refers to the significant differences at P<0.05 The positivity result of human MERSCoV infection by RT-RT-PCR was 5% with AL-Qadisiyah Medical Journal Vol.13 No.23 2017 Oropharyngeal swab 6 1 (16.66%)A AL-Qadisiyah Medical Journal Vol.13 No.23 2017 Oropharyngeal swab 6 1 (16.66%)A ( ) g g p , g in camel by using RT-qPCR technique: AGE groups NO. Samples No. of positive sample Infection Percentage (%) 1month-1 year 9 0 (0%)B 1-5year 41 6 (14.63%)A 5-10 38 6 (15.78%)A >10 12 3 (25%)A Region Aldiwanyah/Al shnifyah 20 7 (35%)A Aldiwanyah/Al shafayah 20 5 (25%)A Aldiwanyah/ Afak 15 1 (6.66%)B Aldiwanyah /Sumer 10 0 (0%)C Babel/Hamza 5 0 (0%)C A-lRumetha/Al sumawa 10 0 (0%)C Slaughterhouse 20 2 (10%)B Month of the year No. Sample No. of positive sample Infection \ Percentage(%) October 20 8 (40%)A November 24 3 (12.5%)B December 32 1 (3.12%)C January 8 0 (0%)D February 16 3 (18.75%)B  Difference letterers refers to the significant differences at P<0.05 g The positivity result of human MERSCoV infection by RT-RT-PCR was 5% with different cycles of threshold (CT) ranging from 13-17 there was no significant difference between gender (Table 5). The positivity result of human MERSCoV infection by RT-RT-PCR was 5% with different cycles of threshold (CT) ranging from 13-17 there was no significant difference between gender (Table 5). g Table (5) Infection rate in sex groups in human by using RT-qPCR technique Table (5) Infection rate in sex groups in human by using RT-qPCR technique Sex No. Sample No. of positive sample Infection Percentage (%) Females 30 2 (6.66%)A Males 70 3 (4.28%)A 154 AL-Qadisiyah Medical Journal Vol.13 No.23 2017 Table (6) Infection rate in human in relation to age groups, type of sample ,month and type of patient by using RT-qPCR technique: Age groups No. Samples No. Results of positive samples Infection Percentage (%) <5year 20 0 (0%)B 5-25 18 0 (0%)B 25-50 25 1 (4%)A 50-80 37 4 (10.81%)A Type of samples Nasal swabs 81 5 (6.17%)A Broncho lavage samples 19 0 (0%)B Months October 30 4 (13.33%)A November 34 1 (2.94%)B December 10 0 (0%)C January 10 0 (0%)C February 16 0 (0%)C Type of patient pilgrim 30 4 (13.33%)A Non_ pilgrim 70 1 (1.42%)B  Difference letterers refers to the significant differences at P<0.05 The result of conventional PCR of camel and human were confirmative Fig.-1, Fig.-2. Fourteen of our clones which were 11 of camel and 3 of human were took their accession number in GenBank-NCBI. (Figure 1): Agarose gel electrophoresis image that show the PCR product analysis of Nucleocapsid -gene of MERSCoV positive Camel Table (6) Infection rate in human in relation to age groups, type of sample ,month and type of patient by using RT-qPCR technique: Table (6) Infection rate in human in relation to age groups, type of sample ,month and type of patient by using RT-qPCR technique: Age groups No. Samples No. of positive samples Infection Percentage (%) <5year 20 0 (0%)B 5-25 18 0 (0%)B 25-50 25 1 (4%)A 50-80 37 4 (10.81%)A Type of samples Nasal swabs 81 5 (6.17%)A Broncho lavage samples 19 0 (0%)B Months October 30 4 (13.33%)A November 34 1 (2.94%)B December 10 0 (0%)C January 10 0 (0%)C February 16 0 (0%)C Type of patient pilgrim 30 4 (13.33%)A Non_ pilgrim 70 1 (1.42%)B ,month and type of patient by using RT-qPCR technique: Age groups No. Samples No. of positive samples Infection Percentage (%) <5year 20 0 (0%)B 5-25 18 0 (0%)B 25-50 25 1 (4%)A 50-80 37 4 (10.81%)A Type of samples Nasal swabs 81 5 (6.17%)A Broncho lavage samples 19 0 (0%)B Months October 30 4 (13.33%)A November 34 1 (2.94%)B December 10 0 (0%)C January 10 0 (0%)C February 16 0 (0%)C Type of patient pilgrim 30 4 (13.33%)A Non_ pilgrim 70 1 (1.42%)B  Difference letterers refers to the significant differences at P<0.05 The result of conventional PCR of camel and human were confirmative Fig.-1, Fig.-2. Fourteen of our clones which were 11 of camel and 3 of human were took their accession number in GenBank-NCBI. Results (Figure 1): Agarose gel electrophoresis image that show the PCR product analysis of Nucleocapsid -gene of MERSCoV positive Camel (Figure 1): Agarose gel electrophoresis image that show the PCR product analysis of Nucleocapsid -gene of MERSCoV positive Camel 155 AL-Qadisiyah Medical Journal Vol.13 No.23 2017 (Figure 2): Agarose gel electrophoresis image that show the PCR product analysis of Nucleocapsid gene in MERSCoV positive Human samples. The result of phylogenetic analysis showed most of Iraqi clones were grouped in clad B and clustered mainly with the same branch of Saudi Arabia and South Korea, only one clone in this study clustered in the same branch of Jordanian in clade A. while bat corona virus, SARSV and neoromica corona virus was out group clustered in separated branch Fig.-3, Fig.-4 AL-Qadisiyah Medical Journal Vol.13 No.23 2017 (Figure 2): Agarose gel electrophoresis image that show the PCR product l i f N l id i MERSC V iti H l (Figure 2): Agarose gel electrophoresis image that show the PCR product analysis of Nucleocapsid gene in MERSCoV positive Human samples. (Figure 2): Agarose gel electrophoresis image that show the PCR product nalysis of Nucleocapsid gene in MERSCoV positive Human samples. The result of phylogenetic analysis showed most of Iraqi clones were grouped in clad B and clustered mainly with the same branch of Saudi Arabia and South Korea, only one clone in this study clustered in the same branch of Jordanian in clade A. while bat corona virus, SARSV and neoromica corona virus was out group clustered in separated branch Fig.-3, Fig.-4 Figure(3)phylogenetic tree of our clones MERS-CoV with references world MERS-CoV and Bat and SARS-Coronavirus and their accession No. Figure(3)phylogenetic tree of our clones MERS-CoV with references world MERS-CoV and Bat and SARS-Coronavirus and their accession No. Figure(3)phylogenetic tree of our clones MERS-CoV with references world MERS-CoV and Bat and SARS-Coronavirus and their accession No. 156 Clade B our clones 156 Figure (4) phylogenetic tree of our clones MERSCoV with references world MERSCoV and Bat and SARS-Coronavirus and the title of strains Discussion and to explain some epidemiological of O clon s Our clonese Figure (4) phylogenetic tree of our clones MERSCoV with references world MERSCoV and Bat and SARS-Coronavirus and the title of strains Discussion MERSC V fi t d d i and to explain some epidemiological of MERS [20 21] and to explain some epidemiological of MERS [20, 21]. Refrences [1] Zaki AM, van Boheemen S, Bestebroer TM, et al. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 2012; 367: 1814–1820 [1] Zaki AM, van Boheemen S, Bestebroer TM, et al. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 2012; 367: 1814–1820 [2] Reusken CB, Farag EA, Jonges M, et al. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RNA and neutralising antibodies in milk collected according to local customs from dromedary camels, Qatar,. Eurosurveillance ; 2014 April,19: 20829. [3] Alexandersen S, Kobinger GP, Soule G, Wernery U. Middle East respiratory Syndrome Coronavirus antibody reactors among camels in Dubai, United Arab Emirates, in 2005. Transbound. Emerg. Dis.; 2014,61:105–8 [4] Reusken CB, Ababneh M, Raj VS, et al. Middle East Respiratory Syndrome coronavirus (MERS-CoV) serology in major livestock species in an affected region in Jordan, June to September. Eurosurveillance : 2013, 18 (50):20662 Phylogenetic analysis demonstrate that most Iraqi variants of MERSCoV of camel and human of this study fell in the clade-B with Saudi strains as well as 2015 South Korea outbreak strains [ 31 ,32, Phylogenetic analysis demonstrate that most Iraqi variants of MERSCoV of camel and human of this study fell in the clade-B with Saudi strains as well as 2015 South Korea outbreak strains [ 31 ,32, 17 ] due to continuous mixing and introduction of camel across Saudi borders in addition to large number of travelers in Hajj season . Furthermore recent recombination and emergence of that novel virus play a role in close relation and high identity among these srrains of MERSCoV. [5] Alagaili AN, Briese T, Mishra N, et al. Middle East Respiratory Syndrome Coronavirus infection in dromedary camels in Saudi Arabia. MBio; 2014, 5(2): e00884-14. [6]Alqabandi S, Al-Qasser, Madi; Detection of MER circulation in Kuwait .Infec. dis. & Threapy 2013 Vol 1 (4) [6]Alqabandi S, Al-Qasser, Madi; Detection of MER circulation in Kuwait .Infec. dis. & Threapy 2013 Vol 1 (4) [6]Alqabandi S, Al-Qasser, Madi; Detection of MER circulation in Kuwait .Infec. dis. & Threapy 2013 Vol 1 (4) [7] Shehata MM,Gomma Mr , Ali AA and Ghai K MERSCoV comprehensive review in Frontiers of Medicine.www.Researchgate.net, 2012 [8] Zhang, Z., Shen, L &Gu,X. Evolutionary dynamics of MERS-CoV :Potential Recombination ,positive Selection and transmission . 2016, Results MERSCoV was first recorded in 2012 in Saudi Arabia, the virus associated with severe respiratory illness, renal failure and high rate of mortality 50% [18, 19] .The emergence of this important infectious pathogen has raised global concerns regarding the current epidemiological features and its future evolution. Camels may considered the potential source of human infection and act as reservoir transmit the virus to human. This study was designed for molecular characterization of the virus High prevalence of infection in camels recorded by using rapid (ICA) and definitive molecular techniques although our results were lower than that revealed by many previous serological surveys [4, 22, 23, 24]. The detection of MERSCoV antigen in combination with viral RNA demonstration indicate likely presence of infectious virus as compare with serological tests [ 14 ] ,in which the positive MERSCoV antibodies may developed from past exposure without virus shedding , as well as most of those 157 Vol.13 No.23 2017 AL-Qadisiyah Medical Journal tree with about 100% identity that indicates the role of camel as a reservoir or intermediate host in zoonotic transmission. Application RT-RT-PCR screening technique in combination with wide seroepidemiological surveys for the disease in aiding in the strict study conducted in endemic locations . The results of this study showed geographical variability in camels infection, the highest rate was at Iraq- Saudi borders in which mixing of grazing camels in same pastures across these borders study conducted in endemic locations . The results of this study showed geographical variability in camels infection, the highest rate was at Iraq- Saudi borders in which mixing of grazing camels in same pastures across these borders Despite of high infection rate of camels[25,26,27,28] and direct contact of camels’ owners including consumption of animals products we did not recorded any positive results , may cause by difficulty in transmission as lower respiratory tract shedding of the virus ,and presence of some degree of immunity ,actually there was lack in active surveillance programs and databases, so further seroepidemiological studies was recommended . On the other hand there was positive results recorded among pilgrims whom they may becoming infected during period of pilgrimage in Mecca / Saudi Arabia [29,30] mentioned the risk of pilgrims returning with MERSCoV from human to human transmission in that large mass gathering of Hajj. Conclusions 6:25049;doi:10.1038/srep25049. MERS was widely distributed in apparently healthy camels at the western borders of Iraq, the risk of pilgrims returning with MERSCoV during the large mass gathering of annual Hajj and minor Umrah must be considered. Genotypicatlly human and camel variants fell in the same branch of phylogenetic [9] Kandil A,Shehata MM,El Shesheny R, Gomma MR ,Ali MA, Kayali Complete genome sequences of MERSCoV isolated from dromedary camel in Egypt . Am. Society For Microbiology 2016 Vol.4, [9] Kandil A,Shehata MM,El Shesheny R, Gomma MR ,Ali MA, Kayali Complete genome sequences of MERSCoV isolated from dromedary camel in Egypt . Am. Society For Microbiology 2016 Vol.4, [10] Chastel C. [Middle East respiratory syndrome (MERS): bats or dromedary, which of them is responsible?]. Bull Soc Pathol Exot 2014; 107(2):69-73. 158 Vol.13 No.23 2017 No.23 2017 AL-Qadisiyah Medical Journal [11] WHO). Middle East respiratory syndrome coronavirus (MERS-CoV) Available at : www.who.int emergencies /mers/en/[accessed January 28,2016] [22] Perera RA, Wang P, Gomaa MR, et al. Seroepidemiology for MERSCoV using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013. Eurosurveillance 2013; 18: 20574. y [12] Cotten M, Lam TT, Watson SJ, Palser AL, Petrova V, Grant P, et al. Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus. Emerg. Infect. Dis..;2013 19:736–742B. [23] Reusken CB, Haagmans BL, Muller MA, et al. Middle East Respiratory Syndrome Voronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. Lancet Infect Dis 2013 Vol.13 No.10. [13] van Boheemen S, de Graaf M, Lauber C, Bestebroer TM, Raj VS, Zaki AM, et al. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. MBio. 2012; 3(6):e00472–12. [24] Gossner C, Danielson N, Gervelmeyer A, et al. Human-dromedary camel interactions and the risk of acquiring zoonotic Middle East Respiratory Syndrome Coronavirus infection. Zoonoses Public Health published online, 2014 Dec 27. DOI:10.1111/zph.12171. [24] Gossner C, Danielson N, Gervelmeyer A, et al. Human-dromedary camel interactions and the risk of acquiring zoonotic Middle East Respiratory Syndrome Coronavirus infection. Zoonoses Public Health published online, 2014 Dec 27. DOI:10.1111/zph.12171. [25] Smits SL, Raj VS, Pas SD, Reusken CBEM , et al. Reliable typing of MERS-CoV variants with a small genome fragment. J Clin Virol.; 2015, 64:83–7. [14]Song D, Ha G, Serhan W, Eltahir Y, et al. Conclusions Development and validation of a rapid immunochromatographic assay for detection of Middle East Respiratory Syndrome Coronavirus antigen in dromedary camels. J Clin Microbiol, 2015, 53:1178 –1182 [25] Smits SL, Raj VS, Pas SD, Reusken CBEM , et al. Reliable typing of MERS-CoV variants with a small genome fragment. J Clin Virol.; 2015, 64:83–7. [15] Mackay IM and K E. Arden, “MERSCoV: diagnostics, epidemiology and transmission,” Virology Journal, 2015 vol. 12, no. 1, article 222, [26] Farag EA, Reusken CB, Haagmans BL, Mohran KA, et al.. High proportion of MERS- CoV shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, Qatar 2014. Infect Ecol Epidemiol. 2015, 5:28305. 19:736–742B. [16] Bhadra S, Jiang YS, Kumar MR, et al. (2015). Real time sequence-validated loop- mediated isothermal amplification assays for detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV). PLoS One.; 10, e0123126. [27] Wernery U, Corman VM, Wong EY, Tsang AK, Muth D, Lau SK, et al.Acute Middle East Respiratory Syndrome Coronavirus infection in livestock Dromedaries, Dubai,. Emerg. Infect. Dis. 2015, 21:1019–22. [17] Kim Y ,Cheon S, Min C, Sohn KM) Spread of Mutant Middle East Respiratory Syndrome Coronavirus with Reduced Affinity to Human CD26 during the South Korean Outbreak .Am. Society for Microbiology 2016 ,Vol.7,2, [18] Hemida MG, Chu DK, Poon LL, et al. MERSCoV in dromedary camel herd, Saudi Arabia. Emerg. Infect. Dis; 2014, 20(7):1231-4 [28] Zumla A, Hui DS, Perlman S. Middle East respiratory syndrome. Lancet; doi: 2015, 10.1016/S0140-6736(15)60454-8 (in press). [29] Zumla A, Mwaba P, Bates M, et al. The Hajj pilgrimage and surveillance for Middle East Respiratory Syndrome Coronavirus in pilgrims from African countries. Trop Med Int Health 2014; 19 (7):838-40 [19] Yusof MF, Eltahir YM, Serhan WS, Hashem FM, et al. Prevalence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in dromedary camels in Abu Dhabi Emirate, United Arab Emirates [cited 2015 Feb Virus Genes. 2015 [Epub ahead of print] http://www.ncbi.nlm.nih. Gov/PubMed/25653016, 2015: Feb 5 [20] Hemida MG, Perera RA, Al Jassim RA, et al. Seroepidemiology of Middle East Respiratory Syndrome (MERSCoV) in Saudi Arabia and Australia (2014) and characterisation of assay [30] Soliman T,Cook AR, and Coker RJ, Pilgrims and MERSCoV : what ʼs the risk ,emerging themes in epidemiology 2015:12 ,3. [31] Oboho IK, Tomczyk SM, Al-Asmari AM, Banjar AA, Al-Mugti H, Aloraini MS, et al. MERS-CoV outbreak in Jeddah–a link to health care facilities. N Engl J Med. Conclusions 2014;2015(372):846–54 [32] Zhang, Z., Shen, L &Gu,X. Evolutionary dynamics of MERS-CoV :Potential Recombination ,positive Selection and transmission g g p gy [31] Oboho IK, Tomczyk SM, Al-Asmari AM, Banjar AA, Al-Mugti H, Aloraini MS, et al. MERS-CoV outbreak in Jeddah–a link to health care facilities. N Engl J Med. 2014;2015(372):846–54 Gov/PubMed/25653016, 2015: Feb 5 [20] Hemida MG, Perera RA, Al Jassim RA, et al. Seroepidemiology of Middle East Respiratory Syndrome (MERSCoV) in Saudi Arabia and Australia (2014) and characterisation of assay specifi city. Eurosurveillance 2014; 19: 20828. [21] Chu DK, Poon LL, Gomaa MM, et al(MERSCoV in dromedary camels, Egypt. Emerg Infect Dis 2014; 20(6): 1049-53. (2016).6:25049;doi:10.1038/srep25049. (2016).6:25049;doi:10.1038/srep25049. 159
https://openalex.org/W1957915213	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0134078&type=printable	English	null	Neural Resolution of Formant Frequencies in the Primary Auditory Cortex of Rats	PloS one	2,015	cc-by	12,009	RESEARCH ARTICLE a11111 Neural Resolution of Formant Frequencies in the Primary Auditory Cortex of Rats Christian Honey1, Jan Schnupp2* Christian Honey1, Jan Schnupp2* 1 Berlin, Germany, 2 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom Christian Honey1, Jan Schnupp2* 1 Berlin, Germany, 2 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom 1 Berlin, Germany, 2 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom * jan.schnupp@dpag.ox.ac.uk Introduction Received: March 6, 2014 Accepted: July 6, 2015 Published: August 7, 2015 Copyright: © 2015 Honey, Schnupp. 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. Pulse-resonance sounds (PRSs) are the backbone of most animals vocalization [1] and vowels in human speech are a prominent member of this sound class. They are richer and more eco- logically relevant than the pure tones or noise bursts more commonly used in physiological studies, but they remain simple enough to be described by a relatively small number of parame- ters. In PRSs, a periodic source produces a regular, click-train like input to a set of resonators. The sound consequently consists of a series of harmonics of unequal amplitude, and the har- monics closest to the peak resonant frequencies (the so-called “formant frequencies”) are larg- est. Most mammalian vocal sounds, including vowels in human speech, are examples of PRSs, in which the fundamental frequency of the harmonics is determined by the glottal pulse rate emanating from the larynx, and the formant frequencies depend on the size of resonant cavities in the vocal tract. Thus, the pitch of such vocal sounds depends on their fundamental fre- quency, but the “identity” of the sound is mostly recognized from its formant frequencies [2]. Received: March 6, 2014 Accepted: July 6, 2015 Published: August 7, 2015 Copyright: © 2015 Honey, Schnupp. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: March 6, 2014 Accepted: July 6, 2015 Published: August 7, 2015 Copyright: © 2015 Honey, Schnupp. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Christian Honey is supported by a Wellcome Trust 4-year doctoral program. Both authors are supported by a BBSRC grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Editor: Sliman J. Bensmaia, University of Chicago, UNITED STATES Editor: Sliman J. Bensmaia, University of Chicago, UNITED STATES Abstract Pulse-resonance sounds play an important role in animal communication and auditory object recognition, yet very little is known about the cortical representation of this class of sounds. In this study we shine light on one simple aspect: how well does the firing rate of cortical neurons resolve resonant (“formant”) frequencies of vowel-like pulse-resonance sounds. We recorded neural responses in the primary auditory cortex (A1) of anesthetized rats to two-formant pulse-resonance sounds, and estimated their formant resolving power using a statistical kernel smoothing method which takes into account the natural variability of cortical responses. While formant-tuning functions were diverse in structure across differ- ent penetrations, most were sensitive to changes in formant frequency, with a frequency resolution comparable to that reported for rat cochlear filters. a1111 OPEN ACCESS Citation: Honey C, Schnupp J (2015) Neural Resolution of Formant Frequencies in the Primary Auditory Cortex of Rats. PLoS ONE 10(8): e0134078. doi:10.1371/journal.pone.0134078 OPEN ACCESS Citation: Honey C, Schnupp J (2015) Neural Resolution of Formant Frequencies in the Primary Auditory Cortex of Rats. PLoS ONE 10(8): e0134078. doi:10.1371/journal.pone.0134078 Editor: Sliman J. Bensmaia, University of Chicago, UNITED STATES * jan.schnupp@dpag.ox.ac.uk Neural Resolution of Formant Frequencies in Rat A1 study to date has used PRSs to explore the sensitivity of cortical responses to small changes in the position of formant peaks, or charted the distribution of such sensitivity. Studying these aspects of tuning to acoustic features of complex sounds may be revealing when one seeks to understand the role particular parts of the auditory pathway play in phonetic tasks. Consider, for example, that neurons involved in recognizing speech sound categories are unlikely to dis- criminate acoustic features at the very physiological limits of frequency selectivity, so that pho- netically unimportant "within phone category" variability of the acoustic parameters does not lead to radically different neural activation patterns [11]. Consequently one might expect to see neural tuning curves to broaden somewhat to reflect acoustic category distributions as one ascends along a neural pathway involved in phonetic processing. To what extent this expecta- tion is borne out is not entirely clear. Cortical pure tone tuning curves are normally consider- ably broader than those seen at the lowest stations of the lemniscal auditory pathway. However, tuning curves or spectro-temporal receptive fields of cortical neurons often predict the same neurons' responses to complex sounds poorly [12–14]. Consequently it may not be advisable to try to understand the neural representation of the formants of pulse-resonance sounds by extrapolating from pure tone tuning functions. However, tuning curves or spectro-temporal receptive fields of cortical neurons often predict the same neurons' responses to complex sounds poorly [12–14]. Consequently it may not be advisable to try to understand the neural representation of the formants of pulse-resonance sounds by extrapolating from pure tone tuning functions. In this study we used simple, synthetic PRSs with two formant peaks (2FPRSs) to map out cortical ‘formant receptive fields’ and to estimate the frequency resolution at which these recep- tive fields operate. We chose two formant sounds because animal vocal sounds are often char- acterized by particular formant combinations. Indeed, the identity of human vowels appears to be determined by their position in “F1/F2 formant space” [15], where F1, F2 refer to the lowest and second lowest formant frequencies. We also wanted to keep the stimuli simple and generic, choosing sounds that are just complex enough to span a “formant space” and to mimic the characteristic pulse-resonance nature of many natural sounds, but simple enough to avoid add- ing unnecessary complexity to the stimuli. We used rats as a model species as they are known to discriminate and categorize PRSs, such as human speech sounds, at high levels of performance both in their behavior [16,17] as well as in their cortical physiological responses [18]. Cortical responses to sounds exhibit considerable trial-to-trial variability [19,20], and both for the experimental observer and for the brain itself the question whether a sensory neuron is able to discriminate between two similar stimuli essentially reduces to the question whether observed response differences are due to more than just the level of chance variability which might be expected if the same stimulus was presented twice. In order to achieve statistically robust esti- mates of the “resolving power” of neural formant tuning functions in the presence of such “phys- iological noise” we devised and applied a novel measure, based on kernel smoothing [21]. Our results reveal highly variable formant receptive fields structures across the cortical pop- ulation, but despite the diversity and variability of the observed response patterns, most neural responses nonetheless appear to resolve formant frequencies with a similar, high resolution of 0.1 octaves or better. Formant resolution tended to be very similar along anatomically neigh- boring recording sites, but varied considerably across different penetrations. Introduction Given the ecological importance of this class of sound, responses of cortical neurons to nat- ural or artificial vocal sounds have been studied in gerbils [3], ferrets [4–6] monkeys [7], guinea pigs [8,9] and humans [10]. However, most studies so far have focused on the strength of the neural response as a func- tion of large changes in stimulus parameters such as fundamental or formant frequencies. No Competing Interests: The authors have declared that no competing interests exist. 1 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Surgery All animal procedures were approved by Oxford University’s “Committee on Animal Care and Ethical Review” and performed under license from the UK Home Office in accordance with the Animal (Scientific Procedures) Act 1986. Four naive adult female rats (Sprague-Dawley, 240–265 g), were used for extracellular recordings from A1 under terminal general anesthesia. Anesthesia was induced by intraperitoneal injection of ketamine (Ketaset; Fort Dodge Animal Health, 2 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Neural Resolution of Formant Frequencies in Rat A1 Overland Park, Kansas, USA) and medetomidine (Domitor, Pfizer, Walton Oaks, Surrey, UK), and maintained with intraperitoneal infusions of ketamine, medetomidine and butorphanol tar- trate (Torbugesic; Fort Dodge Animal Health, Overland Park, Kansas, USA), at typical rates of 4.0 mg/kg/h, 16 μg/kg/h and 0.5 mg/ kg/h, respectively. Medical oxygen was administered at 0.5l/ h to the animals’ snout. The animals' core temperature was monitored and kept at 38°C using a rectal temperature probe and homeothermic blanket. Heart rate was monitored throughout the experiment and the depth of anesthesia was confirmed with regular paw and eye-lid reflex tests. After inducing anesthesia, the skull was exposed and a 3–4 mm diameter craniotomy was performed over auditory cortex at coordinates 5 mm anterior of bregma, 7.2 mm lateral to the right of the midline, and 4.5 mm ventral from the vertex [22]. The coordinates were chosen to center above primary auditory cortex, and were adjusted slightly according to the animals’ individual size. An additional small craniotomy was made over left frontal cortex and a silver wire was placed into the epidural space as an electrical reference for recordings. Overland Park, Kansas, USA) and medetomidine (Domitor, Pfizer, Walton Oaks, Surrey, UK), and maintained with intraperitoneal infusions of ketamine, medetomidine and butorphanol tar- trate (Torbugesic; Fort Dodge Animal Health, Overland Park, Kansas, USA), at typical rates of 4.0 mg/kg/h, 16 μg/kg/h and 0.5 mg/ kg/h, respectively. Medical oxygen was administered at 0.5l/ h to the animals’ snout. The animals' core temperature was monitored and kept at 38°C using a rectal temperature probe and homeothermic blanket. Heart rate was monitored throughout the experiment and the depth of anesthesia was confirmed with regular paw and eye-lid reflex tests. Surgery After inducing anesthesia, the skull was exposed and a 3–4 mm diameter craniotomy was performed over auditory cortex at coordinates 5 mm anterior of bregma, 7.2 mm lateral to the right of the midline, and 4.5 mm ventral from the vertex [22]. The coordinates were chosen to center above primary auditory cortex, and were adjusted slightly according to the animals’ individual size. An additional small craniotomy was made over left frontal cortex and a silver wire was placed into the epidural space as an electrical reference for recordings. Electrophysiology The animals were placed in a soundproofed recording chamber and extracellular signals were recorded using Neuronexus (AnnArbour, Michigan) A-style single-shank silicon probes (16 recording sites at 100 μm spacing, or 32 channels at 50 μm spacing, 177 μm2 surface area) and TDT (Tucker Davies Technologies, Alachua, FL) RA16 measuring amplifiers and RZ5 digital signal processors. Neural signals were recorded with full bandwidth at 24 kHz sampling rate and monitored online using Brainware software (TDT). Recordings continued for a period of between 8 and 25 hours, until a deterioration in the physiological condition of the animal, typi- cally either edema of the auditory cortex or cardiac arrhythmia necessitated termination. Phys- iological measures of auditory responsiveness (thresholds, sharpness of tuning) were monitored both on and off-line. These were comparable in all four animals and showed no deterioration over the course of the experiments. Stimuli Two-formant pulse-resonance sound stimuli of 200 ms length were generated in Matlab, using code adapted from the makeVowel() function of Malcolm Slaney’s Auditory Toolbox (http:// cobweb.ecn.purdue.edu/malcolm/interval/1998-010/). Click trains with a fundamental fre- quency of 151 Hz at a sampling rate of 97656 kHz were passed through a cascade of two "for- mant filters" with center frequencies chosen at two of 11 positions between 10 kHz and 20 kHz logarithmically spaced at 0.1 octave intervals. This resulted in a set of 55 two-formant combi- nations. Positions of formant centers are shown in Fig 1A along with the amplitude spectrum of an example stimulus (B). 5 ms cosine onset and offset ramps were applied to the stimuli, and their amplitudes were scaled to achieve the desired RMS sound level of 75 dB SPL. The digital stimulus waveforms were converted to analog with an RX8 (TDT) digital signal processor and played through a Visaton (Haan, Germany) FRS 5 free field loud speaker. Before the start of the experiment the loud-speaker's impulse response was measured using Golay codes (Zhou et al., 1992) and a Bruel & Kjaer condenser microphone (model 4165) and measuring amplifier, and digital inverse filters were constructed to flatten the transducer output to +/- 3dB over a range of 1–35 kHz. Stimulus sound levels were calibrated to 75 dB SPL irrespective of formant frequency, using the Bruel & Kjaer microphone and measuring amplifier. Neural Resolution of Formant Frequencies in Rat A1 Fig 1. Formant Centers of Two Formant Pulse Resonance Sounds and Example Spectrum. A: Distribution of the 55 formant frequencies used to map out formant receptive fields. Formants are spaced between 10 and 20 kHz at 0.1 octave intervals. B: Amplitude spectrum of the example stimulus marked with a black circle in A. Fig 1. Formant Centers of Two Formant Pulse Resonance Sounds and Example Spectrum. A: Distribution of the 55 formant frequencies used to map out formant receptive fields. Formants are spaced between 10 and 20 kHz at 0.1 octave intervals. B: Amplitude spectrum of the example stimulus marked with a black circle in A. doi:10.1371/journal.pone.0134078.g001 between 200 Hz and 2000 Hz for spike detection. Spikes were detected in each extracellular voltage trace by finding all negative peaks which exceeded a threshold level of -3.5 standard deviations of the signal amplitude distribution. To map out the formant receptive field of each recording site, each of the 55 two-formant stimuli described above was presented 15–20 times in pseudorandom order with a gap of 300 ms after the end of each stimulus, yielding a total of 500 ms interval between subsequent stimulus presentations. The measure of response strength for a stimulus was chosen to be the onset response spike rate, measured in a window of 20–100 ms after stimulus onset, minus the background firing rate, which was estimated by the median spike rate over all stimuli and repeats in a “baseline” time window 400–480 ms after stimulus onset. Receptive fields for formant combinations were constructed by averaging the response strength over all trials per stimulus. To estimate the resolution at which formant receptive fields (FRF) operate each FRF was then analyzed with 2-dimensional Gaussian filtering and cross-validation method, explained based on an example FRF in the Results section. Inclusion criterion. To test for significant responses, we subdivided the pulse-resonance sound stimuli into four separate, approximately equal sized "quadrants" of the F1-F2 formant space as follows: Stimuli with F2<13.2 kHz (the "bottom left portion" of the formant space shown in Fig 1A) belonged to quadrant 1, stimuli with F1 > 13.2 kHz ("top right" part of the sampled formant space) belonged to quadrant 2, stimuli with F1+F2 > 26.4 kHz ("top left") belonged to quadrant 3, and the remaining stimuli ("central" part of the formant space) belonged to quadrant 4. Data Analysis Spike Detection and Construction of FRFs. Offline analysis of the data was performed in Matlab (The MathWorks, Inc., Natick, MA, USA). Neural signals were bandpass filtered 3 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Responses to Two-Formant Stimuli We recorded formant receptive fields in a total of 27 penetrations in 4 animals. Each penetra- tion was performed with a linear multi-electode array, inserted perpendicular to the cortical surface, under visual guidance, so that the most superficial electrode site came to sit just below the cortical surface. The electrode recording sites thus spanned the entire thickness of the cor- tex and sampled a "cortical column" at 16 depths, from the surface to a depth of 1.5 mm in 0.1 mm intervals. Twenty-four penetrations were carried out with this 16 channel linear array elec- trode, and a further 3 penetrations were performed with a 32 channel electrode array, which sampled cortical columns at 32 depths at 0.05 mm intervals. This yielded a data set of multi- unit activity from 480 recording sites in total. Using the inclusion criteria described in the methods, we confirmed that the overwhelming majority of recording sites in our sample (472/ 480 or ca 98%) exhibited statistically significant responses to the stimuli. We excluded the 8 sites which failed to show significant responses from further analysis. For each of the remaining 472 responsive recording sites we constructed formant receptive fields (FRFs), as illustrated in Fig 2. Fig 2A shows, in raster plot format, an example of spikes evoked by a subset of 9 different stimuli. The number of spikes in the evoked response varies substantially as a function of the stimulus: F2 frequencies of 12.3 and 13.2 kHz evoke onset responses which are on average two to four times as large as those seen for other values of F2. However, there is a great deal of trial-to-trial variability in the responses to repeats of identical stimuli. For example, a stimulus with F2 = 15.2 kHz sometimes evoked a strong burst of action potentials, but at other times evoked no response at all. Indeed, for this mutiunit as for many others in our data set, the spike count distributions are “Poisson-like” in the sense that the trial to trail spike count variance is of a similar magnitude to the mean spike count. A fair amount of background activity, which does not appear to be stimulus related, can also be seen in the late part of the recording window. This includes occasional “spontaneous” bursts of action potentials, and in the onset response (20–100 ms) there is no simple way to distinguish stimu- lus evoked activity from background activity. We then performed a Kruskal-Wallis ("non-parametric ANOVA") test comparing median spike counts in the response time windows for the stimuli of each of the four quadrants against each other and against spike counts in the "baseline" time window. Multi-units were thus deemed to exhibit “significant” (at a level of alpha = 0.05), stimulus related modulation of their firing rates if median firing rates in any of the four stimulus quad- rants differed either between each other or from the baseline firing rate. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 4 / 20 Neural Resolution of Formant Frequencies in Rat A1 PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Neural Resolution of Formant Frequencies in Rat A1 Fig 2. Construction of FRFs from spike times. A: Raster plot of spike responses to two formant stimuli with constant F1 of 10.7 kHz and F2 as indicated on the left. Each dot shows the timing of one action potential, each row of dots the response to one stimulus. Bars: blue = stimulus (200 ms), green = onset window (20–100 ms), red = baseline window (400–480 ms). Fifteen responses are shown for each stimulus (demarcated by the red dashed lines). B: Formant receptive field constructed by averaging spike rates in the onset response time window (20–100 ms). Color coded response values are mean spike rates in the onset window in Hertz. The column of data demarcated by the green lines (F1 = 10.7 kHz) was constructed from the raw spike data shown in A. Note that panels A and B share the same abscissa. d i 10 1371/j l 0134078 002 Fig 2. Construction of FRFs from spike times. A: Raster plot of spike responses to two formant stimuli with constant F1 of 10.7 kHz and F2 as indicated on the left. Each dot shows the timing of one action potential, each row of dots the response to one stimulus. Bars: blue = stimulus (200 ms), green = onset window (20–100 ms), red = baseline window (400–480 ms). Fifteen responses are shown for each stimulus (demarcated by the red dashed lines). B: Formant receptive field constructed by averaging spike rates in the onset response time window (20–100 ms). Color coded response values are mean spike rates in the onset window in Hertz. The column of data demarcated by the green lines (F1 = 10.7 kHz) was constructed from the raw spike data shown in A. Note that panels A and B share the same abscissa. doi:10.1371/journal.pone.0134078.g002 to which neural responses follow changes in stimulus features (here formant center frequency). But from the average responses alone it is not clear at all which of the changes in the responses are genuine and which are merely sampling noise. So to estimate neural resolution requires us to take into account the level of sampling noise. In the following we develop and apply a method that manages to do just that. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Responses to Two-Formant Stimuli This level of response variability is of course quite typical for responses of sensory cortical neurons. Fig 2B shows the formant receptive field for the same recording site, constructed by averag- ing the firing rates in the onset response windows for each stimulus and plotting them on a color scale against the corresponding formant frequencies. At this particular recording site, the strongest responses were seen for combinations of relatively low formant frequencies (F1 near 10 kHz, F2 near 13 kHz). The FRF examples in Fig 3 are chosen to illustrate the diversity of formant tuning functions we observed. Each row of FRF examples in Fig 3 comes from a different animal. The receptive field maps exhibit a variety of features: for example Fig 3B exhibits a relatively broad preference for stimuli with F1 below 15 kHz, largely irrespective of F2, while the example in 3C prefers sounds in which both F1 and F2 are relatively high. These are “broad” features or trends in the FRFs, but more fine grained features also occur: for example Fig 3E shows an intriguing and relatively narrow "diagonal stripe" which suggests that neurons at this recording site are not so much tuned to particular frequency ranges for F1 and F2, but are excited if F1 and F2 are spaced close together, within two to three tenths of an octave of each other. Figs 2 and 3 illustrates the "variable" nature of cortical responses to the pulse-resonance sti- muli. The mean responses do not drop off smoothly and monotonically as a function of dis- tance from a best stimulus. Instead, the FRF looks "grainy". This "graininess" is likely to be a consequence of stimulus-unrelated sampling noise (Fig 2A), but some of these “fine grained features” describe the underlying neural tuning. The goal of this study is to estimate the extent 5 / 20 Estimating sensory resolution through kernel smoothing Sampling noise is usually “cleaned up” through the use of smoothing filters. Fig 4 illustrates the effect of smoothing the FRF from Fig 3E by convolution with a 2-dimensional Gaussian filter (or “kernel”) with a width (standard deviation) of either 0.1 or 0.5 octaves. The smoothed FRFs certainly “look less noisy” and may seem easier to interpret in terms of systematic stimulus-response relationships. But the process of smoothing moves us away from the actual, “raw” experimental data, which raises the question whether the smoothed FRF is a more or a less accurate estimate of the “true” underlying neural tuning function. As it turns out, a smoothed FRF can be a more accurate representation of the underlying physiology, but only if the smoothing filter is chosen appropriately. Furthermore, determining the appropriate smoothing filter width is itself revealing about the true physiological resolving power of the neurons. To chose a filter that accounts for the response variability illustrated in Fig 2, we can con- ceptualize an observed response ř to a given stimulus S as a random variable F, which is itself dependent on some deterministic underlying neural tuning function r, i.e. ř(S) = F(r(S)). One popular way to model this is the so-called linear-nonlinear-Poisson (LN-P) model, which seeks to capture the deterministic part r(S) as a linear transformation of the stimulus S followed by an output non-linearity, and the non-deterministic part F with a Poisson random number gen- erator [23,24]. Of course LN-P and other, similar models are only an approximation of the physiology, and rest on a number of simplifying assumptions. However, for our analysis, the 6 / 20 Fig 3. Example FRFs. Examples of formant receptive fields from a variety of recording sites, plotted as in Fig 2B. Color scales give spike rates in Hz. These FRFs are representations of mean spike rates in an onset window (20–100 ms post-stimulus) adjusted by the median spike rate in a baseline window (400–480 ms). It is not immediately clear how much of the observed structure of the FRFs is due to sampling noise, and how much reflects genuine receptive field properties. doi:10.1371/journal.pone.0134078.g003 Neural Resolution of Formant Frequencies in Rat A1 Neural Resolution of Formant Frequencies in Rat A1 Fig 3. Example FRFs. Examples of formant receptive fields from a variety of recording sites, plotted as in Fig 2B. Color scales give spike rates in Hz. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Estimating sensory resolution through kernel smoothing These FRFs are representations of mean spike rates in an onset window (20–100 ms post-stimulus) adjusted by the median spike rate in a baseline window (400–480 ms). It is not immediately clear how much of the observed structure of the FRFs is due to sampling noise, and how much reflects genuine receptive field properties. doi:10.1371/journal.pone.0134078.g003 Fig 3. Example FRFs. Examples of formant receptive fields from a variety of recording sites, plotted as in Fig 2B. Color scales give spike rates in Hz. These FRFs are representations of mean spike rates in an onset window (20–100 ms post-stimulus) adjusted by the median spike rate in a baseline window (400–480 ms). It is not immediately clear how much of the observed structure of the FRFs is due to sampling noise, and how much reflects genuine receptive field properties. Fig 3. Example FRFs. Examples of formant receptive fields from a variety of recording sites, plotted as in Fig 2B. Color scales give spike rates in Hz. These FRFs are representations of mean spike rates in an onset window (20–100 ms post-stimulus) adjusted by the median spike rate in a baseline window (400–480 ms). It is not immediately clear how much of the observed structure of the FRFs is due to sampling noise, and how much reflects genuine receptive field properties. doi:10.1371/journal.pone.0134078.g003 doi:10.1371/journal.pone.0134078.g003 7 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Neural Resolution of Formant Frequencies in Rat A1 Fig 4. Smoothing of an FRF with 2-dimensional Gaussian smoothing filters. A: smoothing with a 0.1 octave wide filter. B: smoothing with a 0.5 octave wide filter. Left panels: The original FRF is same example as the one in Fig 3E. FRFs are plotted as in Figs 2 and 3, with formant frequencies in kHz on the coordinate axes, and the color scale giving spike rates in Hz. Middle panels: 2-dimensional Gaussian filters with σ = 0.1 (A) or σ = 0.5 (B) octaves standard deviation. Right panels: The result of convolving the FRF with the smoothing filter. To determine the ‘best’ σ for individual FRFs we use a cross-validation method described in Fig 5. Fig 4. Smoothing of an FRF with 2-dimensional Gaussian smoothing filters. A: smoothing with a 0.1 octave wide filter. B: smoothing with a 0.5 octave wide filter. Left panels: The original FRF is same example as the one in Fig 3E. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Estimating sensory resolution through kernel smoothing Rectangular smoothing windows are popular choices, as are the Gaussian kernels which we have chosen here. For instance, when smoothing with a σ = 0.1 octave wide Gaussian filter (as depicted in Fig 4A), the responses within +/- 0.1 octave from each stimulus will contribute ca 40% of the weighted average, while those within +/- 0.2 octaves will contribute 98%, so that those further than 0.2 octaves away make only a negligible contribution. Choosing an appropriate value for the width parameter σ, however, is all impor- tant. If it is too small, then the estimated neural tuning function will be noisier than it need be, but if it is too large, then excessive smoothing will blur details of the “true” FRF in our final esti- mate. Most commonly, previous authors have simply used their judgment to pick apparently reasonable but ultimately arbitrary smoothing parameter values [25–27]. A better, but still little known, alternative is to use cross-validation methods to optimize the choice of σ for a particu- lar data set [21]. Estimating the optimal σ in this manner is useful not only because it produces better receptive field estimates, it is also informative because the optimized value of σ provides a direct estimate of the “stimulus resolving power” of the neural responses under study. For an optimally chosen value of σ, r(S)r(S') will hold for all (S, S') such that \|S-S'\|<σ, but not neces- sarily for \|S-S'\|>σ. This is just a more formal way of stating that two stimuli must differ by more than σ for the responses to these stimuli to be reliably different given the expected trial- to-trial response variability. In this sense, σ can serve as a measure of the “stimulus resolution limit” afforded by the neural responses. The process of cross-validation used to determine the optimal value of σ (adapted from Has- tie et al. 2009) is illustrated in Fig 5. The data set is repeatedly randomly split into two subsets, usually referred to as “training sets” and “test sets”. The principle is to smooth the training sets with a range of varying width parameters σ, and to find the value of σ that offers the best com- promise between insufficient and excessive smoothing through trial and error by determining which smoothed training sets most accurately predicts the test sets. The number of samples used to construct the training and test sets is not critical. Estimating sensory resolution through kernel smoothing FRFs are plotted as in Figs 2 and 3, with formant frequencies in kHz on the coordinate axes, and the color scale giving spike rates in Hz. Middle panels: 2-dimensional Gaussian filters with σ = 0.1 (A) or σ = 0.5 (B) octaves standard deviation. Right panels: The result of convolving the FRF with the smoothing filter. To determine the ‘best’ σ for individual FRFs we use a cross-validation method described in Fig 5 doi:10.1371/journal.pone.0134078.g004 doi:10.1371/journal.pone.0134078.g004 precise form of the noise process F and of the deterministic neural tuning function rare unimportant. It is common practice to use the averaged observed responses ř(S) as an estimate of the “true” underlying tuning function r(S), and repetition and averaging are used to minimize the influence of the noise process F. Such repetition is useful because, according to the central limit theorem of statistics, the expected error in the estimate declines with the square root of the number of repeats that we have available to average over. Additional smoothing in the manner illustrated in Fig 4 may improve the estimate further because it effectively includes responses to presentations of a set of “neighboring” stimuli ř(S') into the averaging process (possibly with some weighting). This increases the effective number of observations that con- tribute to each data point and thereby reduces “noise”, but it genuinely improves the final esti- mate only if S and S' are “closely neighboring stimuli” in the sense that they can be expected to produce very similar responses. If that was not the case, the smoothing would “flatten out” gen- uine differences in stimulus evoked firing more than it would suppress physiological noise in the responses. In other words, smoothing relies on the fact that, to within expected measure- ment error, r(S)r(S') provided that the distance between the stimuli \|S-S'\| in the relevant parameter space is “small enough”, smaller than some appropriately chosen smoothing param- eter σ. In practice this is usually implemented with a nearest neighbor weighting filter which weights the contributions of stimuli S' heavily if \|S-S'\|< σ, but the weight of these contributions should decline sharply if \|S-S'\|> σ. The precise shape of this weighting function is not critical. Most functions which heavily discount stimuli that are "far away" (\|S-S'\|>σ) are suitable and PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 8 / 20 Neural Resolution of Formant Frequencies in Rat A1 will generally yield very similar results. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Estimating sensory resolution through kernel smoothing In the results shown here, our total data set typically comprised 15 presentations for each stimulus, and the training and test sets were made up of random subsamples (without replacement) of 10 and 5 repeats each, but a 14:1 split (“leave-one-out cross-validation”) gave essentially the same results. Fig 5B shows the effect of smoothing at varying values of σ. The mean squared difference between the “predictor FRF” (the smoothed training set) and the test set FRF serves as a measure of the prediction error. The process of randomly splitting the data and calculating prediction errors was repeated 500 times for each value of σ to generate a bootstrap estimate of the prediction errors. In this manner we calculated prediction errors for σ 2 {0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56, 5.12} octaves, and interpolated between these values with cubic splines. The value of σ associ- ated with the lowest prediction error optimally smoothes the FRF data, and at the same time provides an estimate of the “formant frequency resolution limit” afforded by the neural responses. When applying this kernel smoothing analysis to a large sample of FRFs, one observes that the formant frequency resolution afforded by the neural responses differs substantially between recording sites. This is illustrated in Fig 6. The top row of Fig 6A shows examples of four raw FRFs, and just below them, the FRFs are shown after smoothing with Gaussian kernels of opti- mal σ. Fig 6B shows the prediction error (the mean squared error M.S.E.) associated with dif- fering σ. The value of σ associated with the lowest prediction error is indicated above each plot. The FRF plotted in the leftmost panels has the poorest underlying formant frequency reso- lution (best σ = 0.33 octaves) among the sample shown in Fig 6. Smoothing with kernels as wide as a fifth of an octave or more substantially increases the “predictive power” of the FRF, and prediction errors do not increase substantially when the kernel widths are increased fur- ther to very large values of an octave or more. This indicates that, for this multi-unit, trial-to- 9 / 20 Neural Resolution of Formant Frequencies in Rat A1 Fig 5. Using cross-validation to estimate optimal FRF smoothing filters. A: The original raw FRF, estimated by averaging over 15 repeats. Estimating sensory resolution through kernel smoothing The data se is randomly split into subsets, a “training” set, shown in B, comprising 10 repeats for each stimulus, which are smoothed with a variety of smoothing filters of varying width, and a test set, shown in C, comprising the remaining 5 repeats. D: The differences between the smoothed training sets and the test set (residuals) are calculated. E: Mean squared residual values serve as a measure of the prediction error associated with each σ. Mean and standard error of prediction errors were estimated using “bootstrap” repeated random subsampling of the data as shown in A to D for 500 iterations. Mean and SEM predictio errors are plotted connected by a spline interpolation between the sampled values. The minimum of the interpolated curve serves as the estimated optimal σ in all subsequent analysis. doi:10 1371/journal pone 0134078 g005 eu a eso u o o o a eque c es a Fig 5. Using cross-validation to estimate optimal FRF smoothing filters. A: The original raw FRF, estimated by averaging over 15 repeats. The data set is randomly split into subsets, a “training” set, shown in B, comprising 10 repeats for each stimulus, which are smoothed with a variety of smoothing filters of varying width, and a test set, shown in C, comprising the remaining 5 repeats. D: The differences between the smoothed training sets and the test set (residuals) are calculated. E: Mean squared residual values serve as a measure of the prediction error associated with each σ. Mean and standard error of prediction errors were estimated using “bootstrap” repeated random subsampling of the data as shown in A to D for 500 iterations. Mean and SEM prediction errors are plotted connected by a spline interpolation between the sampled values. The minimum of the interpolated curve serves as the estimated optimal σ in all subsequent analysis. doi:10.1371/journal.pone.0134078.g005 doi:10.1371/journal.pone.0134078.g005 trial variability of neural responses is very large and stimulus related changes in response rate are comparatively small, so that the grand average response over all stimuli (produced by filters with very large σ) provides about as good a prediction for the response to any particular stimu- lus as one is going to get. doi:10.1371/journal.pone.0134078.g005 Estimating sensory resolution through kernel smoothing The multi-unit's responses are therefore unable to support reliable trial variability of neural responses is very large and stimulus related changes in response rate are comparatively small, so that the grand average response over all stimuli (produced by filters with very large σ) provides about as good a prediction for the response to any particular stimu- lus as one is going to get. The multi-unit's responses are therefore unable to support reliable PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 10 / 20 Neural Resolution of Formant Frequencies in Rat A1 Fig 6. Effects of Smoothing and Determination of Optimal Filter Widths. A: Four example multiunit FRFs, without smoothing (top row) and after optimal smoothing (below). B: Bootstrap estimates of prediction errors associated with various levels of smoothing for the FRFs shown in A. Error bars are s.e.m. at the respective σ. Blue lines are at values interpolated (using a spline interpolation) between the sampled σ values. Red lines show the minima of the interpolated curves that were chosen as estimators for optimal smoothing and formant resolution and are summarized in Fig 7. doi:10 1371/journal pone 0134078 g006 Fig 6. Effects of Smoothing and Determination of Optimal Filter Widths. A: Four example multiunit FRFs, without smoothing (top row) and after optimal smoothing (below). B: Bootstrap estimates of prediction errors associated with various levels of smoothing for the FRFs shown in A. Error bars are s.e.m. at the respective σ. Blue lines are at values interpolated (using a spline interpolation) between the sampled σ values. Red lines show the minima of the interpolated curves that were chosen as estimators for optimal smoothing and formant resolution and are summarized in Fig 7. doi:10.1371/journal.pone.0134078.g006 doi:10.1371/journal.pone.0134078.g006 discrimination between different formant frequencies, and the fine grained details in the raw FRF are predominantly attributable to sampling noise. Contrast this with the multi-unit shown in the third column in Fig 6. The initial decrease of residuals is very small, indicating that smoothing the raw FRF leads to only very modest increases in predictive power, while smoothing with σ greater than a very modest 0.03 octaves substantially and significantly impairs the predictive power of the smoothed FRF. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Neural Resolution of Formant Frequencies in Rat A1 Before we move on to consider the statistical and anatomical distribution of σ resolution values observed across our data set, it is worth pausing to elaborate on how σ values should be interpreted. For example, the reader might wonder whether it is sensible for us to even consider σ values smaller than the stimulus step size of 0.1 octaves used during the data collection. Does the inherent resolution limit of 0.1 octaves in our experiment not preclude the possibility of investigating σ resolution values smaller than 0.1 octaves? When interpreting σ values it is important to bear in mind that these are “standard deviations” of a Gaussian filter function, and stimuli as far as ±2σ or further away from the filter's center frequency will make non-negli- gible contributions to the filter output. For example, when we set σ = 0.08, responses to stimuli 0.1 octaves removed from the center of the filter will contribute to the spatially averaged output with a sizable weight, corresponding to 45% of the weight of the response at the center of the smoothing filter. For σ = 0.04, the weighting of stimuli 0.1 octaves away from the center drops to a modest 4.4%, and at σ = 0.02 it becomes a completely negligible 3.7 parts per million. Bear- ing this in mind, if we analyze an FRF with Gaussian filters as illustrated in Fig 6 and find the optimal value of σ to be 0.04 octaves but no smaller, then that indicates that the smoothing afforded by adding 4.4% weighted contributions from the responses to each of the 0.1 octave distant “nearest neighbor” stimuli tested to the weighted average response improves the predic- tive power of the smoothed FRF in the cross-validation test. Thus, an “optimal σ” of 0.04 there- fore does not imply that a multiunit can necessarily reliably signal a 0.04 octave change in formants. It merely implies that the influence of stimuli 0.1 octaves away is small but greater than zero, otherwise factoring in responses to those stimuli in the smoothed FRF would lead to a degradation, rather than an improvement, of the FRF's power to predict the responses in the cross-validation set. The contribution made by stimuli away from the center of the filter falls to less than 1% at ~3σ, and rapidly asymptotes to zero for increasing σ. It is therefore reasonable to interpret a particular value of σ as evidence that responses to frequencies more than 3σ apart are independent, but frequencies less than 2σ apart are not. Furthermore, if we were to find the best σ for a particular multiunit to be 0.02, the smallest value tested, then that would imply that the multiunit's “frequency resolving power” is too small to measure with the 0.1 octave spaced stimulus set used in this experiment. As we will see, none of the multiunits in our data set had optimal values of σ below 0.04. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Estimating sensory resolution through kernel smoothing The cross validation analysis thus indicates that the complex, fine grained structure of this FRF is a “real” feature of the underlying neural tuning function, and this multiunit therefore “resolves” for- mant frequency steps which are only a small fraction of an octave wide because such small changes in formant frequency can lead to reliable changes in the evoked firing rate. 11 / 20 Neural Resolution of Formant Frequencies The substantial differences in formant frequency resolution which are illustrated in Fig 6 raise the question: how is formant frequency resolution distributed across the cortical population, both statistically and anatomically? Are some formant frequency resolutions found more fre- quently than others? Do highly or poorly resolving multi-units tend to cluster in cortical layers or cortical columns? Figs 7–9 address these questions. Fig 7 shows the distribution of best filter widths for all 472 multi-units which responded to the pulse-resonance sounds. The panel on the left shows them as a scatter plot against time after the start of the electrophysiological recordings, with data from each animal plotted with a different symbol. Plotting the data in this manner visualizes the distribution obtained from each multielectrode penetration, shows that the distributions were similar in each of the four animals, and that there were no obvious trends for the quality of the data deteriorating during the course of the experiment. The panel to the left summarizes the observed formant resolution values in histogram form (it shows the “marginal” distribution of the left panel). While many multielectrode penetrations exhibit a wide spread of formant frequency resolutions, resolutions of around one tenth of an octave are clearly the most numerous. Approximately two thirds of the multi-units have a formant fre- quency resolution between 0.05 and 0.2 octaves. The median best filter width over all 472 PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 12 / 20 Neural Resolution of Formant Frequencies in Rat A1 Fig 7. Distribution of best filter width, as estimated with the kernel smoothing and cross-validation method. A: observed multiunit resolutions plotted as a function of time since the start of each recording experiment. Each symbol shows the formant resolution of one multiunit. Different symbols and colors are used to distinguish data from different animals. A random Gaussian jitter with a standard deviation of 0.2 has been added to the time coordinate to spread out overlapping data points from simultaneously recorded multiunits. B: Distribution of filter widths summarized in histogram form. The median filter width lies at 0.1161. doi:10.1371/journal.pone.0134078.g007 Fig 7. Distribution of best filter width, as estimated with the kernel smoothing and cross-validation method. A: observed multiunit resolutions plotted as a function of time since the start of each recording experiment. Each symbol shows the formant resolution of one multiunit. Different symbols and colors are used to distinguish data from different animals. doi:10.1371/journal.pone.0134078.g008 Neural Resolution of Formant Frequencies A random Gaussian jitter with a standard deviation of 0.2 has been added to the time coordinate to spread out overlapping data points from simultaneously recorded multiunits. B: Distribution of filter widths summarized in histogram form. The median filter width lies at 0.1161. doi:10.1371/journal.pone.0134078.g007 Fig 8. Development of filter width with cortical depth. Left is a schematic of the 16-channel probe used to perpendicularly penetrate rat auditory cortex. Channel 1 represents the pial surface and channels are 100 microns apart. In the middle panel FRFs from 3 animals (left columns: raw data, right columns: smoothed at optimal σ) at each of the 16 channels are shown. The right panel shows box plots of optimal σ at the respective cortical depth over all 27 penetrations (total 472 data points). doi:10.1371/journal.pone.0134078.g008 Fig 8. Development of filter width with cortical depth. Left is a schematic of the 16-channel probe used to perpendicularly penetrate rat auditory cortex. Channel 1 represents the pial surface and channels are 100 microns apart. In the middle panel FRFs from 3 animals (left columns: raw data, right columns: smoothed at optimal σ) at each of the 16 channels are shown. The right panel shows box plots of optimal σ at the respective cortical depth over all 27 penetrations (total 472 data points). doi:10.1371/journal.pone.0134078.g008 13 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Neural Resolution of Formant Frequencies in Rat A1 Fig 9. Difference of filter width with cortical location. The box plot shows the distribution of best σ values sorted by median for each array electrode penetration. Penetrations 1–9, 10–18, 19–24 and 25–27 are from different animals. Median σ values differ highly significantly from one penetration to the nex in all 4 animals (Kruskal-Wallis Test: p << 10−12). Fig 9. Difference of filter width with cortical location. The box plot shows the distribution of best σ values sorted by median for each array electrode penetration. Penetrations 1–9, 10–18, 19–24 and 25–27 are from different animals. Median σ values differ highly significantly from one penetration to the next in all 4 animals (Kruskal-Wallis Test: p << 10−12). doi:10.1371/journal.pone.0134078.g009 doi:10.1371/journal.pone.0134078.g009 multi-units is 0.1116 octaves. This corresponds to a shift in formant frequency of approxi- mately 8%. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Neural Resolution of Formant Frequencies We observed no obvious relationship between optimal filter width and the median response strength of multi-units (data not shown), and even relatively weakly responding multi-units sometimes exhibited the reliable dependence of firing on stimulus parameters that is necessary for FRFs which resolve formant frequencies with high acuity. We also examined whether cortical depth or location (or both) had a systematic influence on formant frequency resolution of two-formant PRSs. Fig 8 shows examples of FRFs recorded from three different “cortical columns” (i.e. three penetrations with electrode arrays orthogonal to the cortical surface which record neural activity at regular 100 μm intervals). Both raw and optimally smoothed FRFs are shown. The examples illustrate that FRFs recorded at one partic- ular depth often, but not always, closely resemble those recorded in the sites immediately 14 / 20 Neural Resolution of Formant Frequencies in Rat A1 above or below. Thus there appears to be a clustering of formant tuning properties within corti- cal columns which is somewhat reminiscent of “orientation tuning columns” described in pri- mary visual cortex [28], even if a particular shape of FRF is not usually sustained through the entire depth of the cortical column. Also, formant frequency resolution (best σ) can vary con- siderably as a function of cortical depth, but there is no clear systematic relationship between formant resolution and cortical depth. The right panel of Fig 8 shows the distribution of for- mant resolution as a function of cortical depth. Data at odd numbered electrode sites from the three 50 μm spaced 32 channel electrode penetrations were omitted here for clarity to yield a sample of 432 sites sampled at 100 μm intervals in 27 penetrations. The anatomical distribution of optimal σ along the three example penetrations is highlighted by the three red traces in the same panel. While some penetrations may exhibit a wider spread of formant resolution values throughout the depth of penetration, this appears not to be the general case, and there is no very clear dependence of formant resolution on cortical depth. Median resolution values are similar at all depths in most penetrations, and they exhibit no statistically significant depen- dence on depth (Kruskal-Wallis test, p>0.2). But while cortical resolution of formant frequencies did not depend systematically on depth, there were substantial and statistically highly significant variations from one penetration to the next. Discussion In this study we examined the tuning of neural responses in A1 of rats to formant frequencies of two-formant pulse resonance sounds. The stimuli used here provide a simple, experimen- tally tractable approximation of the types of pulse-resonance sounds that are widely used in human and animal communication. That neurons in A1 are clearly very sensitive to these types of sounds can be inferred from the fact that we observed statistically significant responses at 98% of recording sites in our sample. The structure of the observed FRFs was, however, highly diverse, and the fact that many neurons exhibit large trial to trial response variability makes it difficult to distinguish “real” detail in the FRFs from sampling noise in the raw data. To deal with this diversity and variability we deployed a kernel-smoothing method [21], which uses cross validation to discover an optimal neighbor weighting function (or "kernel") which maximizes the predictive power of the smoothed receptive field data. This allowed us to opti- mize our FRF estimates and at the same time quantify the formant frequency resolution afforded by the neural responses. Neural Resolution of Formant Frequencies In other words, while some cortical columns appear to resolve formant frequencies in fine detail, others do not. This is illustrated in Fig 9, which shows the distribution of optimal σ for each of the 27 penetrations, sorted by the median optimal σ. The apparent, large fre- quency resolution differences from one column to another were statistically highly significant (Kruskal-Wallis Test: p << 10−12). Neural Resolution of Formant Frequencies in Rat A1 has unique strengths which make it particularly suitable for certain types of study, and sec- ondly, while an exact comparison of different types of neural or psychoacoustic measures of bandwidth or frequency resolution is difficult, one can nevertheless discern quite easily whether the different methods give results which are in approximate, qualitative agreement. For example, Barbary ([29] reports Q20 factors for rat auditory nerve fiber tuning at charac- teristic frequencies near 10 kHz to be about 4, implying a bandwidth of 10/4 = 2.5 kHz. Mean- while, Ruggero & Temchin [30] argue that Q10 values are more readily comparable to psychoacoustically determined measures of frequency tuning, and calculate these to be about 6 for rats at 10 kHz, corresponding to a bandwidth of 10/6 = 1.66 kHz. How does that compare to the median best σ values of ca. 0.11 octaves we observed? As already mentioned in the results, these values specify the standard deviations of a 2-D Gaussian filter, and as such are possibly better through of as an estimate of the “half-bandwidth”, rather than the bandwidth, of the underlying “sensory filters”. If we consider values of 2σ as approximate “cortical band- width” estimates, then our median “cortical bandwidth” in the rat would be approximately 0.22 octaves, corresponding to ca 1.6 kHz bandwidth at a 10 kHz center frequency. The “corti- cal formant resolution”we have observed is therefore quite similar, and apparently no larger, than auditory nerve fiber frequency resolution reported in the literature, which suggests that A1 makes efficient use of the available cochlear frequency resolution in its representation of formant frequencies of vocal sounds. Comparing the values we obtained against psychoacoustic thresholds might be similarly interesting. Sadly there are no behavioral studies of formant discrimination thresholds in rats, but pure tone discrimination thresholds have been published by Syka et al. [31], who report Weber ratios of 5.7% on average, and by Talwar and Gerstein [32], who observed similar, if slightly better, Weber ratios in the range of 3.06 ± 0.44%. In comparison, the auditory nerve fiber and cortical formant resolution “bandwidths” just discussed, in the order of about 16%, appear rather broad. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Cortical Resolution of Formant Combinations We found that the majority of best smoothing filters had a width of less than 0.2 octaves, with a median best σ of 0.11 octaves. The method we used to generate these estimates of the “neural resolution” has, to our knowledge, not been previously used in sensory neurophysiology, and some comments on how these σ values should be interpreted and compared to results obtained from previous studies with very different methodologies are clearly called for. A variety of methods used to quantify neural tuning bandwidths is in use in the auditory literature, each incorporating different conventions, which make exact comparison of the values obtained with the various method difficult, and one might wonder whether the new method we introduce here does not just add to the confusion. However, firstly, as we will discuss below, our method PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 15 / 20 If we assume that behavioral formant discrimination thresholds are simi- lar to those seen in pure tones, and that these behavioral thresholds need to be achieved from a readout of cortical responses, then it appears that this readout may need to achieve a degree of “hyper-resolution” to reach observed psychoacoustic thresholds. Of course, how such a readout would operate, and how much any one cortical neuron or multiunit would contribute to it, is still largely unknown, although Bizley and colleagues [33] have demonstrated that even relatively naïve vector decoding of responses from small ensem- bles of auditory cortical neurons can be sufficient to substantially improve the tuning of a neu- ral ensemble to the fundamental frequency of an artificial vowel relative to the tuning of the individual neurons which constitute that ensemble, and that the improvements achieved by reading responses from modest sized ensembles of less than 20 neurons are often sufficient to account for behavioral thresholds. Thus, achieving highly accurate readouts is not difficult if ensembles are large and the activ- ity of individual responses of the neurons in the ensemble is “informative”, but the readout accuracy is limited by the degree of redundancy in the information inherent in the tuning of individual neurons [34,35] as well as by “noise correlations” in the firing of the neural ensemble [36] which would further reduce the amount of independent information which each neuron can contribute. While the neural resolution measure we developed here is sensitive to the noise inherent in trial-to-trial variability of discharges, it contributes nothing to our understanding of such informational redundancy in neural population codes. Furthermore, there are other measures which are in widespread use in central auditory physiology which quantify neural coding in the light of response variability, such as Shannon information [6,17,37,38] or Fisher information [39,40]. So why, or when, is the use of a neural resolution estimate through ker- nel-smoothing of the type developed here more appropriate than the use of alternative, already PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 16 / 20 Neural Resolution of Formant Frequencies in Rat A1 well established methods for the quantification of neural tuning or coding? Information-theo- retic measures are by design agnostic about the value or significance of individual “signals” and merely quantify their average discriminability. In contrast, in the perceptual interpretation of sound signals, some discriminable differences between signals may be highly relevant, and oth- ers not, and we might expect the shape of the tuning function to reflect which distinctions “matter” to the neurons. The fact that our kernel-smoothing method, rather than disregarding the shape of the underlying neural tuning function, instead produces an optimized estimate of it, can therefore be advantageous. To give a specific example, consider the FRF shown in Fig 3D, which features a prominent “diagonal”, which indicates that this multiunit may have a preference for pulse-resonance sounds whose formants have a fixed relationship to one- another, but irrespective of the precise value of the lower of the two formants. Such tuning properties would be compatible with the intriguing hypothesis put forward by Patterson and others [41] that the higher levels of the auditory system construct a “scale invariant” represen- tation of vocal communication sounds which normalizes out the length of the vocal tract of the vocalizing animal. Or alternatively, the diagonal feature could just be noise artefact. Given that we found the formant resolution value σ for this particular multiunit to be as small as 0.031 octaves (compare Fig 6), that banal explanation can be discounted. The diagonal feature in this particular example is “robust”, as well as intriguing, all the more so since this multiunit shares that diagonal feature with neighboring multiunits in the deeper layers of the same cortical col- umn (compare Fig 8). It is tempting to interpret this example as preliminary evidence for the emergence of scale-invariant representations of formant structure in the deeper layers of audi- tory cortex, but one must of course be must be careful not to read too much into a small num- ber of examples, and a comprehensive assessment of the likely functional significance of FRF shapes will have to await future work with substantially larger data sets. But in any event, kernel smoothing of the observed receptive fields is bound to provide a valuable, if not essential, pre- processing step. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Acknowledgments Funded by the Wellcome Trust grant 083132/Z/07/A and BBSRC grant BB/H008608/1. Author Contributions Conceived and designed the experiments: CH JS. Performed the experiments: CH. Analyzed the data: CH JS. Wrote the paper: CH. Neural Resolution of Formant Frequencies in Rat A1 field size and sensitivity to spatial phase [48]. In contrast, in auditory cortex, the evidence for columnar transformation of acoustic information has been described as “contradictory and inconclusive” [49] and until recently the only simple neural response features which were known to differ systematically across layers were response latency [50,51] and perhaps also stimulus specific adaptation [52]. Our results similarly reveal no widespread, systematic dependence of formant resolution on depth. Fig 8 suggests a trend that formant tuning properties may be more heterogenous in the superficial layer, which would be consistent with very recent investigations of pure tone tuning properties in the auditory cortex of the mouse [53]. However, while we found little evidence for any systematic depth dependence, individual penetrations appearently can have particular depth profiles. This observation is supported by the highly significant differences in median resolution between penetrations illustrated in Fig 9. This is indicative of a patchy organization of cortex, where each cortical column may be processing sounds in a slightly different manner, and while many cortical columns are quite sharply tuned for formants, others are not. Such a patchwork-like organization is in agreement with recent reports that units at different cortical locations integrate the features of vowel-like sounds in a very diverse manner [4,6]. In conclusion, the evidence for information processing within columns of auditory cortex remains largely inconclusive today, 57 years after Mountcastle coined the term “cortical col- umn” to describe his observations in cat V1. Our results similarly indicate a distributed, patch- work-like anatomical organization of formant frequency resolution in auditory cortex which is diverse both within and across cortical columns. Yet, while cortical locations vary widely in their resolution of formant frequencies, most exhibit resolutions of around 0.11 octaves, a value which is broadly comparable with behaviorally determined pure tone discrimination thresholds in rats. PLOS ONE \| DOI:10.1371/journal.pone.0134078 August 7, 2015 Anatomical Distribution of Cortical Formant Resolution Values Our investigation of the anatomical distribution of formant tuning parameters led to three main observations. First, there is no consistent depth profile for formant resolution across our penetrations. Second, while changes across cortical layers revealed no significant canonical pat- tern, there may be a trend towards more homogeneous resolutions in layers 5/6. And third, overall formant resolution varies highly significantly from one penetration to the next, in other words there appear to be “patches” of sharper and broader formant frequency tuning. Within each penetration we found that formant resolution (and indeed the FRF structure) is homogeneous along neighboring recording sites. There is no obvious systematic depth pro- file across penetrations, and roughly two thirds of all recorded sites give σ values between 0.05 and 0.2 octaves (median ~0.11). Similar trends for tuning properties to remain similar across depth for individual penetrations normal to the surface of auditory cortex have been observed for pure tones [42,43], intensity tuning [44], frequency sweep tuning [45] as well as binaural integration properties [46,47], and these observations have often been interpreted as evidence for a "columnar" functional organization of auditory cortex. 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https://openalex.org/W2272996378	https://www.frontiersin.org/articles/10.3389/fmicb.2015.01486/pdf	English	null	Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem	Frontiers in microbiology	2,016	cc-by	14,070	ORIGINAL RESEARCH published: 05 January 2016 doi: 10.3389/fmicb.2015.01486 Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem Jorge A. Valdivia-Anistro1, Luis E. Eguiarte-Fruns1, Gabriela Delgado-Sapién2, Pedro Márquez-Zacarías3, Jaime Gasca-Pineda1, Jennifer Learned4, James J. Elser4, Gabriela Olmedo-Alvarez5 and Valeria Souza1* 1 Laboratorio de Evolución Molecular y Experimental, Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Coyoacán, Mexico, 2 Laboratorio de Genómica Bacteriana, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Coyoacán, Mexico, 3 School of Biology, Georgia Institute of Technology, Atlanta, GA, USA, 4 School of Life Sciences, Arizona State University, Tempe, AZ, USA, 5 Laboratorio de Bacteriología Molecular, Departamento de Ingeniería Genética, CINVESTAV – Unidad Irapuato, Irapuato, Mexico Keywords: rRNA operon copies, oligotrophy, bacterial growth, Bacillus, Cuatro Ciénegas Citation: Valdivia-Anistro JA, Eguiarte-Fruns LE, Delgado-Sapién G, Márquez-Zacarías P, Gasca-Pineda J, Learned J, Elser JJ, Olmedo-Alvarez G and Souza V (2016) Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem. Front. Microbiol. 6:1486. doi: 10.3389/fmicb.2015.01486 The ribosomal RNA (rrn) operon is a key suite of genes related to the production of protein synthesis machinery and thus to bacterial growth physiology. Experimental evidence has suggested an intrinsic relationship between the number of copies of this operon and environmental resource availability, especially the availability of phosphorus (P), because bacteria that live in oligotrophic ecosystems usually have few rrn operons and a slow growth rate. The Cuatro Ciénegas Basin (CCB) is a complex aquatic ecosystem that contains an unusually high microbial diversity that is able to persist under highly oligotrophic conditions. These environmental conditions impose a variety of strong selective pressures that shape the genome dynamics of their inhabitants. The genus Bacillus is one of the most abundant cultivable bacterial groups in the CCB and usually possesses a relatively large number of rrn operon copies (6–15 copies). The main goal of this study was to analyze the variation in the number of rrn operon copies of Bacillus in the CCB and to assess their growth-related properties as well as their stoichiometric balance (N and P content). We deﬁned 18 phylogenetic groups within the Bacilli clade and documented a range of from six to 14 copies of the rrn operon. The growth dynamic of these Bacilli was heterogeneous and did not show a direct relation to the number of operon copies. Physiologically, our results were not consistent with the Growth Rate Hypothesis, since the copies of the rrn operon were decoupled from growth rate. However, we speculate that the diversity of the growth properties of these Bacilli as well as the low P content of their cells in an ample range of rrn copy number is an adaptive response to oligotrophy of the CCB and could represent an ecological mechanism that allows these taxa to coexist. These ﬁndings increase the knowledge of the variability in the number of copies of the rrn operon in the genus Bacillus and give insights about the physiology of this bacterial group under extreme oligotrophic conditions. Edited by: Roland Hatzenpichler, California Institute of Technology, USA Edited by: Roland Hatzenpichler, California Institute of Technology, USA Reviewed by: Sebastian Kopf, Princeton University, USA Albert Leopold Mueller, Stanford University, USA Correspondence: Valeria Souza souza@unam.mx Correspondence: Valeria Souza souza@unam.mx Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Received: 16 September 2015 Accepted: 09 December 2015 Published: 05 January 2016 Edited by: Edited by: Roland Hatzenpichler, California Institute of Technology, USA INTRODUCTION Functional traits can aid in the study of population genetics, because they help to deﬁne species in terms of their ecological roles, such as how they use environmental resources or how they interact with other species (McGill et al., 2006; Hughes et al., 2008). These functional traits are often considered to be ecological strategies because they are useful in understanding why certain bacteria live in a particular environment and how they respond to environmental challenges (Green et al., 2008). The ribosomal RNA operon (rrn hereafter) is the key genetic structure for protein synthesis and thus a functional trait related to bacterial life history (Stevenson and Schmidt, 2004). Ecologically, the rrn operon has been related with the bacterial capacity to respond to changes in environmental conditions (Codon et al., 1995; Prüβ et al., 1999; Green et al., 2008). In particular, the variation in the number of copies of the rrn operon has been considered an ecological strategy related to resource availability, with physiological implications associated with bacterial growth rate and ﬁtness (Klappenbach et al., 2000; Shrestha et al., 2007). The rrn operon is comprised of three genes (5S, 16S, and 23S rDNA) and its copy number varies from 1 to 15 among bacterial genomes (Klappenbach et al., 2001; Acinas et al., 2004; Stoddard et al., 2015) and even more dramatically among eukaryotes (Elser et al., 2000). Experimentally, it has been shown that deletions of one or more copies of the rrn operon have a considerable impact on growth rate, aﬀecting various stress-response mechanisms (Nanamiya et al., 2010; Yano et al., 2013). Hence, it has been suggested that the multiplicity of the rrn operon is a potential mechanism for adaptation to diﬀerent environmental conditions (Elser et al., 2000; Green et al., 2008). In general terms, bacteria that possess more rrn operon copies may cope better with ﬂuctuating nutrient inputs than bacteria with fewer rrn operon copies, which tend to live in environments where nutrients are scarce (Klappenbach et al., 2001; Elser, 2003; Jeyasingh and Weider, 2007). Moreover, the relationship between rrn operon copy number and the bacterial biotic potential for the cellular allocation of key resources could be analogous to the ecological strategies described in other macro-biota (r- and K-strategies), (Pianka, 1970; Elser et al., 2000; Dethlefsen and Schmidt, 2007; Shrestha et al., 2007; Lipowsky et al., 2012). INTRODUCTION copy number may be favored because the rich P supply could then support the rapid production of P-rich rrn to meet the protein demands of rapid growth, as stated by the “Growth Rate Hypothesis” (GRH), a core idea within the theory of biological stoichiometry (Elser et al., 2000; Elser, 2006). However, in environments with low phosphorus availability, multiple rrn operon copies could represent a competitive cost if a high rrn operon copy number leads to the over-production of P-rich rrn (Sterner and Elser, 2002; Maciá, 2005; Jeyasingh and Weider, 2007). Indeed, aquatic bacteria isolated from oligotrophic environments usually have low rrn operon copy numbers (Fegatella et al., 1998; Strehl et al., 1999; Lauro et al., 2009), as well as various other adaptations to decrease cellular phosphorus demand (Cavicchioli et al., 2003; Alcaraz et al., 2008; Martiny et al., 2009; Van Mooy et al., 2009). Thus, it has been proposed that there is a connection between the number of rrn operon copies and environmental P availability (Elser et al., 2000; Weider et al., 2005; Jeyasingh and Weider, 2007). However, to our knowledge, we lack extensive studies that document this variation in rrn operon copy number and other associated ecological strategies employed by bacteria coexisting in oligotrophic environments, especially those characterized by severe P limitation. Population genetics is the most direct tool for use in understanding adaptation to the ecological challenges imposed upon microbial communities by the environment (Whitaker et al., 2003; Xu, 2006; Spencer et al., 2008). Functional traits can aid in the study of population genetics, because they help to deﬁne species in terms of their ecological roles, such as how they use environmental resources or how they interact with other species (McGill et al., 2006; Hughes et al., 2008). These functional traits are often considered to be ecological strategies because they are useful in understanding why certain bacteria live in a particular environment and how they respond to environmental challenges (Green et al., 2008). Population genetics is the most direct tool for use in understanding adaptation to the ecological challenges imposed upon microbial communities by the environment (Whitaker et al., 2003; Xu, 2006; Spencer et al., 2008). INTRODUCTION The aims of this study were to describe rrn operon copy number variation in diﬀerent lineages of Bacillus strains isolated from an extremely oligotrophic ecosystem and to analyze the possible association between the copy number and its physiological implications for growth rate and chemical composition (P content and N:P stoichiometry). Severe P limitation is considered a primary selective pressure that drives bacterial evolution in this environment (Souza et al., 2008, 2012). For example, we have previously reported on an endemic and moderately halophilic Bacillus (type strain of B. coahuilensis: m4-4 = NRRL B-41737T), (Cerritos et al., 2008) that has a typical number of rrn copies (nine) but also clear adaptations to the extreme oligotrophic conditions, including a small genome (3.5 Mb), a diversity of phosphate acquisition genes (Moreno- Letelier et al., 2011), and a cellular membrane composed of sulfolipids (Alcaraz et al., 2008). Similar adaptations to low P levels have been reported only from oligotrophic marine cyanobacteria with low rrn copy numbers (Cavicchioli et al., 2003; Lauro et al., 2009; Martiny et al., 2009; Van Mooy et al., 2009). Hence, the present study represents the ﬁrst attempt to link biological stoichiometry to Bacillus diversity and rrn operon copy number, as well as the ﬁrst report in which the numbers of rrn copies are analyzed in several members of this genus that coexist in the same habitats. Bacillus is a genus that is well-known because of its ecological versatility (Feldgarden et al., 2003). The fact that it can sporulate increases its long-range dispersal and allows it to explore diverse environments (Nanamiya et al., 2010; Yano et al., 2013). Coincidentally, Bacilli have a relatively high number of rrn operon copies per genome, ranging from six to 15 (rrnDB, Stoddard et al., 2015), a degree of variation that may aid in this lifestyle strategy of colonizing new environments and provide great adaptability in response to stress, as well as being able to uptake a wide variety of resources (Feldgarden et al., 2003; Connor et al., 2010). If the new environment is rich in phosphorus (P), high rrn operon Citation: Citation: Valdivia-Anistro JA, Eguiarte-Fruns LE, Delgado-Sapién G, Márquez-Zacarías P, Gasca-Pineda J, Learned J, Elser JJ, Olmedo-Alvarez G and Souza V (2016) Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem. Front. Microbiol. 6:1486. doi: 10.3389/fmicb.2015.01486 January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org 1 rrn Operon and Growth Dynamics Valdivia-Anistro et al. Site Description and Selection of Bacillus Strains The Cuatro Ciénegas Basin (CCB hereafter) is a hydrologic system in the Chihuahuan Desert in northeastern México (Souza et al., 2006), (Figure 1A, yellow triangle). This basin represents an oasis with a high microbial diversity in extremely oligotrophic January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 2 rrn Operon and Growth Dynamics Valdivia-Anistro et al. FIGURE 1 \| The Cuatro Ciénegas Basin (CCB) in the Chihuahuan Desert, in northeastern México (A,B) and the sites where Bacillus strains were previously isolated. (C) The Churince system, (D) Pozas Rojas (Los Hundidos), and (E) Río Mesquites. (F) Maximum-Likelihood (ML) tree of the 19 phylogenetic groups identiﬁed using the 5′ HV region of the 16S rDNA. Bootstrap values higher than 70% are shown. Symbols represent the sample type of isolation: □= Top section of sediment, ■= Bottom section of sediment, ▲= Water sediment adjacent to a plant, and ♦= Water. F ti i Mi bi l \| f ti i 3 J 2016 \| V l 6 \| A ti l 1486 FIGURE 1 \| The Cuatro Ciénegas Basin (CCB) in the Chihuahuan Desert, in northeastern México (A,B) and the sites where Bacillus strains were previously isolated. (C) The Churince system, (D) Pozas Rojas (Los Hundidos), and (E) Río Mesquites. (F) Maximum-Likelihood (ML) tree of the 19 phylogenetic groups identiﬁed using the 5′ HV region of the 16S rDNA. Bootstrap values higher than 70% are shown. Symbols represent the sample type of isolation: □= Top section of sediment, ■= Bottom section of sediment, ▲= Water sediment adjacent to a plant, and ♦= Water. January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org 3 rrn Operon and Growth Dynamics Valdivia-Anistro et al. conditions (<1 μmol PO43−; Peimbert et al., 2012; Souza et al., 2012). Interestingly, ca. 50% of the bacterial communities in the CCB are most closely related to marine relatives (Souza et al., 2006). Isolates related to the genus Bacillus were identiﬁed from samples collected at various sites in the CCB during 15 years of ﬁeld work (Souza et al., 2006; Alcaraz et al., 2008, 2010; Cerritos et al., 2010; Pérez-Gutiérrez et al., 2013), (Figure 1B). fragments usually representing the number of rrn operons. Pulsed-ﬁeld gel electrophoresis (PFGE) was used to construct the rrn proﬁle of the chromosome from the Bacillus isolates. Phylogenetic Analysis y g y To obtain biomass for DNA extraction, Bacillus isolates were grown in the standard medium used for their isolation in the ﬁeld (Marine agar, DifcoTM 2216). Genomic DNA extractions were performed using the QIAmp R⃝DNA Mini Kit (USA), according to the manufacturer’s instructions. The 5′ hypervariant (HV) region of the 16S rDNA was ampliﬁed (275 bp; 70–344 position), following Goto et al., (2000). This region has a high level of conservation and is useful for the clustering of Bacillus species. The polymerase chain reaction (PCR) products were conﬁrmed via 1.5% agarose gel electrophoresis. The sequencing of the HV region was performed by the High Throughput Genomics Center (htSEQ), University of Washington (USA), and compared with the GenBank database using BLAST (NCBI). Sequences were aligned using CLUSTAL W (Thompson et al., 2002), and a maximum-likelihood tree was constructed using MEGA5, with a bootstrap of 1000 replicates (Tamura et al., 2011). The sequences of the 5′ HV region of the 16S rDNA were submitted to GenBank with the following accession numbers: KT781592–KT781661. The agarose gels were radiated with UV light for 1 min in a UV Crosslinker (UVP) to ﬁx the DNA. The gels were washed in 250 mM HCl solution for 15 min with moderate shaking. Next, the gels were washed in denaturing buﬀer (1.5 M NaCl, 0.5 M NaOH) for 2 h and later washed in a neutralization buﬀer (0.5 M Tris/HCl, 1.5 M NaCl; pH 8.0) for 2 h. The DNA fragments were then transferred onto N+nylon membrane (Amersham Biosciences) via Southern blotting as described previously (Sambrook et al., 1989). Finally, the membrane was radiated with UV light to ﬁx the DNA (1 min; UV Crosslinker, UVP). Site Description and Selection of Bacillus Strains Bacterial genomic DNA from Salmonella enterica serovar Typhimurium LT2 cleaved with CeuI and the 0.1–200 kb Sigma Plus Marker were used as molecular weight markers. Because the size of S. enterica Typhimurium LT2 identifying fragments had been determined previously (Liu et al., 1993), their inclusion improved the precision of the band-size estimation. Our isolates are from three primary sampling sites within the basin: (i) the Churince site consists of a freshwater spring that connects to an intermediate shallow pond via a small stream and eventually terminates in a shallow desiccated lagoon (Figure 1C); (ii) the Río Mesquites is a stable system composed of a river and some lateral desiccated ponds (Figure 1D) with low nutrient concentrations and highly imbalanced C:N:P ratios [C:N:P, 900:150:1 (molar); Souza et al., 2012]; and (iii) the Pozas Rojas site (Figure 1E) is located in a system called Los Hundidos and consists of a shallow lake and nine to 12 small semi- permanent ponds with strongly ﬂuctuating conditions (high salinity and temperature in summer, both decrease in winter). Preparation of DNA Probes and Hybridization The rrn proﬁles of Bacillus isolates were validated by probing the Southern blotting membranes with PCR products of the 16S and 23S rrn operon from the Bacillus horikoshii ATCC 700161 strain. The primer sets used to amplify the rrs gene were designed using the 5′ HV region (described above), and an internal region of the rrl gene was designed from the 2283 to 2696 position (23S3) of the rrn. Then, the 23S3 region (413 bp) was ampliﬁed using the Preparation and Digestion of Genomic DNA in Agarose Blocks Bacillus isolates were cultured aerobically in DifcoTM Marine Broth 2216 (BD & Co.) and incubated overnight at 35◦C. The genomic DNA of each strain was prepared in agarose blocks using a previously described method, with some modiﬁcations (Nakasone et al., 2000; Delgado et al., 2013). Two processes of incubation in proteinase K solution (12 h at 37◦C) were carried out to increase the purity of the DNA. Agarose blocks were pre- incubated in 1X NEBuﬀer 4 (New England Biolabs) for 30 min at 4◦C. Finally, the digestion of the genomic DNA was achieved with 100 μl fresh 1X NEBuﬀer 4 containing 15 U of I-CeuI restriction enzyme, and it was incubated overnight at 37◦C. y p The Bacillus strains isolated from the CCB are part of a larger collection (several thousands of isolates) of microbes that is maintained at the Molecular Evolution and Experimental Laboratory at the Instituto de Ecología, UNAM (Valeria Souza) and at the Molecular Bacteriology Laboratory in the CINVESTAV, Irapuato (Gabriela Olmedo); cultures are available upon request. We selected 71 of these isolates and classiﬁed them according to the site of isolation and sample type (plant root, sediment, or water). Sixty-seven Bacillus isolates were sampled from Churince, one was from Río Mesquites and three were from Pozas Rojas. Pulsed-Field Gel Electrophoresis (PFGE) and DNA Fragments Transfer g The CeuI fragments were separated by a CHEF-DR II electrophoresis system (Bio-Rad). Electrophoresis was performed on a 1% agarose (Seakem Gold agarose, BioWhittaker Molecular Applications) gel and 0.5X TBE buﬀer (Bio-Rad) at 11◦C. The electrophoresis conditions were divided into two stages to separate the DNA fragments of various sizes: First stage, pulse time ramped from 6.75 s to 2 min for 20 h at 4 V cm−1 and in a second stage, pulse time ramped from 0.22 to 5.10 s for 15 h at 6 V cm−1. I-CeuI Cleavage Map of the Bacillus Strains The I-CeuI (CeuI hereafter) restriction endonuclease recognizes a 26-bp sequence from position 1911–1936 of the 23S rRNA gene in rrn operons, with the number of CeuI (New England Biolabs) January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org 4 Valdivia-Anistro et al. rrn Operon and Growth Dynamics we estimated the maximum speciﬁc growth rate (μmax; units: hours−1) as follows: we estimated the maximum speciﬁc growth rate (μmax; units: hours−1) as follows: forward primer F23S3 5′-ACG GAG GCG CCC AAA GGT T- 3′ and the reverse primer R23S3 5′-CCA GCG GTG CGT CCA TCC-3′. The primer set used to amplify the 23S3, was designed based on previously sequenced genomes using the Primer Select program of the DNASTAR Lasergene 7 package (DNASTAR, Inc., Madison, WI, USA). μmax = ln (Ne −N0)/(te −t0) and the bacterial doubling (generation) time as follows: The PCR ampliﬁcation conditions were as follows: 95◦C for 5 min for the initial denaturation, 30 cycles of denaturation at 95◦C for 40 s, annealing at 60◦C for 40 s, an extension of 1 min at 72◦C and a ﬁnal extension of 5 min at 72◦C (Gene Amp, PCR System 9700). The presence and size of PCR products were subsequently conﬁrmed via 1.5% agarose gel electrophoresis. The PCR products were puriﬁed with the PCR Clean-up Gel Extraction Kit (Macherey–Nagel products) and then DIG-labeled using the random primer method of the DIG High Prime DNA Labeling system (Roche). The membrane was incubated in 10 ml hybridization solution (DIG Easy Hyb buﬀer). Incubation was carried out at 58◦C with constant, gentle shaking for 1 h. The labeled probe was then added to fresh hybridization solution and hybridization was carried out overnight at 58◦C with constant and gentle shaking. The membrane was exposed to X-ray ﬁlm after being washed at high astringency (64◦C). td = ln 2/μmax N0 and Ne are the cell densities reached at the beginning and at the end of the exponential phase, respectively, while t0 and te are the times (h) at which the exponential phase started and ended, respectively. Growth Parameter Estimations Genotypes of Bacillus with diﬀerent numbers of copies of the rrn operon were chosen from among the groups described in the phylogenetic analysis. Prior to growth parameter estimation, all the strains were pre-cultured in fresh marine broth for 24 h to homogenize their metabolic condition. All cultures were incubated at 35◦C, the maximum water temperature during summer at the CCB (Pérez-Gutiérrez et al., 2013), with shaking at 150 rpm. Additionally, experiments were carried out for nutritional conditions similar to CCB; for this, we inoculated the strains into sterile water collected from the Churince ﬁeld site but supplemented with tryptone (5 g per liter; BactoTM Tryptone, BD and Company; hereafter, CCBwt) and incubated for 12 h (overnight), (Supplementary Figure S1A). At ASU total phosphorus content was measured using a modiﬁed ascorbic acid method with persulfate digestion (APHA, 2005). The dried biomass samples were weighed and treated with a potassium persulfate and sulfuric acid solution and then autoclaved for 30 min at 121◦C and 15–20 psi. The samples were allowed to cool and then neutralized before the addition of the color reagent. After 30 min, the absorbance was read on a spectrophotometer at 880 nm. The samples were analyzed with a triplicate standard curve and triplicate NIST reference material. The total carbon and nitrogen content was measured via combustion in a Perkin Elmer model 2400 elemental analyzer. The samples were combusted at 1760◦C. Elemental detection was conducted via a thermal conductivity detector. The growth parameters were then determined using the previously described overnight culture. Three new 50 ml ﬂask of fresh CCBwt medium were inoculated to reach an optical density of 0.05 (600 nm wavelength; BioPhotometer Plus, Eppendorf), which corresponded to ∼107 colony-forming units (CFU) ml−1. CFU counts were made taking at least seven samples distributed through a period of 12 h to cover all the phases of the growth curve, the samples were diluted appropriately in 0.85% NaCl to perform a plate count analysis. The C, N, and P data were expressed as percentages of dry mass and referred to as “C content,” “N content,” and “P content,” respectively (Supplementary Table S2). Cell Contents of Carbon (C), Nitrogen (N), and Phosphorus (P) During the Exponential Phase The samples of bacterial biomass were harvested in the exponential phase during the determination of growth dynamics. Biomass samples were spin in a centrifuge for the removal of the growth medium. To avoid the inﬂuence of remains of the growth medium in the elemental composition analysis, the biomass samples were washed three times with 250 μl water (Mili-Q) R⃝and after of each washing, supernatant was removed by centrifuge. Finally, the biomass samples were vacuum dried to be shipped frozen to Arizona State University for analysis. Statistical Analysis All statistical analyses, including the estimation of Pearson correlation coeﬃcients (r) and principal component analysis (PCA) were performed with the R Statistic program Version 3.3.1 (24-07-10). We estimated the lag phase period (λ; units: hours), the tangential growth rate (Gtan; units: cells/h), and maximal biomass reached [A; units: Ln(CFU/CFU(t = 0))] from our data using a non-linear regression (CurveExpert Professional 2.0.3 software) to ﬁt a Gompertz equation according to Zwietering et al. (1990), (Supplementary Figure S1B). We obtained the ﬁnal parameters from the predicted curve, deﬁning Gtan as the tangent of the inﬂection point of the curve, λ as the X intercept of the tangential line through the inﬂection point (where the X-axis is time), and A as the Y-value of the asymptote [where the Y-axis is Ln(CFU/CFU(t = 0))] for plate-count assays. In addition, Frontiers in Microbiology \| www.frontiersin.org Identiﬁcation and Clustering of Bacillus Isolates from the CCB The 71 isolates used for this study were clustered into 19 phylogenetic groups, forming a large “marine” cluster (30 isolates; i.e., formed by strains with marine aﬃnities). Most of the lineages January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org 5 rrn Operon and Growth Dynamics Valdivia-Anistro et al. include representatives from a variety of habitats (e.g., soil, water, sediment); however, some “marine” groups included strains only sampled from water, while the B. atrophaeus lineage was only found in the top layers of sediment. Strains related to B. pumilus were also only found in sediment (Figure 1F). samples; B. idriensis isolates (group XVI), were related to strains from soil samples with halotolerance. Finally, the B. horikoshii cluster (group XVII) has as a representative a strain isolated from a ﬁsh pond as well as a lineage related to a halotolerant B. nanhaiensis (group XVIII), isolated from a non-saline soil sample. Unexpectedly, the 169A 5R isolate was closely related to the strain Staphylococcus sp. Mg48 (JQ399818), isolated from a saline lake (group XIX), but it is not unusual to isolate a Staphylococcus strain when aiming to select Bacillus. y The “marine” cluster is composed of nine phylogenetic groups (arbitrarily numbered I to IX) composed of Bacillus strains isolated mostly from CCB aquatic samples, such as B. sp. m2- 34 (group II) and B. coahuilensis (group V). In addition, three CCB phylogenetic groups of Bacillus were related to type strains isolated from water: B. endophyticus (group III), related to a pollutant-degrading strain isolated from industrial eﬄuent, as well as B. sp. NRRLB-14911 (group IV) and B. marisﬂavi (group IX), both isolated from seawater. The B. aquamaris (group I), B. sp. SP61 (group VI), and B. vietnamensis (group VIII) groups are related to strains isolated from hypersaline environments (salterns and a microbial mat). Finally, group VII is related to a strain isolated from a bioﬁlm of a lake (B. sp. BA-117). The small B. cereus cluster is related to type strains isolated from marine sediments (group X). The B. subtilis cluster (group XIII), was similar to soil type Bacillus. Group XI is most closely related to B. sonorensis isolated from the soil of the Sonoran desert. Organisms in group XII are related to a B. atrophaeus strain isolated from soil and water samples, and group XIII is related to a B. Phylogenetic Variability of the rrn Operon Copy Number The rrn operon copy number was determined for every isolate described above in the maximum-likelihood tree via PFGE and hybridization analyses (Figure 2 and Supplementary Figure S2). To obtain a benchmark for the rrn copy number in the Bacillus diversity from the CCB, we analyzed the type strain of B. coahuilensis (m4-4 = NRRL B-41737T) that was isolated from the Churince site (Cerritos et al., 2008) and that has already been sequenced (Alcaraz et al., 2008). After genomic digestion and hybridization analysis, eight rrn operons were quantiﬁed (Figure 2 and Supplementary Figure S2; group V). The 70 strains of Bacillus from the CCB showed a range of between six and 14 rrn operon copies (Figure 2 and Supplementary Figure S2). Some groups showed intraspeciﬁc variation from one to four copies. Interestingly, we quantiﬁed only six copies of this operon in some phylogenetic groups, such FIGURE 2 \| Variability of the rRNA operon copy number in the Bacillus diversity from the CCB. ML tree of the 5′ HV region of the 16S rDNA with a 50% of cut-off value. The black circles represent the type strain of Bacillus for every phylogenetic group. The squares, triangles and rhomboids symbols correspond to the environment as described in Figure 1. The dotted red line represents the low number of the rrn operon in Bacillus according to the rrnDB. The black bars represent the number of operon copies in the isolates from the CCB, and the green bars represent the number of copies in Bacillus species reported in the rrnDB. Gray bar represents the number of copies in the isolate related to the genus Staphylococcus. The light purple bars represent the number of operon copies in different Staphylococcus species analyzed by the rrnDB. The orange bar represents the number of copies quantiﬁed in the type strain of B. coahuilensis (m4-4 = NRRL B-41737T), group V. The yellow bars represent the number of copies in the type strains of B. marisﬂavi (JCM 11544 = KCCM 41588) and B. horikoshii (ATTCC 700161), groups IX and XVII, respectively. FIGURE 2 \| Variability of the rRNA operon copy number in the Bacillus diversity from the CCB. ML tree of the 5′ HV region of the 16S rDNA with a 50% of cut-off value. The black circles represent the type strain of Bacillus for every phylogenetic group. Identiﬁcation and Clustering of Bacillus Isolates from the CCB subtilis strain from marine samples, although this is a well-known cosmopolitan species. Strains related to a B. altitudinis strain (group XV), were present in soil and water Phylogenetic Variability of the rrn Operon Copy Number atrophaeus (XII), and B. nanhaiensis (XVIII); the lowest number of copies quantiﬁed in other strains of the genus (rrnDB, Stoddard et al., 2015). The highest number of copies was observed in the B. cereus group (X) (14 copies). p To further increase knowledge about the number of copies of the rrn operon in the genus Bacillus, we analyzed two type strains similar to those observed in the CCB, B. marisﬂavi (JCM 11544 = KCCM 41588), and B. horikoshii (ATTCC 700161). The genomes of these species have not yet been sequenced and the number of copies of this functional gene is unknown. The B. marisﬂavi and B. horikoshii type strains showed ten and eight copies of the rrn, respectively (Figure 2 and Supplementary Figure S2, groups IX and XVII). Thus, the number of copies quantiﬁed in these Bacillus type species was similar to the number described in the strains isolated from the CCB (Figure 2). Homogeneity in rrn operon copy number was observed in the B. sp. m2-34 (II; eight copies) and B. sonorensis (XI; six copies) groups. However, considerable heterogeneity and intraspeciﬁc variation were observed in several other groups: B. aquamaris (I; seven to nine copies), B. sp. NRRLB-14911 (IV; eight, nine, eleven and twelve copies), B. vietnamiensis (VIII; eight to ten copies), B subtilis (XIII; seven to eleven copies), B. pumilus (XIV; eight, eleven, and twelve copies) and B. horikoshii (XVII; eight, nine and eleven copies). In addition, the phylogenetic groups composed of only one isolate showed diﬀerent numbers of rrn operon copies: B. endophyticus (III; seven copies), B. sp. SP61 (VI; ten copies), B. sp. BA-117 (VII; nine copies), B. altitudinis (XV; eight copies), B. idriensis (XVI; 13 copies) and B. nanhaiensis (XVIII; six copies; Figure 2). The Staphylococcus isolate had ﬁve copies of the rrn. A = maximum biomass reached [Ln(CFU/CFU(t = 0))]; λ = lag phase; µmax = maximum speciﬁc growth rate; td = doubling (generation) time. reached (A). Component 2 explained 26.87% of the variance, and was principally deﬁned by adaptation time (λ) and the rrn operon copies (Supplementary Figure S4). Meanwhile, isolates with the highest copy numbers showed greater dispersion, having the most extreme parameter values (Table 1). These results may indicate that the rrn operon copy number in the Bacillus from the CCB may be related to the integrated suite of growth dynamics parameters, but not exclusively to the growth rate. Growth Parameters Growth parameters were estimated for a subsample of 15 Bacillus isolates from the CCB representative of the phylogenetic diversity present and the range of rrn operon copy numbers observed. We also characterized the isolate related to the Staphylococcus genus (Table 1). Not surprisingly, given the large diversity in this genus within the CCB, the results show a high heterogeneity in the growth parameters estimated. In agreement with these results, the lag phase period of these Bacilli is variable and it is not related with their growth rate (μmax; Supplementary Figure S3A). In addition, the maximum biomass reached was correlated with the maximum speciﬁc growth rate (μmax; Supplementary Figure S3B). Phylogenetic Variability of the rrn Operon Copy Number Then, the heterogeneity in the growth dynamics of the isolates of Bacillus from the CCB could be a response to the low availability of nutrients and the competitive cost that represents the high number of copies of the rrn operon. Phylogenetic Variability of the rrn Operon Copy Number The squares, triangles and rhomboids symbols correspond to the environment as described in Figure 1. The dotted red line represents the low number of the rrn operon in Bacillus according to the rrnDB. The black bars represent the number of operon copies in the isolates from the CCB, and the green bars represent the number of copies in Bacillus species reported in the rrnDB. Gray bar represents the number of copies in the isolate related to the genus Staphylococcus. The light purple bars represent the number of operon copies in different Staphylococcus species analyzed by the rrnDB. The orange bar represents the number of copies quantiﬁed in the type strain of B. coahuilensis (m4-4 = NRRL B-41737T), group V. The yellow bars represent the number of copies in the type strains of B. marisﬂavi (JCM 11544 = KCCM 41588) and B. horikoshii (ATTCC 700161), groups IX and XVII, respectively. January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org rrn Operon and Growth Dynamics Valdivia-Anistro et al. TABLE 1 \| Growth parameters estimated in the Bacillus isolates in the CCB. Growth parameters rrn copies Isolate Phylogenetic group A λ(h) μmax(h−1) td(h) A 14 155B_5T X. B. cereus 9.98 0.92 0.26 2.66 B 6 118_4C XI. B. sonorensis 9.89 1.13 0.24 2.88 C 8 m3-18 II. B. sp. m2-34 9.78 2.89 0.24 2.88 D 9 m2-9 V. B. coahuilensis 7.95 1.08 0.22 3.15 E 10 m2-6 V. B. coahuilensis 7.68 1.45 0.21 3.30 F 12 112B_4D XIV. B. pumilus 7.35 1.70 0.31 2.23 G 5 169A_5R XIX. Staphylococcus 7.30 0.92 0.26 2.66 H 12 107_3D IX. B. marisﬂavi 7.28 0.25 0.19 3.64 I 13 178_5C XVI. B. idriensis 7.02 2.56 0.57 1.21 J 11 126_4D X. B. cereus 6.69 2.78 0.23 3.01 K 7 152A_5R I. B. aquamaris 6.48 0.22 0.16 4.33 L 8 44_1T XVII. B. horikoshii 5.20 4.12 0.19 3.64 M 10 315_11T IX. B. marisﬂavi 4.44 1.44 0.12 5.77 N 11 144b_14T XIV. B. pumilus 3.67 2.79 0.11 6.30 O 6 108 XII. B. atrophaeus 3.38 2.06 0.08 8.66 P 11 127B_4D XVII. B. horikoshii 3.22 1.70 0.09 7.70 A = maximum biomass reached [Ln(CFU/CFU(t = 0))]; λ = lag phase; µmax = maximum speciﬁc growth rate; td = doubling (generation) time. TABLE 1 \| Growth parameters estimated in the Bacillus isolates in the CCB. as B. sonorensis (XI), B. Frontiers in Microbiology \| www.frontiersin.org P and N Contents and N:P Ratios in the Exponential Phase To assess potential eco-physiological implications associated with the oligotrophic conditions of the CCB regarding the genus Bacillus, biomass C, N, and P contents were estimated during the exponential phase of growth for all strains (Supplementary Table S2). All the isolates showed a relatively low but variable P (%) content (Mean = 0.496; SD = 0.616; Median = 0.258). While C (%) and N (%) content also showed high variability among the isolates [N (%): Mean = 7.14; SD = 5.61; Median = 5.49; C (%): Mean = 64.64; SD = 26.63; Median = 59.63]. Both the C:N ratio (Mean = 13.18; SD = 7.18; Median = 12.32) and the N:P ratio (Mean = 126.7; SD = 235.1; Median = 52.1) showed a considerable range (Table 2). Finally, the N:P ratios estimated in the Bacillus from the CCB were substantially higher than the ratios reported for other Bacilli (B. subtilis 10.6; Loladze and Elser, 2011). Interestingly, an exploratory analysis showed no overall correlation between the number of copies of the rrn operon and the growth parameters estimated (Supplementary Table S1). However, an arbitrary categorization of this copy number (where “low” was from ﬁve to seven copies, “mid” was from eight to ten copies and “high” was from 11 to 14 copies) showed that the isolates with the fewest copies had lower levels of dispersion in their growth parameters (Figure 3). Principal component analysis (PCA) was performed to describe the inﬂuence of the various growth parameters in these categories in the isolates from the CCB (Figure 3). It seems that 42.71% of the variance was explained by Component 1, which was deﬁned by doubling time (td), maximum growth rate (μmax) and the maximum biomass January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 7 rrn Operon and Growth Dynamics Valdivia-Anistro et al. FIGURE 3 \| Principal component analysis (PCA) of the growth parameters estimated in the Bacillus isolates from the CCB. The names of isolates (letters, from A to P) were in agreement with the maximum biomass reached (A) in Table 1. DISCUSSION Isolate G, with a low copy number (ﬁve copies; Staphylococcus), had the highest cellular content of both elements. In addition, isolates B and O, with six rrn operon copies, had high P-content levels TABLE 2 \| C:N and N:P ratio during the exponential phase of growth in the isolates of Bacillus from the CCB. rrn copies Isolate Phylogenetic group C:N N:P A 14 155B_5T X. B. cereus 4.94 965.38 B 6 118_4C XI. B. sonorensis 19.28 10 C 8 m3-18 II. B. sp. m2-34 8.35 286.47 D 9 m2-9 V. B. coahuilensis 24.82 79.09 E 10 m2-6 V. B. coahuilensis 18.18 52.91 F 12 112B_4D XIV. B. pumilus 18.77 194.61 G 5 169A_5R XIX. Staphylococcus 5.75 25.83 H 12 107_3D IX. B. marisﬂavi 8.87 19.55 I 13 178_5C XVI. B. idriensis 17.85 40.67 J 11 126_4D X. B. cereus 24.73 67.41 K 7 152A_5R I. B. aquamaris 0.62 72.58 L 8 44_1T XVII. B. horikoshii 13.79 4.61 M 10 315_11T IX. B. marisﬂavi 16.7 82.72 N 11 144b_14T XIV. B. pumilus 6.59 50.75 O 6 108 XII. B. atrophaeus 10.77 23.6 P 11 127B_4D XVII. B. horikoshii 10.85 51.32 Mean ± SD 13.18 ± 7.18 126.72 ± 235.13 TABLE 2 \| C:N and N:P ratio during the exponential phase of growth in the isolates of Bacillus from the CCB. DISCUSSION FIGURE 3 \| Principal component analysis (PCA) of the growth parameters estimated in the Bacillus isolates from the CCB. The names of isolates (letters, from A to P) were in agreement with the maximum biomass reached (A) in Table 1. Color labels represent the isolates of the arbitrary categorization of the number of copies of the rrn operon (upper left box), where red labels are the isolates with “low copy numbers” (ﬁve to seven), green labels are the isolates with “intermediate copy numbers” (8 to 10) and black labels are the isolates with “high copy numbers” (from 11 to 14). FIGURE 3 \| Principal component analysis (PCA) of the growth parameters estimated in the Bacillus isolates from the CCB. The names of isolates (letters, from A to P) were in agreement with the maximum biomass reached (A) in Table 1. Color labels represent the isolates of the arbitrary categorization of the number of copies of the rrn operon (upper left box), where red labels are the isolates with “low copy numbers” (ﬁve to seven), green labels are the isolates with “intermediate copy numbers” (8 to 10) and black labels are the isolates with “high copy numbers” (from 11 to 14). The general objectives of this work were to assess the variability of a particular ecological trait, the rrn operon copy number in Bacillus strains isolated from extremely oligotrophic aquatic ecosystems, and to evaluate whether there is any association between the variation of this trait and strain physiology. We analyzed 71 isolates of this ecosystem and found considerable variation in the ribosomal operon copy number with a tendency toward an intermediate number of rrn operon copies. We also documented variation in growth rate dynamics and elemental composition. While we did observe physiological associations consistent with the GRH (e.g., the isolate with the slower- growth showed the lowest P content, and a high ratio of N:P). However, there were no consistent associations between copy number and growth parameters. Instead, it is likely that a variety of genomic strategies beyond variation in rrn copy number are employed to modulate growth in this clade of Bacillus, potentially allowing for their coexistence in diﬀerent niches. Despite the growth rate not being related to cellular P content (Figure 4A), the correlation between rrn operon copy number and P (%) content is negative and signiﬁcant (Figure 4B). P and N Contents and N:P Ratios in the Exponential Phase Color labels represent the isolates of the arbitrary categorization of the number of copies of the rrn operon (upper left box), where red labels are the isolates with “low copy numbers” (ﬁve to seven), green labels are the isolates with “intermediate copy numbers” (8 to 10) and black labels are the isolates with “high copy numbers” (from 11 to 14). (B. sonorensis and B. atrophaeus, respectively). Meanwhile, isolate A (14 rrn operon copies; B. cereus) had the lowest P content of all the isolates analyzed; this isolate also had the second- highest N content. The isolates D and E (B. coahuilensis; nine and ten rrn operon copies, respectively) showed low P contents and had the lowest N content. Moreover, the previous isolates with low rrn operon copies (B, G, and O) showed low N:P ratios, while isolate A, with a high number of rrn operon copies had the highest estimated N:P ratio. In addition, the two isolates related to B. coahuilensis showed intermediate values of this elemental ratio (Table 2). These results were not consistent with the GRH and it seems that the isolates with low number of copies of the rrn operon may cope better in this oligotrophy of the CCB; although the number of operon copies quantiﬁed is high in comparison with other bacterial groups that live in other oligotrophic environments (e.g., cyanobacteria; Fegatella et al., 1998). Phylogenetic Clustering of Bacillus Isolates Our phylogenetic reconstruction showed a large diversity of species with isolates obtained from diﬀerent habitats generally widespread in the tree. This suggests that each sampling site, as well as the overall system, contains several coexisting taxa. The microbial diversity in the Churince ecosystem has been deeply documented, particularly its Bacillus population, yielding 55 thermo-resistant strains and several extremely halotolerant strains (Cerritos et al., 2010). Moreover, endemic Bacillus strains have been described and genotyped. For instance, the genomes of B. coahuilensis and Bacillus m3-13 show several interesting low-nutrient adaptations (Alcaraz et al., 2008, 2010), as well as an ancient ancestry (Moreno-Letelier et al., 2011). Further sampling of pond sediments in Churince led to a demonstration of how antagonistic interactions between the Bacilli contribute to the large observed diversity, while maintaining a large local January 2016 \| Volume 6 \| Article 1486 8 Frontiers in Microbiology \| www.frontiersin.org Valdivia-Anistro et al. rrn Operon and Growth Dynamics FIGURE 4 \| Relationship between the maximum speciﬁc growth rate (μmax) (A) and the number of copies of the rrn operon (B), along P (%) content, during the exponential phase. The isolates names (letters, from A to P) are within the circles in black and were in agreement with the maximum biomass reached (A) in Table 1. FIGURE 4 \| Relationship between the maximum speciﬁc growth rate (μmax) (A) and the number of copies of the rrn operon (B), along P (%) content, during the exponential phase. The isolates names (letters, from A to P) are within the circles in black and were in agreement with the maximum biomass reached (A) in Table 1. Previous work has described the genomic properties of some Bacillus isolated from the CCB (Alcaraz et al., 2008, 2010). For example, the genome analysis of B. coahuilensis documented nine rrn operon copies (Alcaraz et al., 2010; unpublished results). However, in our analysis, we quantiﬁed eight copies, perhaps because the diﬀerences in size among operons were too small to be detected via the pulse ﬁeld method. Nevertheless, such discrepancies are common when these types of data are compared (Vishnivetskaya et al., 2009). Bacillus groups isolated from the CCB showed from six to 14 rrn operon copies, which is consistent with previous quantiﬁcations of the rrn operon (from six to 15 copies reported in rrnDB, Stoddard et al., 2015). Phylogenetic Clustering of Bacillus Isolates This range of operon copies is not what would be expected for isolates from the CCB because the idea is that they should match a slower (i.e., “K-selected”) life history in an oligotrophic environment such as the CCB. This could be the case for all of the CCB isolates related to B. sonorensis, a soil strain that was ﬁrst described in a desert sample with similar environmental conditions to those of the CCB (Palmisano et al., 2001; Souza et al., 2006, 2012). To evaluate this further, we compared our results with data from the rrnDB database. B. atrophaeus strain 1942 has seven rrn operon copies, but as mentioned above, in our analysis, we found a related strain with six copies. The rrnDB B. subtilis strains from the database showed from eight to ten copies; in our analysis, we observed a wider range (seven to eleven copies). On the other hand, seven copies of the rrn operon were observed in the B. pumilus genome, while B. pumilus relatives isolated in the CCB have eight, eleven, and twelve copies. Such intraspeciﬁc variability was evaluated by Acinas et al., (2004) for diﬀerent bacterial genomes. They documented three species of Bacillus that had normal variation from one to three copies. Subsequent analysis with a larger number of Bacillus genomes showed similar variability (Rastogi et al., 2009). Accordingly, the CCB’s closely related, pumilus-like Bacillus showed a similar level of intraspeciﬁc variation, from one diﬀerentiation via either resistance or avoidance as in a paper- rock-scissors model (Pérez-Gutiérrez et al., 2013; Aguirre-von- Wobeser et al., 2014). Most of our isolates came from Churince, whose large terminal lake is now mostly dry due to water overexploitation (Souza et al., 2006, 2012). Interestingly, several times during this sampling period, we recovered the same phylogroups in the sediments in the same sampling sites. It has been argued that sediment is the “native” habitat of Bacillus in the CCB because many phylogenetic groups coexist there and can be recovered consistently at diﬀerent sampling times (Pérez-Gutiérrez et al., 2013). Our results support what has been observed previously: the considerable levels of bacterial diversity in this basin are the outcome of complex biotic interactions within the community, CCB’s ancient geological history, low nutrient availability, and considerable spatial and seasonal variability in environmental conditions (Souza et al., 2012). Frontiers in Microbiology \| www.frontiersin.org Variation in rrn Operon Numbers p We found no simple answer regarding variations in rrn operon copy number as a response on the part of bacteria in a low- nutrient environment. This was somewhat unexpected because rrn operon copy number is a well-studied functional trait that has been reported to be associated with bacterial lifestyle and represents an ecological strategy for nutrient use (Klappenbach et al., 2000; Stevenson and Schmidt, 2004; Green et al., 2008). A variety of studies have quantiﬁed the copy number of this operon in strains isolated from environmental samples, including some for which some Bacillus strains were analyzed (Klappenbach et al., 2000; Shrestha et al., 2007; Vieria-Silva and Rocha, 2015). However, no previous studies have focused on rrn operon copy number in such a diverse group of coexisting species within the genus Bacillus, much less in a shared environment with extremely low nutrient availability. January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 9 rrn Operon and Growth Dynamics Valdivia-Anistro et al. Antolinos et al., 2011, 2012; Bren et al., 2013). Furthermore, estimates of maximum biomass reached can be quite variable because of the inﬂuence of overall nutrient availability and sensitivity to the waste material that accumulates during the exponential phase (Buchanan et al., 1997; Chorin et al., 1997). In addition, long doubling times are characteristic of bacteria in oligotrophic environments (Vieria-Silva and Rocha, 2015). Indeed, bacteria from other ecosystems that are extremely limited in terms of nutrients can achieve generation times of thousands of years (Jørgensen and Boetius, 2007; Labonté et al., 2015). Thus, the bacterial growth properties of Bacillus that live in the extremely oligotrophic ecosystems of the CCB likely involve a complex response to environmental and nutritional conditions acting in concert with genomic potential. to four copies. Overall, the number of copies of the rrn operon in the genus Bacillus at the CCB shows considerable variability, but it does not show evidence of any clear directional change from previously published values for various taxa. This could be due to the fact that very large ranges of taxa within the Bacilli are being selected for a wide variety of responses to cope with the oligotrophic environment. rrn Operon Copy Number and Phosphorus Availability in the CCB rrn Operon Copy Number and Phosphorus Availability in the CCB As previously mentioned, ecosystems in the CCB are characterized by very low P availability in water, soil, and sediments (Elser et al., 2005; Peimbert et al., 2012), a condition that makes its high variation in rrn operon copy number somewhat surprising because we expected that such habitats would be dominated by taxa with low operon copy numbers. Previous work has suggested that rrn operon copy number is related to nutrient availability and especially with P because the copy number is linked to the growth rate, which is associated with the production of P-rich rrn (Elser, 2003; Jeyasingh and Weider, 2007). Indeed, it has been shown that bacteria that live in oligotrophic environments do tend to have very low rrn operon copy numbers (< two copies; Fegatella et al., 1998; Strehl et al., 1999; Lauro et al., 2009). In theory, multiple copies could allow a growth rate advantage when resources are abundant but would impose a competitive cost when resources are limited due to the costs of the over-production of rrn (Weider et al., 2005; Dethlefsen and Schmidt, 2007; Jeyasingh and Weider, 2007). While we detected many Bacillus isolates with six rrn operon copies, even this number is relatively high in comparison with other bacterial lineages that live under low nutrient conditions (Fegatella et al., 1998; Strehl et al., 1999; Lauro et al., 2009). Thus, it seems that the number of copies of the rrn operon is a functional trait related with the evolutionary history of the genus Bacillus, and deﬁnes its ecological versatility and adaptability to diﬀerent environmental conditions (Klappenbach et al., 2000; Feldgarden et al., 2003; Stevenson and Schmidt, 2004; Connor et al., 2010). Among the various growth dynamics of the strains from the CCB, we observed not only that many parameters were out of range when compared with those estimated for other previously studied Bacillus but also that they presented high variability that at ﬁrst inspection, does not seem to relate either to evolutionary history, isolation site, or rrn operon copy number. We hypothesize that the oligotrophic condition in the CCB may have had a signiﬁcant eﬀect on the Bacillus growth dynamics that contribute to this variability. Variation in rrn Operon Numbers For example, while some save P in their ribosomes with a slow growth rate (“K strategists”), others may maintain a high growth capacity (“r strategists”) that is compensated by other tactics, such as phospholipid to sulfolipid substitution, the presence of high-aﬃnity P-uptake systems, and small genomes (Alcaraz et al., 2008; Moreno-Letelier et al., 2011). Frontiers in Microbiology \| www.frontiersin.org Growth Parameters and rrn Operon Copy Number The analysis of bacterial growth is considered to be an important tool in understanding and characterizing an organism because it describes potential bacterial response to changes in environmental conditions, as well as ecological responses to other microorganisms (Monod, 1949; Neidhardt, 1999). As mentioned in the results section, the growth dynamics of Bacillus inhabitants of the CCB showed a high degree of variability in various parameters. This heterogeneity in growth rate has been previously observed for environmental strains with diﬀerent numbers of rrn operons (Dethlefsen and Schmidt, 2007). In addition, several of the estimated parameters were similar to those described in other Bacillus strains under extreme experimental growth conditions (Supplementary Table S3); however, many of these previous studies were performed with model or economically important species. Previous work has considered the ecological importance of the rrn operon copy numbers in bacterial adaptation to diﬀerent environmental conditions (Elser et al., 2000; Klappenbach et al., 2000; Shrestha et al., 2007; Green et al., 2008). Variation in the number of rrn operon copies is potentially related to the bacterial growth rate because of the need to sustain high levels of rRNA synthesis (Codon et al., 1995; Nanamiya et al., 2010; Yano et al., 2013). However, in our analysis, rrn operon copy number was not correlated with the estimated growth rate parameters. Instead, the growth dynamics of the Bacillus from the CCB may be dictated by a combination of diﬀerent physiological responses that are uncoupled from the rrn operon copies, such as diﬀerences in transcription rates or intra-cellular allocation processes. rrn Operon Copy Number and Phosphorus Availability in the CCB For example, long adaptation times are related to stressful conditions aﬀecting the speed of bacterial growth, like limited nutrient availability (Chorin et al., 1997; Schaechter, 2006; A simple explanation of this incongruity is that the genus Bacillus does not pay for the full cost of high copy number under nutrient limitations due to its ability to escape from scarcity by forming spores and then germinating under better conditions. Indeed, stress response capacity has been shown to be related to rrn operon multiplicity (Nanamiya et al., 2010; Yano et al., 2013). However, spore formation in Bacillus of the CCB is not a given, due to the loss of many of the genes of the spore- forming complex in the sequenced genomes (Alcaraz et al., 2010) January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org 10 rrn Operon and Growth Dynamics Valdivia-Anistro et al. and the diﬃculty of obtaining spores experimentally in all the isolated strains. Nevertheless, B. coahuilensis m4-4 and B. sp. m3- 13 have eight and nine rrn operon copies, respectively, like other spore-forming Bacilli (B. halodurans C-125, eight rrn operon copies; B. amyloliquefaciens CC178, 9 rrn operon copies). Thus, the variability in the rrn operon copy number could reﬂect broader life history strategies in which each taxa uses diﬀerent sets of resources or inhabits distinct microhabitats in structured sediments, potentially decreasing competition. In agreement with this view, various bacterial communities have been shown to have similar functional heterogeneity regarding other important ecological traits (Martínez-Alonso et al., 2004; Giovannoni and Stingl, 2005; Martiny et al., 2006). Additional work is needed to understand the importance of conserving high rrn operon copy numbers in CCB Bacillus. persist in this oligotrophic ecosystem. Analogous strategies have been described in other organisms whose growth parameters are also aﬀected by various environmental and biotic factors (Pianka, 1970; Page, 2002; Lipowsky et al., 2012). Variable lag phases may help in adapting to changing environmental conditions to reach optimum growth with long generation times (Crooks, 2005; Wangen and Webster, 2006; Daehler, 2009). We also note that the retardation of growth is a common result of intense interaction with other organisms, as well as stressful conditions (Gao et al., 2013; Tsugama et al., 2014). To date, these ecological strategies in bacteria have been largely related only to nutrient availability (Klappenbach et al., 2000; Fierer et al., 2007; Shrestha et al., 2007). rrn Operon Copy Number and Phosphorus Availability in the CCB However, the intensity of direct inter-speciﬁc interactions (such as chemical antagonisms) can also establish coexistence, and these interactions are known to be particularly intense in the CCB (Souza et al., 2012; Pérez-Gutiérrez et al., 2013; Aguirre- von-Wobeser et al., 2014). These inferences suggest a bet-hedging strategy on the part of CCB bacteria in which expression of RNA genes is tightly controlled due to low P-conditions (reﬂected in their overall low P content and high N:P ratio), but when chemical antagonism is successful, resources suddenly arrive, and the rrn operons are activated to grow rapidly under the nutrient bounty. Our overall ﬁndings about diversity in Bacillus rrn operon number in the CCB do not seem to conform to the broader context of the GRH (Elser et al., 2000; Elser, 2006). For example, isolate A (B. cereus) had the highest number of copies of the rrn operon (14) and also had the slowest growth rate and highest N:P ratio (965) when growing on media produced using the CCB’s natural waters. Furthermore, several isolates with low rrn operon numbers showed some of the highest P-content values, contrary to the GRH. However, the high N:P ratio and slow growth rate of isolate A is consistent with the development of severe P limitations for this high-copy-number strain, resulting in its high biomass N:P ratio. Then, it seems that the isolates with lower number of copies of the rrn operon may cope better in this oligotrophy of the CCB. To more eﬀectively test the genetic dimension of the GRH, each strain would need to be raised under optimal conditions at its genetically constrained maximal growth rate. Indeed, this inference is supported by the higher dispersion of growth parameters seen for high- copy-number strains. That is, low-copy-number strains may have a limited range of growth variation, regardless of media, while high-copy-number strains may have a considerable range of growth, depending on whether or not the environment is well-matched to their needs. Another possible explanation for the decoupling of rrn operon number from growth rate and stoichiometric properties is that CCB Bacillus are selected for ﬁne-tuned signaling with resource supplies or for variation in the rates of rRNA genes expression, thus disconnecting copy number from RNA production and growth rate (i.e., low-copy-strains may have high levels of transcription for each copy, while high- copy strains may more stringently express each copy). rrn Operon Copy Number and Phosphorus Availability in the CCB In any case, our data do not provide a clear resolution regarding the validity of the genetic components of the GRH within the Bacillus of the CCB. It is possible that the consideration of a broader range of bacterial taxa, as well as the more extensive testing of growth conditions, are needed in order to more rigorously test the GRH in the microbial realm, using approaches that can overcome the possible impacts of physiological conditions, phylogenetic inertia, and taxon-speciﬁc lifecycle strategies (e.g., sporulation) in terms of confounding the interpretations. W t th t th th tt d ib d i th B ill The main results of our work indicate that the rrn operon copy number exhibits considerable variation among ﬁeld-isolated Bacilli and that considerable variation also exist in their growth properties and chemical composition. However, rrn operon copy number appears to be largely uncoupled from growth and chemical properties in this clade. Further investigation is needed to understand the ecological and physiological importance of this rrn operon variability, as mediated by gene transcriptional regulation, and its inﬂuence on ribosome and protein content and thus N:P stoichiometry (Gourse et al., 1996; Fegatella and Cavicchioli, 2000; Dethlefsen and Schmidt, 2007; Scott et al., 2010; Piir et al., 2011). It may be that the extreme oligotrophic conditions in the CCB have imposed important physiological constraints on resource allocation and growth rate, as well as the expression of the rRNA genes, and thus, the rate of production of ribosomes per rrn operon copy diﬀers considerably among strains. More detailed studies, including competition experiments involving Bacillus strains isolated from diﬀerent CCB environments and subject to various environmental limitations (such as diﬀerences in nutrient supply concentrations, ratios, and supply schedules), may be needed to identify the ecological and evolutionary signiﬁcance of rrn operon copy number variation among microbes in the habitats of Cuatro Ciénegas and similar nutrient-deﬁcient habitats. Frontiers in Microbiology \| www.frontiersin.org ACKNOWLEDGMENTS FIGURE S1 \| Growth conditions assays and Gompertz growth curves of the Bacillus isolates from the CCB. (A) Different nutritional conditions were analyzed to deﬁne the optimum conditions and thus describe the growth dynamics of the Bacillus: A = Marine broth (DifcoTM 2216); B = CCB water; C = CCB water + P (KH2PO4, 5 g per liter); and D = CCB water + N (tryptone, 5 g per liter). Growth was quantiﬁed via culture turbidity (OD600; BioPhotometer Plus, Eppendorf) after 12 h of growth, and the data were normalized to deﬁne the optimal conditions for growth. (B) Growth curves were described after linear regression analysis according to the Gompertz equation. Isolate names were in agreement with the maximum growth reached (A), followed by the number of copies of the rrn operon for every isolate. Growth curves were plotted with the data of the plate count assays [Ln(CFU/CFU(t = 0))]. FIGURE S1 \| Growth conditions assays and Gompertz growth curves of the Bacillus isolates from the CCB. (A) Different nutritional conditions were analyzed to deﬁne the optimum conditions and thus describe the growth dynamics of the Bacillus: A = Marine broth (DifcoTM 2216); B = CCB water; C = CCB water + P (KH2PO4, 5 g per liter); and D = CCB water + N (tryptone, 5 g per liter). Growth was quantiﬁed via culture turbidity (OD600; BioPhotometer Plus, Eppendorf) after 12 h of growth, and the data were normalized to deﬁne the optimal conditions for growth. (B) Growth curves were described after linear regression analysis according to the Gompertz equation. Isolate names were in agreement with the maximum growth reached (A), followed by the number of copies of the rrn operon for every isolate. Growth curves were plotted with the data of the plate count assays [Ln(CFU/CFU(t = 0))]. Thanks are due to Posgrado en Ciencias Biológicas of the Universidad Nacional Autónoma de México (UNAM). This work is the doctoral research of JV-A in the Doctorado en Ciencias Biológicas program (CVU: 216032; CONACyT fellow number: 207187). We would like to thank Ariadnna del C. REFERENCES APHA (2005). Standard Methods for the Examination of Water and Wastewater. Washington, DC: American Publish Health Association. Bren, A., Hart, Y., Dekel, E., Koster, D., and Alon, U. (2013). 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E., Avitia, M., Siefert, J., Travisano, M., Rodríguez- Verdugo, A., et al. (2010). Diversity of culturable thermo-resistant aquatic bacteria along an environmental gradient in Cuatro Ciénegas, Coahuila, México. Antonie Van Leeuwenhoek 98, 1–16. doi: 10.1007/s10482-010- 9490-9 Antolinos, V., Muñoz, M., Ros-Chumillas, M., Aznar, A., Periago, P. M., and Fernández, P. S. (2011). Combined eﬀect of lysozyme and nisin at diﬀerent incubation temperature and mild heat treatment on the probability of time growth of Bacillus cereus. Food Microbiol. 28, 305–310. doi: 10.1016/j.fm.2010. 07.021 Cerritos, R., Vinuesa, P., Eguiarte, L. E., Herrera-Estrella, L., Alcaraz-Peraza, L. D., Arvizu-Gómez, J. L., et al. (2008). Bacillus coahuilensis sp. SUPPLEMENTARY MATERIAL Experimental design, analysis of data. GO-A: Experimental design, analysis of data. VS: Group leader, experimental design, analysis of data. Experimental design, analysis of data. GO-A: Experimental design, analysis of data. VS: Group leader, experimental design, analysis of data. Experimental design, analysis of data. GO-A: Experimental design, analysis of data. VS: Group leader, experimental design, analysis of data. The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.01486 The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.01486 FIGURE S4 \| Relative contribution (%) per variable in the PCA of the growth parameters estimated in the Bacillus isolates from the CCB. FIGURE S4 \| Relative contribution (%) per variable in the PCA of the growth parameters estimated in the Bacillus isolates from the CCB. A = maximum growth reached; λ = lag phase; td = doubling time; and μmax = maximum speciﬁc growth rate. AUTHOR CONTRIBUTIONS JV-A: Primary author, experimental design, ampliﬁcation and analysis of genetic material, PFGE, analysis of data. LE-F: Experimental design, analysis of data. GD-S: PFGE standardization, analysis of data. PM-Z: Growth curves standardization, parameter estimations. JG-P: Statistical analysis. JL: Cell contents of phosphorus (P) and nitrogen (N). JE: We suggest that the growth patterns described in the Bacillus isolates from the CCB, as well as the high variability in rrn operon copy number, represent ecological strategies that allow them to January 2016 \| Volume 6 \| Article 1486 Frontiers in Microbiology \| www.frontiersin.org 11 rrn Operon and Growth Dynamics Valdivia-Anistro et al. ACKNOWLEDGMENTS Cruz- Córdoba and Juan Xicohtencatl-Cortés of the Laboratorio de Bacteriología Intestinal (Hospital Infantil de México “Federico Gómez”), and José Luis Méndez and Rosario Morales of the Laboratorio de Genómica Bacteriana (UNAM), because of their technical assistance in PFGE standardization, Africa Islas of the Laboratorio de Bacteriología Molecular (CINVESTAV-Unidad Irapuato) for providing the strains needed to perform this work, Felipe García-Oliva (Biogeoquímica de Suelos; CIEco, UNAM) and Mario Soberón-Chávez (Departamento de Microbiología Molecular; IBT, UNAM) for their advice and input of ideas throughout the development of this work, Ana Gutiérrez- Preciado (Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València) for her comments and observations that contributed to the improvement of the manuscript. Grants from Alianza WWF-Fundación Carlos Slim to VS and LE-F and U.S. NSF (DEB-0950179) to JE supported this study. Laura Espinosa- Asuar and Erika Aguirre-Planter helped in the general technical logistics during the entire project. FIGURE S2 \| rRNA operon copy number in the genus Bacillus isolated in the CCB. For every isolate, the separation of fragments with PFGE is shown on the left and Southern-blot conﬁrmation is shown on the right. At the bottom of the ﬁgure is the total number of rRNA operon copies quantiﬁed. The red asterisk represents the fragments identiﬁed with hybridization analysis. FIGURE S3 \| Relationship between the growth parameters estimated in the Bacillus isolates from the CCB. (A) λ vs μmax and, (B) μmax vs A. Isolate names (letters, from A to P) are within the circles in black and were in agreement with the maximum biomass reached (A) in Table 1. A = maximum biomass reached [Ln(CFU/CFU(t = 0))]; λ = lag phase; μmax = maximum speciﬁc growth rate. 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https://openalex.org/W4281621325	https://www.frontiersin.org/articles/10.3389/fonc.2022.896904/pdf	English	null	β-Boswellic Acid Suppresses Breast Precancerous Lesions via GLUT1 Targeting-Mediated Glycolysis Inhibition and AMPK Pathway Activation	Frontiers in oncology	2,022	cc-by	8,284	ORIGINAL RESEARCH published: 31 May 2022 doi: 10.3389/fonc.2022.896904 Keywords: b-boswellic acid, breast precancerous lesions, glycolysis, GLUT1, AMPK Fengjie Bie 1†, Guijuan Zhang 2†, Xianxin Yan 1†, Xinyi Ma 3, Sha Zhan 1, Yebei Qiu 4 Jingyu Cao 4, Yi Ma 5 and Min Ma 1,4* Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncology Received: 15 March 2022 Accepted: 25 April 2022 Published: 31 May 2022 Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncology Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncology Received: 15 March 2022 Accepted: 25 April 2022 Published: 31 May 2022 Fengjie Bie 1†, Guijuan Zhang 2†, Xianxin Yan 1†, Xinyi Ma 3, Sha Zhan 1, Yebei Qiu 4 Jingyu Cao 4, Yi Ma 5 and Min Ma 1,4* Reviewed by: Manoj Garg, Amity University, India Chakrabhavi Mohan, Chakrabhavi, India Ravi Manoharan, University of Madras, India Reviewed by: Manoj Garg, Amity University, India Chakrabhavi Mohan, Chakrabhavi, India Ravi Manoharan, University of Madras, India Correspondence: Min Ma tmamin@jnu.edu.cn †These authors have contributed equally to this work and share ﬁrst authorship 1 School of Traditional Chinese Medicine, Jinan University, Guangzhou, China, 2 School of Nursing, Jinan University, Guangzhou, China, 3 The First Clinical Medical College, Southern Medical University, Guangzhou, China, 4 The Oncology Department, The First Afﬁliated Hospital of Jinan University, Guangzhou, China, 5 Department of Cellular Biology, Institute of Biomedicine, National Engineering, Research Center of Genetic Medicine, Key Laboratory of Bioengineering Medicine of Guangdong Province, The National Demonstration Center for Experimental Education of Life Science and Technology, Jinan University, Guangzhou, China Correspondence: Min Ma tmamin@jnu.edu.cn †These authors have contributed equally to this work and share ﬁrst authorship Breast carcinoma is a multistep progressive disease. Precancerous prevention seems to be crucial. b-Boswellic acid (b-BA), the main component of the folk medicine Boswellia serrata (B. serrata), has been reported to be effective in various diseases including tumors. In this work, we demonstrated that b-BA could inhibit breast precancerous lesions in rat disease models. Consistently, b-BA could suppress proliferation and induce apoptosis on MCF-10AT without signiﬁcantly inﬂuencing MCF-10A. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that b-BA may interfere with the metabolic pathway. Metabolism-related assays showed that b-BA suppressed glycolysis and reduced ATP production, which then activated the AMPK pathway and inhibited the mTOR pathway to limit MCF-10AT proliferation. Further molecular docking analysis suggested that GLUT1 might be the target of b-BA. Forced expression of GLUT1 could rescue the glycolysis suppression and survival limitation induced by b-BA on MCF-10AT. Taken together, b-BA could relieve precancerous lesions in vivo and in vitro through GLUT1 targeting-induced glycolysis suppression and AMPK/mTOR pathway alterations. Here, we offered a molecular basis for b-BA to be developed as a promising drug candidate for the prevention of breast precancerous lesions. ORIGINAL RESEARCH published: 31 May 2022 doi: 10.3389/fonc.2022.896904 Edited by: Gautam Sethi, National University of Singapore, Singapore Fengjie Bie 1†, Guijuan Zhang 2†, Xianxin Yan 1†, Xinyi Ma 3, Sha Zhan 1, Yebei Qiu 4, Jingyu Cao 4, Yi Ma 5 and Min Ma 1,4* Fengjie Bie 1†, Guijuan Zhang 2†, Xianxin Yan 1†, Xinyi Ma 3, Sha Zhan 1, Yebei Qiu 4, Jingyu Cao 4, Yi Ma 5 and Min Ma 1,4* Abbreviations: BA, boswellic acid; Tam, tamoxifen; DAVID, Database Visualization and Integrated Discovery; KEGG, Kyoto Encyclopedia of Genes and Genomes; AMPK, adenosine 5’-monophosphate (AMP)-activated protein kinase; mTOR, mammalian target of rapamycin; FBS, fetal bovine serum; USPSTF, the United States Preventive Services Task Force; ATCC, American Type Culture Collection; KCI, Karmanos Cancer Institute; ECAR, extracellular acidiﬁcation rate; DMBA, 7,12-dimethyltetraphene. 1 INTRODUCTION past decades (14), while the potential mechanism of BAs on treating breast precancerous lesions is still elusive. The latest research shows that female breast cancer has surpassed lung cancer as the most commonly diagnosed (11.7%) and the ﬁfth most lethal (6.9%) cancer in the world (1). Due to insufﬁcient ﬁnancial and medical resources, the sharply increasing incidence of female breast cancer results in enormous social and economic burden not only to individuals but also to nations. Breast carcinogenesis is a multistep, multipath, and multiyear disease of progressive gene-associated tissue damage (2). The positive interventions to arrest these steps may impede or delay canceration. Therefore, efforts to develop precancerous therapy and prevention seem to be extremely important. In this article, we found that b-BA could prevent precancerous lesions in rat models, and exert an inhibition effect on MCF-10AT. Pathway enrichment indicated that the effect of b-BA may focus on the metabolism. Further experiment showed that the glycolysis process of MCF-10AT was inhibited via targeting GLUT1. Our work may provide evidence for the potential clinical use of b-BA in breast precancerous prevention. Citation: Bie F, Zhang G, Yan X, Ma X, Zhan S, Qiu Y, Cao J, Ma Y and Ma M (2022) b-Boswellic Acid Suppresses Breast Precancerous Lesions via GLUT1 Targeting- Mediated Glycolysis Inhibition and AMPK Pathway Activation. Front. Oncol. 12:896904. doi: 10.3389/fonc.2022.896904 May 2022 \| Volume 12 \| Article 896904 1 Frontiers in Oncology \| www.frontiersin.org b-BA for Breast Precancerous Prevention Bie et al. past decades (14), while the potential mechanism of BAs on treating breast precancerous lesions is still elusive. In this article, we found that b-BA could prevent precancerous lesions in rat models, and exert an inhibition effect on MCF-10AT. Pathway enrichment indicated that the effect of b-BA may focus on the metabolism. Further experiment showed that the glycolysis process of MCF-10AT was inhibited via targeting GLUT1. Our work may provide evidence for the potential clinical use of b-BA in breast precancerous prevention. 2.4 Apoptosis Assay by Flow Cytometry 2.4 Apoptosis Assay by Flow Cytometry The apoptosis assay was determined using an Annexin V-APC/ PI detection kit (DOJINDO, Japanese) following the manufacturer’s protocol. The cells under detection were harvested, washed, and suspended in the binding buffer. After staining with the Annexin V/PI staining mixture for 15 min at room temperature, the samples were analyzed by ﬂow cytometry (Beckman, USA) within 1 h. 2.2 Colony Formation Assay Cells were seeded into six-well plates at the concentration of 500 cells per well and maintained with or without b-BA for 2 weeks. For analysis, the plates were washed with PBS twice, ﬁxed with methanol for 30 min, stained by 0.1% crystal violet, and then counted via ImageJ. 2.1 Cell Culture Currently, no publicly recognized breast precancerous therapy has been achieved. Tamoxifen (Tam) as the most effective and widely used antiestrogen therapy is applied to reduce the incidence of breast cancer in high-risk women. Due to its principal toxicities like endometrial cancers and thromboembolic events (3), USPSTF announcedthatTamwasnotsuggestedforpreventioninnon-high- risk populations. An effective and well-tolerated remedy is urgently needed to ﬁll up the therapeutic gap between precancerous lesion and breast cancer. The Warburg effect is one of the main characteristics of cancer, which describes a phenomenon in which cancer cells metabolize more glucose by glycolysis than normal tissuesdespitethepresenceofoxygen,producingsubstantiallactate. There are lines ofevidencethat showed thatcellmetabolism may be reprogrammed at the precancerous stage with the involvement of glycolysis increase (4, 5), which created a potential therapeutic window between normal tissues and premalignant lesions. Glucose transporters (GLUTs) play critical roles in the alteration of metabolism. GLUTs are membrane proteins regulating glucose uptake and maintaining intracellular glucose concentration, which act on the upstream of glycolysis (6). Glucose transport 1 (GLUT1) is upregulated in many types of cancers. Targeting GLUT1 could reduce glucose uptake and induce apoptosis in cancer cells (7–9). Some small molecules derived from natural compounds have been proven to be GLUT1 inhibitors with anti- cancer effects (10–12). The MCF-10A and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s directions. MCF-10AT was obtained from the Barbara Ann Karmanos Cancer Institute. MCF-10A was derived from spontaneous immortalized breast epithelial cells of a patient with ﬁbrocystic disease. MCF-10AT was obtained by H-ras-transfected MCF-10A cells. MDA-MB-231 was an estrogen receptor-negative breast cancer cell line. MCF-10A and MCF-10AT were maintained in DMEM/F12 medium supplemented with 10% horse serum, 20 ng/ml EGF, 10 mg/ml insulin, and 50 mg/ml hydrocortisone. MDA-MB-231 was cultured with Leibovitz’s L-15 medium containing 10% FBS. All cells were incubated in the 37°C incubator with 5% CO2 and 95% humidity. 2.3 Cell Viability Assay The effect of b-BA on cells was assessed by CCK8 assay. Twenty- four hours before treatment, 4 × 103 cells were seeded into each well, followed by adding various concentrations of b-BA. On the day of detection, the supernatant was replaced by 10% CCK8 mixture (10 ml of CCK8 with 90 ml of basic medium for each well). With additional 2 h incubation at 37°C, the plate was read at 450 nm by a microplate spectrophotometer. Nowadays, about 50% of pharmaceuticals are derived from natural compounds and their derivatives. Phytochemicals such as ﬂavonoids, alkaloids, and diterpenoids function well in diverse ﬁelds with higher safe dose and fewer side effects (13). Boswellic acids (BAs) are pentacyclic triterpenes, the main active components of Boswellia serrata Roxb. BAs and their derivatives have been proven to possess signiﬁcant potential in treating inﬂammation diseases concerning asthma, rheumatoid arthritis, hydrocephalus, and, more importantly, a variety of cancers. The results of several studies indicated that BAs exert suppressing actions against tumor survival, proliferation, angiogenesis, and metastasis with outstanding tolerance in the 2.14.1 Establishment of the Breast Precancerous Rat Model The breast precancerous lesion was induced by the combination of DMBA, estrogen, and progesterone as described previously. Brieﬂy, the rats received DMBA sesame oil (1 ml/100 g body weight) via gavage and benzoate estradiol (0.5 mg/kg body weight) via intramuscular injection from Day 1 to Day 3. Then, progesterone (4 mg/kg body weight) was injected on Day 4. The rats were observed on Day 5. Continuous 12 cycles (5 days per cycle) were performed. 2.9 Real-Time PCR Assay RNA was extracted from control and drug-treated cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed (Takara, Japan). Then, the cDNA was submitted to quantitative PCR using 2× SYBR green master mix (Bio-Rad laboratories, USA) on a CFX96 detection system according to the manufacturer’s instructions. Data of samples were normalized by the levels of ACTB. The sequences of primers were acquired from PrimerBank. 2.5 Target Prediction and KEGG Enrichment Analysis of b-BA To identify potential targets and signaling pathways, b-BA (PubChem CID:168928) was submitted to Bioinformatics May 2022 \| Volume 12 \| Article 896904 Frontiers in Oncology \| www.frontiersin.org 2 b-BA for Breast Precancerous Prevention Bie et al. USA) according to the protocol of the manufacturer. Twenty- four hours after transfection, the cells were treated with vehicle or b-BA 48 h before downstream assays. Analysis Tool for Molecular mechanism of TCM (BATMAN- TCM, http://bionet.ncpsb.org/batman-tcm/). The predicted targets were submitted for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on the DAVID website to enrich related signaling pathways. USA) according to the protocol of the manufacturer. Twenty- four hours after transfection, the cells were treated with vehicle or b-BA 48 h before downstream assays. 2.11 Preparation of DMBA Solution DMBA was precisely weighted, dissolved in sesame oil, and ultrasonic processed in a water bath at a constant temperature of 60°C to get a homogeneous mixture. The concentration of DMBA was 7 mg/ml. 2.11 Preparation of DMBA Solution DMBA was precisely weighted, dissolved in sesame oil, and ultrasonic processed in a water bath at a constant temperature of 60°C to get a homogeneous mixture. The concentration of DMBA was 7 mg/ml. 2.14 Establishment and Treatment of the Rat Model With Precancerous Breast Lesions 2.14 Establishment and Treatment of the Rat Model With Precancerous Breast Lesions Forty female SD rats that were 6 weeks old were fed in the SPF- grade fostering environment of Jinan University Animal Center. Then, the rats were randomly divided into four groups (10 per group): the vehicle group, the disease model group, the Tam- treated group, and the b-BA acid-treated group. One-week post arrival, the hair around the breast (1 cm diameter) of these rats was removed by depilatory paste for follow-up treatment. Finally, the rats were sacriﬁced and tested in the 14th week. This animal experiment was approved by the Laboratory Animal Ethics Committee of Jinan University. The experimental procedure and animal welfare strictly followed the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and related regulations of Jinan University. .7 2-NBDG Glucose Uptake Detection 2-NBDG (Med Chem Express, USA), a ﬂuorescent D-glucose analog, was used to detect glucose uptake level of cells following treatment with b-BA. The stock solution was diluted with basic medium to yield 20 mM working solution in advance. After treatment with 40 mM b-BA for 48 h, 2-NBDG working solution was added to cover the surface of culture cells sufﬁciently and incubated at 37°C for 30 min. Then, the cells were washed with PBS twice to remove residual reagent and detected as soon as possible. The intensity of ﬂuorescence was detected using ﬂow cytometry (Beckman, USA) with FL1. The blank matrix was prepared as previously described. Brieﬂy, the water phase was prepared by dissolving 7 g of KOH in an appropriate amount of distilled water and heated to 80°C. Stearic acid (140 g) was heated, melted, and cooled down to 80°C. KOH water solution (100 g) and glycerol were added and stirred constantly. Then, an appropriate amount of distilled water was added, and the volume was metered to 1,000 ml to obtain the blank matrix (Mat). The matrix was continuously stirred and cooled down to 40°C. Then, a deﬁnite dose of b-BA (MedChemExpress, USA) was added, thoroughly mixed, and cooled down to room temperature to obtain the b-BA cream. The concentration of b-BA in cream was 5 mg/g. 2.12 Preparation of Tamoxifen Cream The blank matrix was heated to melt and slowly mixed with Tam via constant stirring. Then, the mix was cooled down to room temperature. The concentration of Tam in cream was 5 mg/g. The blank matrix was heated to melt and slowly mixed with Tam via constant stirring. Then, the mix was cooled down to room temperature. The concentration of Tam in cream was 5 mg/g. 2.6 ECAR Detection Extracellular acidiﬁcation rate (ECAR) was analyzed using Glycolysis Stress Test Kit via Seahorse XF96 instrument (Agilent Technologies, USA). Brieﬂy, cells were pre-treated with or without b-BA for 48 h and then seeded into a plate one day before testing. During the experiment, cells were treated with glucose, oligomycin, and 2-deoxy-glucose sequentially with ECAR value quantiﬁed at different time points. 2.8 Western Blot Assay For cells, reasonable RIPA lysis buffer was added to the dishes following twice washing with PBS. Then, cells were harvested by gentle scraping and stored at −80°C if not processed immediately. The well-prepared total protein was separated by 12% or 10% SDS-PAGE and transferred to a 0.22-mm polyvinylidene ﬂuoride (PVDF) membrane (Millipore, Billerica, USA). After blocking with 5% skimmed milk at room temperature, the membrane was washed 3 times and incubated with a certain ﬁrst antibody overnight at 4°C. HRP-conjugated secondary antibody (Cell Signaling Technology, Inc., Boston, USA) was used with bands visualized by the ECL detection kit. 2.10 Plasmid Construction and Transfection The CDS sequence of GLUT1 was successfully cloned into pcDNA3.1 plasmid and validated by sequencing. Then, the plasmid pcDNA3.1-GLUT1 and its control were transfected into MCF-10AT using Lipofectamine 2000 (Invitrogen, CA, May 2022 \| Volume 12 \| Article 896904 Frontiers in Oncology \| www.frontiersin.org 3 b-BA for Breast Precancerous Prevention Bie et al. 2.14.2 Therapeutic Intervention of Tamoxifen From Day 1 of model establishment to the end of the 14th week, 0.2 g of Tam cream (5 mg/g cream) was smeared with cotton swabs on the exposed skin of breast of each rat once a day. multiple groups using GraphPad Prism v. 8.0 (La Jolla, CA). p < 0.05 was considered statistically signiﬁcant. 2.15 Hematoxylin and Eosin Staining Once separated from the bodies of rats, organ tissues were ﬁxed in formalin immediately, and later embedded in parafﬁn. The blocks were cut into 5-mm sections. Before staining, the slides were baked, dewaxed, and hydrated. For HE staining, the slides were stained with hematoxylin ﬁrst for 2 min followed by hydrochloric acid alcohol differentiation and then eosin stain. 3.1 b-BA Suppresses Cell Growth With Different Effects From Day 1 of model establishment to the end of the 14th week, 0.2 g of b-BA cream (5 mg/g cream) was smeared with cotton swabs on the exposed skin of breast of each rat once a day. The chemical structure of cordycepin is shown in Figure 1A. Cell lines used in this experiment involve MCF-10A, MCF-10AT, and MDA-MB-231. Due to the various similarities between precancerous lesions and tumors, a commonly used triple- negative breast cancer cell line, MDA-MB-231, was used as a control. These cells were treated with b-BA at various concentrations for 24, 48, or 72 h to investigate the toxic effect of b-BA on these cells. As shown in Figure 1B, the CCK-8 assay showed that b-BA displayed a signiﬁcant inhibition effect on MCF-10AT and MDA-MB-231, and a mild effect on MCF-10A. The IC50 values of b-BA at 48 and 72 h were 89.04 and 91.41 mM in MCF-10A cells, 37.2 and 30.3 mM in MCF-10AT cells, and 39.4 and 26.23 mM in MDA-MB-231 cells, respectively. In addition, a colony formation assay was performed to assess the inhibitory effect of b-BA. The results revealed that MCF-10AT and MDA-MB-231 treated with 5, 10, and 20 mM b-BA for 2 2.16 Statistical Analysis Error bars represent the S.D., as indicated in ﬁgure legends. All statistical tests were analyzed by Student’s two-tailed t-test for comparison of two groups and by analysis of variance (with post- hoc comparisons using Dunnett’s test) for comparison of B A B C FIGURE 1 \| b-BA inhibits cell proliferation in MCF-10AT and MDA-MB-231. (A) Chemical structure of b-BA. (B) Cell viability was detected by CCK8 assay following treatment with different doses of b-BA. Cells were treated with 0, 10, 20, 40, 80, 160, and 320 mM of b-BA for 24, 36 and 48 h, respectively. IC50 values for each cell line at different time points were determined. (C) Colony formation assay for MCF-10A, MCF-10AT, and MDA-MB-231 treated with 0, 20, 40, and 60 mM of b- BA, respectively. Numbers of colonies were counted and shown below. p < 0.05, p < 0.01, and *p < 0.001. B A A C C FIGURE 1 \| b-BA inhibits cell proliferation in MCF-10AT and MDA-MB-231. (A) Chemical structure of b-BA. (B) Cell viability was detected by CCK8 assay following treatment with different doses of b-BA. Cells were treated with 0, 10, 20, 40, 80, 160, and 320 mM of b-BA for 24, 36 and 48 h, respectively. IC50 values for each cell line at different time points were determined. (C) Colony formation assay for MCF-10A, MCF-10AT, and MDA-MB-231 treated with 0, 20, 40, and 60 mM of b- BA, respectively. Numbers of colonies were counted and shown below. p < 0.05, p < 0.01, and p < 0.001. 4 May 2022 \| Volume 12 \| Article 896904 Frontiers in Oncology \| www.frontiersin.org b-BA for Breast Precancerous Prevention Bie et al. weeks formed fewer and smaller colonies than control (Figure 1C), indicating that b-BA inhibited the growth of these cells in a dose-dependent manner, while MCF-10A was suppressed in a relatively milder way. aspartic acid proteases (caspases) plays an important role in cell apoptosis. As shown in Figure 2C, the cleaved form of caspase 3 was dominantly increased in MCF-10AT. Bax, the core regulator of the intrinsic pathway of apoptosis, also increased following b- BA treatment in MCF-10AT. Altogether, b-BA markedly promoted apoptosis in MCF-10AT with a relatively milder effect on MCF-10A. 3.2 b-BA Promotes Apoptosis in MCF-10AT We further explored whether b-BA had an effect on the apoptosis of MCF-10A and MCF-10AT. After treating with b-BA for 48 h, MCF-10A and MCF-10AT cells were analyzed via Annexin V/PI staining. As shown in Figures 2A, B, the Q3 area indicating early apoptosis and the Q2 area indicating late apoptosis were both dramatically increased for MCF-10AT, suggesting that b-BA induced signiﬁcant apoptosis, while b-BA induced apoptosis on MCF-10A in a relative milder way. To further conﬁrm these results, we performed Western blot to examine protein expression of apoptosis-related genes. Activation of cysteine- 3.3 b-BA Suppresses Glycolysis and Activates AMPK Pathway in MCF-10AT To further explore the potential mechanism of b-BA in inhibiting proliferation and inducing apoptosis on MCF-10AT, we searched the BATMAN-TCM website to predict potential targets. A total of 45 genes were obtained, including COX1, ADH1B, and CYP17A1. To further characterize the potential affected pathways, these genes were submitted for KEGG analysis. The top enriched pathway was the metabolic pathway (Figure 3A). Considering glycolysis A B C FIGURE 2 \| b-BA promotes apoptosis in MCF-10AT. (A) FACS analysis Annexin V/PI staining was used to determine apoptosis induced by b-BA. (B) The population of early apoptosis and that of late apoptosis were counted. (C) The expression levels of cleaved-caspase 3, pro-caspase 3, and Bax were analyzed in MCF-10AT following a 48-h treatment with b-BA at designated concentrations. Protein levels were quantiﬁed by Image J and normalized with that of b-actin in the right panel. Data were shown as the mean ± S.D from independent experiments. p < 0.001. A B A B B C C FIGURE 2 \| b-BA promotes apoptosis in MCF-10AT. (A) FACS analysis Annexin V/PI staining was used to determine apoptosis induced by b-BA. (B) The population of early apoptosis and that of late apoptosis were counted. (C) The expression levels of cleaved-caspase 3, pro-caspase 3, and Bax were analyzed in MCF-10AT following a 48-h treatment with b-BA at designated concentrations. Protein levels were quantiﬁed by Image J and normalized with that of b-actin in the right panel. Data were shown as the mean ± S.D from independent experiments. p < 0.001. May 2022 \| Volume 12 \| Article 896904 Frontiers in Oncology \| www.frontiersin.org 5 b-BA for Breast Precancerous Prevention Bie et al. pathways. Here, we found that the phosphorylation of AMPK a-subunit was enhanced following b-BA treatment with the total expression of AMPK not inﬂuenced. The activation of AMPK downstream target ACC further conﬁrmed these. mTORC1 as a reported target of AMPK functions to promote cell growth and proliferation. Here, the results of Western blot showed that p- mTOR as well as the downstream target p-p70S6k of MCF-10AT were reduced following b-BA treatment, with the pan-protein expression not affected (Figure 3E). Altogether, b-BA could suppress the glycolysis process and induce lactate and ATP reduction in MCF-10AT. The energy stresses would activate the AMPK pathway and inhibit the mTORC1 pathway, which may partially explain the inhibition effects of b-BA on MCF-10AT. 3.3 b-BA Suppresses Glycolysis and Activates AMPK Pathway in MCF-10AT increase is a key character of precancerous lesions compared to normal tissues, and we detected glycolysis changes of MCF-10A and MCF-10AT via seahorse assays. Following treatment with b- BA, MCF-10AT displayed decreased extracellular acidiﬁcation rate (ECAR) compared to control cells, while the change of ECAR in MCF-10A was mild (Figure 3B). Consistently, decreased lactate production was observed in b-BA-treated MCF-10AT (Figure 3C), and the ATP level was also decreased (Figure 3D). Since ATP is the main source of energy required by most in vivo biochemical processes, its reduction may limit cell survival. The AMPK system is a key player in regulating energy balance, which could be activated by increased AMP/ATP ratio. Once activated, it switches anabolic processes to catabolic A B D E C FIGURE 3 \| b-BA suppresses glycolysis and activates the AMPK pathway in MCF-10AT. (A) KEGG pathway enrichment analysis was performed with predicted targets derived from BATMAN-TCM. (B) The cellular ECAR was measured in MCF-10A and MCF-10AT cells with or without b-BA treatment. Statistics of glycolysis ECAR values are shown in the lower panel. The lactate production (C) and ATP levels (D) were measured in MCF-10AT cells with or without b-BA treatment. (E) The expression levels of AMPK, p-AMPK, ACC, p-ACC, mTOR, p-mTOR, p70S6k, and p-p70S6k were analyzed in MCF-10A and MCF-10AT following a 48-h treatment with 40 mM b-BA. Protein levels were quantiﬁed by ImageJ and normalized with that of b-actin in the lower panel. Data were shown as the mean ± S.D from independent experiments. p < 0.001. D C A B B B FIGURE 3 \| b-BA suppresses glycolysis and activates the AMPK pathway in MCF-10AT. (A) KEGG pathway enrichment analysis was performed with predicted targets derived from BATMAN-TCM. (B) The cellular ECAR was measured in MCF-10A and MCF-10AT cells with or without b-BA treatment. Statistics of glycolysis ECAR values are shown in the lower panel. The lactate production (C) and ATP levels (D) were measured in MCF-10AT cells with or without b-BA treatment. (E) The expression levels of AMPK, p-AMPK, ACC, p-ACC, mTOR, p-mTOR, p70S6k, and p-p70S6k were analyzed in MCF-10A and MCF-10AT following a 48-h treatment with 40 mM b-BA. Protein levels were quantiﬁed by ImageJ and normalized with that of b-actin in the lower panel. Data were shown as the mean ± S.D from independent experiments. p < 0.001. 3.3 b-BA Suppresses Glycolysis and Activates AMPK Pathway in MCF-10AT May 2022 \| Volume 12 \| Article 896904 6 Frontiers in Oncology \| www.frontiersin.org Frontiers in Oncology \| www.frontiersin.org b-BA for Breast Precancerous Prevention Bie et al. 3.4 b-BA Interacts With GLUT1 to Block Glucose Uptake in MCF-10AT proteins in the glycolysis pathway. The molecular docking analysis was based on vital parameters like binding site and strength of molecular interactions. The initial structures were taken from the Protein Data Bank (PDB). Substrates were docked into the active site of protein using the AutoDock Vina tool (15) in Chimera (16). The docked poses with the highest docking scores were used for the subsequent studies. As a result, the highest afﬁnity was reached between b-BA and GLUT1 (−10.5 kcal/mol), which was higher than the redocking score between natural substrate and GLUT1 (−6.9 kcal/mol) (Table 1). GLUT1 is a key transporter that regulates glucose absorbance and maintains intracellular glucose level, which governs the fuel of glycolysis. It was reported that Phe379, Trp388, and Trp412 played essential roles in GLUT1 function and glucose uptake (17–19). Notably, our results showed that b-BA bonded to the same pocket and interacted with these reported residues as natural substrate glucose (Figures 4A, B), which indicated that it might be a competent inhibitor for GLUT1. To validate the effect of b-BA on GLUT1, we checked the expression of GLUT1. As a result, neither the protein nor the mRNA level of GLUT1 in MCF-10AT was inﬂuenced following b-BA treatment (Figures 4C, D), while the results of 2-NBDG glucose uptake assay indicated that b-BA suppressed glucose transport (Figure 4E). In summary, b-BA could bind with GLUT1 to block glucose uptake, without inﬂuencing GLUT1 expression in MCF-10AT. b-BA Interacts With GLUT1 to Block ucose Uptake in MCF-10AT To obtain further insight into the mechanisms of b-BA in regulating the glycolysis process, we used docking analysis to search potential targets of b-BA. To focus on the glycolysis process, we predicted the binding between b-BA and the key TABLE 1 \| Molecular docking scores (kcal/mol) of hit molecules with PDB IDs. Ligand Receptor PDB ID Afﬁnity (kcal/mol) b-BA Aldolase 1QO5 −7.6 b-BA Enolase 4ZA0 −8.6 b-BA Glucose transporter 1 4PYP −10.5 b-BA Glyceraldehyde 3- phosphatedehydrogenase 1ZNQ −8.4 b-BA Hexokinase 2NZT −8.3 b-BA Lactate dehydrogenase 6SBV −8.2 b-BA Phosphoglycerate kinase 2X13 −8.0 b-BA Phosphofructokinase l 4XYK −6.2 b-BA Phosphoglycerate mutase 5Y2I −8.2 b-BA Phosphohexose isomerase 6XUH −5.6 b-BA Pyruvate kinase 3ME3 −8.6 b-BA Triose phosphate isomerase 4ZVJ −8.1 Nonyl D- glucopyranoside Glucose transporter 1 4PYP TABLE 1 \| Molecular docking scores (kcal/mol) of hit molecules with PDB IDs. D A B D E C FIGURE 4 \| b-BA interacts with GLUT1 to block glucose uptake in MCF-10AT. (A) Visualization of 3D interaction of docked complex composed of GLUT1 and b- BA. (B) Ligand interactions between b-BA and GLUT1. (C) The protein expression of GLUT1 in MCF-10AT cell with or without b-BA treatment was measured by Western blot. (D) The gene expression of GLUT1 in MCF-10AT cell with or without b-BA treatment was measured by real-time PCR. (E) The 2-NBDG glucose uptake in GLUT1 overexpression MCF-10AT and its corresponding control cells was measured by FACS. Statistics are shown in the right panel as mean ﬂuorescent intensity ± S.D. C A B A B C A B B E E FIGURE 4 \| b-BA interacts with GLUT1 to block glucose uptake in MCF-10AT. (A) Visualization of 3D interaction of docked complex composed of GLUT1 and b- BA. (B) Ligand interactions between b-BA and GLUT1. (C) The protein expression of GLUT1 in MCF-10AT cell with or without b-BA treatment was measured by Western blot. (D) The gene expression of GLUT1 in MCF-10AT cell with or without b-BA treatment was measured by real-time PCR. (E) The 2-NBDG glucose uptake in GLUT1 overexpression MCF-10AT and its corresponding control cells was measured by FACS. Statistics are shown in the right panel as mean ﬂuorescent intensity ± S D May 2022 \| Volume 12 \| Article 896904 7 Frontiers in Oncology \| www.frontiersin.org b-BA for Breast Precancerous Prevention Bie et al. 3.5 GLUT1 Mediates b-BA Regulated Glycolysis Suppression To further evaluate the therapy value of b-BA in breast precancerous prevention, b-BA was given to a precancerous rat model in the form of cream once a day. The animal model was established by DMBA combined with estrogen and progestin induction in SD female rats, with Tam used as a positive control. After 14 weeks of treatment, all the rats were sacriﬁced, and the morphology of mammary glands was checked by HE staining (Figure 6). As shown in Table 2, the rats in the normal group hardly showed abnormal hyperplasia, while in the model group, most animals developed atypical hyperplasia and invasive carcinoma (77.5%). After treating with b-BA cream, the dysplasia and carcinoma phenotypes were signiﬁcantly prevented (44.2%), the effect of which was comparable to the Tam-positive treatment group (36.6%). These results further validated that b-BA could relieve precancerous breast lesion. GLUT1 was reported to be upregulated in various types of carcinomas, while its function in breast precancerous cells remains to be elusive. We overexpressed GLUT1 by transfecting plasmid pcDNA3.1-GLUT1 into MCF-10AT and then tested the efﬁciency (Figure 5A). As shown in Figures 5B, C, forced expression of GLUT1 promoted cell proliferation and increased glucose uptake. We further checked whether b-BA-regulated glycolysis alteration was related to GLUT1. The results suggested that overexpression of GLUT1 could predominantly reverse the proliferation inhibition induced by b-BA (Figure 5G). Consistently, overexpression of GLUT1 can also rescue the glycolysis phenotypes in MCF-10AT cells (Figures 5D–F). These results conﬁrmed that GLUT1 was involved in b-BA- regulated glycolysis of MCF-10AT. A B D E F G C FIGURE 5 \| GLUT1 mediates b-BA regulated glycolysis suppression. (A) The overexpression level of GLUT1 protein was detected by Western blot. (B) The 2- NBDG glucose uptake levels of GLUT1 overexpressing and its corresponding control cells were detected by FACS. Statistics are shown in the right panel as mean ﬂuorescent intensity ± S.D. (C) Cell viability was detected by CCK8 assay in GLUT1 overexpressing and its corresponding control cells. The glucose uptake level (D) ATP levels (E), lactate production (F), and cell viability (G) were measured in the group of MCF-10AT+pcDNA3.1, MCF-10AT+pcDNA3.1+BA, and MCF-10AT+ GLUT1+BA, respectively. Data were shown as the mean ± S.D from independent experiments. p < 0.01 and p < 0.001. A B C D D D D C E G F G E F FIGURE 5 \| GLUT1 mediates b-BA regulated glycolysis suppression. 3.5 GLUT1 Mediates b-BA Regulated Glycolysis Suppression (A) The overexpression level of GLUT1 protein was detected by Western blot. (B) The 2- NBDG glucose uptake levels of GLUT1 overexpressing and its corresponding control cells were detected by FACS. Statistics are shown in the right panel as mean ﬂuorescent intensity ± S.D. (C) Cell viability was detected by CCK8 assay in GLUT1 overexpressing and its corresponding control cells. The glucose uptake level (D) ATP levels (E), lactate production (F), and cell viability (G) were measured in the group of MCF-10AT+pcDNA3.1, MCF-10AT+pcDNA3.1+BA, and MCF-10AT+ GLUT1+BA, respectively. Data were shown as the mean ± S.D from independent experiments. p < 0.01 and ***p < 0.001. May 2022 \| Volume 12 \| Article 896904 Frontiers in Oncology \| www.frontiersin.org 8 b-BA for Breast Precancerous Prevention Bie et al. FIGURE 6 \| b-BA could inhibit the breast precancerous lesions in vivo This panel shows the hematoxylin–eosin staining of the control group, the disease model group, the tamoxifen-treated group, and the b-BA-treated group, respectively. differences between normal tissues and precancerous lesions. Our results indicated that b-BA-mediated glycolysis inhibition and subsequent proliferation suppression may play a positive role in breast precancerous prevention. B. serrata as an herbal medicine has been widely used in folk medicine for centuries in Asian countries (like China, India, and Korea) to treat diverse disorders, such as inﬂammation, anxiety, and diabetes (22–26). BAs are the main active components of B. serrata. Several studies have proven that BAs could treat inﬂammation diseases and more importantly a variety of cancers. A study showed that topical application of Boswellin (a methanolic extract of B. serrata) could signiﬁcantly inhibit skin inﬂammation, epidermal proliferation, and tumor promotion in the phorbol ester-induced rat model (27). A pre- clinical study reported that B. serrata extracts could alleviate the symptoms in patients with intracranial tumors while no side effects were observed during application (27). Many in vitro assays reported that BA could inhibit cell proliferation and induce apoptosis in cancer cells, including leukemic cells, melanoma cells, and glioblastoma cells (28–32). Our results suggested that b-BA could inhibit atypical hyperplasia in a rat model in vivo, and in vitro b-BA could suppress the proliferation and induce the apoptosis of MCF-10AT without a remarkable effect on MCF-10A. The detection of related proteins veriﬁed that b-BA could induce apoptosis in MCF-10AT but not in MCF-10A. 3.5 GLUT1 Mediates b-BA Regulated Glycolysis Suppression FIGURE 6 \| b-BA could inhibit the breast precancerous lesions in vivo This panel shows the hematoxylin–eosin staining of the control group, the disease model group, the tamoxifen-treated group, and the b-BA-treated group, respectively. TABLE 2 \| The pathological changes of mammary gland tissue in rats in each group. Groups Breast number No hyperplasia General hyperplasia Precancerous lesions Invasive carcinoma Normal 120 115 5 0 0 Model 120 0 27 86 7 Tam 120 13 63 42 2 b-BA 120 10 57 50 3 TABLE 2 \| The pathological changes of mammary gland tissue in rats in each group. The Warburg effect is a well-known feature of various types of cancers. It is deﬁned as an increase in the rate of glucose uptake and production of lactate anaerobically. It was found by Otto Warburg early in the 1920s, and extensively studied over the past 10 years to establish its causes and functions. Recently, some studies unveiled that due to its rapid conversion rate, the Warburg effect played an important role in biosynthesis and ATP production (33), while compared with the intensive interest in cancer, the function of the Warburg effect in precancerous tissues remains elusive. Lately, Chen et al. found that glucose consumption and lactate production increased in precancerous lesions through metabolomics analysis (5). In this work, we obtained possible targets of b-BA via the BATMAN-TCM website and submitted them for KEGG pathway enrichment. The results revealed that b-BA may exert an inhibition effect on MCF-10AT by targeting metabolism. Further metabolic-related assays suggested that b-BA suppressed glycolysis and ATP production. The AMPK pathway plays an important role in cell growth and metabolism as a fuel sensor and regulator. AMPK as a heterotrimeric complex is composed of a catalytic a-subunit and regulatory b- and g-subunits. Cellular stress- induced AMP/ATP ratio increase activates AMPK by phosphorylating a-subunits. The activated AMPK will inhibit ATP-consuming pathways, like mTOR (34). The mammalian target of rapamycin (mTOR) is a central controller of cell growth and proliferation. There are two forms of mTOR complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1, which is activated by multiple signals such as growth factors, amino acids, and cellular energy, regulates numerous essential cellular processes. It is reported Frontiers in Oncology \| www.frontiersin.org 3. Cuzick J, Powles T, Veronesi U, Forbes J, Edwards R, Ashley S, et al. Overview of the Main Outcomes in Breast-Cancer Prevention Trials. Lancet (2003) 361(9354):296–300. doi: 10.1016/s0140-6736(03) 12342-2 5. Chen X, Yi C, Yang M-J, Sun X, Liu X, Ma H, et al. Metabolomics Study Reveals the Potential Evidence of Metabolic Reprogramming Towards the Warburg Effect in Precancerous Lesions. J Cancer (2021) 12(5):1563–74. doi: 10.7150/jca.54252 4. Dela Cruz M, Ledbetter S, Chowdhury S, Tiwari AK, Momi N, Wali RK, et al. MetabolicReprogrammingofthePremalignantColonicMucosaisanEarlyEvent in Carcinogenesis. Oncotarget (2017) 8(13):20543–57. doi: 10.18632/ oncotarget.16129 4 DISCUSSION Precancerous lesions mainly consist of atypical hyperplasia (atypical ductal or lobular hyperplasia), ductal carcinoma in situ, ductal carcinoma, and papillary tumor, which result in approximately 50% probability of deterioration (20). However, with positive intervention, the possibility of canceration may be reduced, while there is no consistent therapy for precancerous lesions. Tam was approved by the FDA in 1978 for the treatment of ER-positive breast cancer and then further applied to reduce the incidence of breast cancer in high-risk women. An overview of the main outcomes in breast cancer prevention trials indicated about 30% to 40% reduction in ER-positive breast cancer incidence following 5-year Tam therapy versus placebo, while principal toxicities like endometrial cancers and thromboembolic events could not be avoided. Moreover, chemoresistance that appeared following Tam treatment may complicate the situation, which could be explained by the mutation and expression changes of ERa (21). In 2002, USPSTF announced that Tam was not suggested in non-high-risk populations unless risk and beneﬁt were fully weighted. To avoid potential side effects, therapy targets should further focus on the signiﬁcant May 2022 \| Volume 12 \| Article 896904 Frontiers in Oncology \| www.frontiersin.org 9 Bie et al. b-BA for Breast Precancerous Prevention DATA AVAILABILITY STATEMENT that these two pathways serve as a signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth (35). Our study further revealed that b-BA-mediated glycolysis inhibition and ATP reduction would lead to AMPK a-subunit activation and mTOR pathway suppression, which might partly explain why b-BA could inhibit the proliferation and induce apoptosis on MCF-10AT. The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author. 5 CONCLUSION Overall, b-BA could suppress the glycolysis pathway and reduce ATP production in MCF-10AT cells without an obvious effect on normal MCF-10A. The induced metabolic stress activated the AMPK pathway and inhibited the mTOR pathway, which limited cell proliferation and promoted apoptosis. The consistent precancerous prevention effect of b-BA could be seen in the rat model. Mechanically, b-BA could combine with Glut1 and impair its glucose transporter function, which is critical for providing fuel to glycolysis. Taken together, b-BA is suggested to be a potential candidate for the treatment of breast precancerous lesions. FUNDING This work was supported by the National Natural Science Foundation of China (nos. 82074430, 81803979, 81673979, 82073748, and 81741130); the Natural Science Foundation of Guangdong Province, China (nos. 2022A1515011674, 2018A030313393 and 2016A030313114); the Science and Technology Program of Guangzhou, China (nos. 201803010051, 201707010245, and 201704020117); the Fourth Batch of TCM Clinical Outstanding Talent Program of China (no. 444258); the Guangdong Basic and Applied Basic Research Foundation (nos. 2019A1515011866 and 2021A1515010993); the Guangdong Science and Technology Innovation Strategy Special fund (International Science and Technology Cooperation Projects, no. 2021A0505030034); and Jinan University’s National Collegiate Innovation and Startups Training Program (no. 202110559093). Thanks to the General Project of Natural Science Foundation of Guangdong Province, China in 2022. ETHICS STATEMENT p p The changes of glycolysis are often accompanied with alterations of related enzymes. To ﬁnd the potential targets, we analyzed the interaction between b-BA and glycolytic enzymes via molecular docking. The highest score was reached between b-BA and GLUT1. Glucose transporter 1 (GLUT1) is a facilitative GLUT overexpressed in various types of tumors. It has been considered as an important target for cancer therapy. A previous study reported that the transformed mammary cells isolated from Glut1 conditional deletion mice grew slower in immunodeﬁcient mice compared to control tumors (36). Another study presented that compared with the control breast cancer model group, even the Glut1 heterozygous group behaved like both allele deletion mice with occasional tumor development, which indicated the crucial role of Glut1 in breast tumor development (37). In this work, we identiﬁed b-BA to be a potential Glut1 inhibitor, which was not reported before. The 2-NBDG glucose uptake assay revealed that the combination of b-BA to Glut1 inhibited the intracellular glucose concentration and triggered acute metabolic stress. Although it has been reported that some small molecules derived from natural compounds could be GLUT1 inhibitors, most of them were not tested in actual disease models in vivo. In this article, we successfully constructed a breast precancerous model and proved that b-BA could suppress precancerous cell proliferation in vivo. The animal study was reviewed and approved by the Laboratory Animal Ethics Committee of Jinan University. Written informed consent was obtained from the owners for the participation of their animals in this study. AUTHOR CONTRIBUTIONS FB and MM contributed to conception and wrote the article. GZ and XY performed the target prediction and KEGG enrichment analysis. XM and SZ performed in vitro experiments. 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https://openalex.org/W3021715419	https://europepmc.org/articles/pmc7259675?pdf=render	English	null	mstree: A Multispecies Coalescent Approach for Estimating Ancestral Population Size and Divergence Time during Speciation with Gene Flow	Genome biology and evolution	2,020	cc-by	5,732	GBE GBE The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. 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. Abstract Gene ﬂow between species may cause variations in branch length and topology of gene tree, which are beyond the expected variations from ancestral processes. These additional variations make it difﬁcult to estimate parameters during speciation with gene ﬂow, as the pattern of these additional variations differs with the relationship between isolation and migration. As far as we know, mostmethodsrelyontheassumptionabouttherelationshipbetweenisolationandmigrationbyagivenmodel,suchastheisolation- with-migration model, when estimating parameters during speciation with gene ﬂow. In this article, we develop a multispecies coalescent approach which does not rely on any assumption about the relationship between isolation and migration when esti- matingparametersandiscalledmstree.mstreeisavailableathttps://github.com/liujunfengtop/MStree/andusessomemathematical inequalitiesamongseveralfactors,whichincludethespeciesdivergencetime,theancestralpopulationsize,andthenumberofgene trees, to estimate parameters during speciation with gene ﬂow. Using simulations, we show that the estimated values of ancestral population sizes and species divergence times are close to the true values when analyzing the simulation data sets, which are generated based on the isolation-with-initial-migration model, secondary contact model, and isolation-with-migration model. Therefore, our method is able to estimate ancestral population sizes and speciation times in the presence of different modes of gene ﬂow and may be helpful to test different theories of speciation. Key words: coalescent, gene tree, mathematical inequalities. mstree: A Multispecies Coalescent Approach for Estimating Ancestral Population Size and Divergence Time during Speciation with Gene Flow Junfeng Liu 1,, Qiao Liu2, and Qingzhu Yang1,2, 1Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China 2Department of Automation, Tsinghua University, Beijing, China Junfeng Liu 1,, Qiao Liu2, and Qingzhu Yang1,2, 1Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China 2Department of Automation, Tsinghua University, Beijing, China Corresponding authors: E-mails: liujunfeng@big.ac.cn; yangqz@mail.tsinghua.edu.cn. Corresponding authors: E-mails: liujunfeng@big.ac.cn; yangqz@mail.tsinghua.edu.cn. Accepted: April 27, 2020 *Corresponding authors: E-mails: liujunfeng@big.ac.cn; yangqz@mail.tsinghua.edu.cn. Accepted: April 27, 2020 The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommon distribution, and reproduction in any medium, provided the original work is properly cited. Materials and Methods Th Th ti l M d l G2–G5 are possible. In this study, the term “loci” refers to independent or loosely linked short segments of the genome, and we assume that there is no recombination within a locus while different loci are free recombining. For tens of thou- sands of loci, there are some mathematical inequalities among the species divergence time, the ancestral species pop- ulation size, and the number of gene trees, of which t1 is larger than s1. Based on the coalescent theory with no gene ﬂow under given species, the probability of gene tree, of which t1 belongs to s1;s0 , is 1 e2 s0s1 ð Þ=h1; and the probability of gene tree, of which t0 belongs to s0; s 0 0 and t1 is less than s0, is 1 e2 s 0 0s0 ð Þ=h0. We use gi([a, b],[c, d]) as the number of gene trees with category Gi (ﬁg. 2) and with t0 is in [a, b] and t1 in [c, d] for i¼ 1, 2, ..., 5. Moreover, to simplify notation, let gi denote the number of gene trees with category Gi (ﬁg. 2) for i¼ 1, ..., 5. Then, the formulas of cases A and C are as follows: G2–G5 are possible. In this study, the term “loci” refers to independent or loosely linked short segments of the genome, and we assume that there is no recombination within a locus while different loci are free recombining. For tens of thou- sands of loci, there are some mathematical inequalities among the species divergence time, the ancestral species pop- ulation size, and the number of gene trees, of which t1 is larger than s1. Based on the coalescent theory with no gene ﬂow under given species, the probability of gene tree, of which t1 belongs to s1;s0 , is 1 e2 s0s1 ð Þ=h1; and the probability of gene tree, of which t0 belongs to s0; s 0 0 and t1 The Theoretical Model Consider two closely related species (1 and 2) with an out- group species 3. We assume that there is only gene ﬂow between two closely related species (ﬁg. 1). We use s0 and s1 to denote the two species divergence times, scaled by mutation rate. Let h0 ¼ 4N0l and h1 ¼ 4N1l measure the two ancestral species population sizes. Here, l is the mutation rate per site and generation, and the N0 and N1 denote the effective population sizes. There are ﬁve possible gene trees for a locus with three sequences (k, l, and m), which are from species 1, species 2, and species 3, respectively (ﬁg. 2). For any locus, t0 is the coalescent time among three sequences and t1 is the coalescent time between two sequences. In the pres- ence of gene ﬂow between species 1 and species 2, the gene tree G1 may be possible at a locus. Otherwise, only gene trees is less than s0, is 1 e2 s 0 0s0 ð Þ=h0. We use gi([a, b],[c, d]) as the number of gene trees with category Gi (ﬁg. 2) and with t0 is in [a, b] and t1 in [c, d] for i¼ 1, 2, ..., 5. Moreover, to simplify notation, let gi denote the number of gene trees with category Gi (ﬁg. 2) for i¼ 1, ..., 5. Then, the formulas of cases A and C are as follows: Case A: If s1 s 0 1 < s0, then g3þg4þg5 g2ð s0;1 ½ ;½s0 1;s0Þþg3þg4þg5 e2 s0s 0 1 ð Þ=h1 for the category G2–G5 (ﬁg. 2). Case A: If s1 s 0 1 < s0, then g3þg4þg5 g2ð s0;1 ½ ;½s0 1;s0Þþg3þg4þg5 e2 s0s 0 1 ð Þ=h1 for the category G2–G5 (ﬁg. 2). Case A: If s1 s 0 1 < s0, then g3þg4þg5 g2ð s0;1 ½ ;½s0 1;s0Þþg3þg4þg5 e2 s0s 0 1 ð Þ=h1 for the category G2–G5 (ﬁg. 2). FIG. 1.—Species tree ((1, 2), 3) for three species. The species diver- gence times are denoted as s0 and s1. The ancestral species population sizes are denoted as h0 and h1. GBE GBE Liu et al. Introduction others on an isolation-with-initial-migration (IIM) model (Mailund et al. 2012; Costa and Wilkinson-Herbots 2017). However, the above two models include an assumption about the relationship between isolation and migration. Here, we use the properties of coalescent-based model in gene tree data for estimating the important parameters such as ances- tral population sizes and divergence times without any as- sumption of the relationship between isolation and migration. Furthermore, we conduct simulations to examine the accuracy of the estimates of parameters. The simulation results show that our method can accurately estimate the parameters. At last, we compared mstree with the program 3s (Dalquen et al. 2017) and IMa3 (Hey et al. 2018) with simulation data; the simulation results show that mstree is faster than 3s and IMa3. The role of gene ﬂow in speciation is a fundamental issue in evolutionary biology. Allopatric speciation considers complete lack of gene ﬂow as prerequisite to the formation of new species. However, parapatric and sympatric speciation allow gene ﬂow during speciation. Although allopatric speciation has been historically taken as the paramount mode of speci- ation (Futuyma and Mayer 1980), theoretical modeling and empirical evidence increasingly support that speciation can occur with gene ﬂow (Gourbiere and Mallet 2010; Smadja and Butlin 2011; Feder et al. 2012). There are usually two kinds of models to make inferences about gene ﬂow during speciation. Some methods are based on an isolation-with-migration (IM) model (Wang and Hey 2010; Tian and Kubatko 2016; Dalquen et al. 2017) and 715 Genome Biol. Evol. 12(5):715–719. doi:10.1093/gbe/evaa087 Advance Access publication May 4, 2020 The Theoretical Model Case B: If s0 s 0 0 < s 00 0, then g1 s 00 0 ;1 ½ ; 0;s0 ½ ð Þþg2ð s 00 0 ;1 ½ ;½0;s0Þ g1 s0 0;1 ½ ; 0;s0 ½ ð Þþg2ð s0 0;1 ½ ;½0;s0Þ e2 s 00 0 s 0 0 ð Þ=h0 for the cate- gory G1–G2 (ﬁg. 2). Case C: g4þg5 g2þg3þg4þg5 2 3 e2 s0s1 ð Þ=h1 for the category G2– G5 (ﬁg. 2). The above gene tree distributions allow us to compute the parameters h0, s0, h1, and s1. The approach of estimating the parameters is called mstree and the strategies are as follows. First, we can estimate the value of s0 based on the fact that the shape of gene tree may be ((k, l),m), ((k, m),l) or ((l, m),k) FIG. 1.—Species tree ((1, 2), 3) for three species. The species diver- gence times are denoted as s0 and s1. The ancestral species population sizes are denoted as h0 and h1. FIG. 2.—For three species (1–3) with gene ﬂow between species 1 and 2, there are ﬁve categories of gene trees for any locus with three sequences (k, l, and m), which are from species 1, species 2, and species 3, respectively. h i ( ) i h ﬂ b i d h ﬁ i f f l i h h (k FIG. 2.—For three species (1–3) with gene ﬂow between species 1 and 2, there are ﬁve categories of gene trees for any locus with three sequences (k, l, and m), which are from species 1, species 2, and species 3, respectively. 716 Genome Biol. Evol. Results than s0 and assume that t 0 is larger than s1 when t 0 is closed to s0, the estimated value of h1 approximates 2 s0 t 0 =log a=a þ b 0 , where a is the number of gene trees, of which t1 is larger than s0 and b 0 is the number of gene trees, of which t1 belongs to t 0; s0 . Lastly, we estimate the value of s1 based on the values of s0 and h1 by using the formula 2 3 e2 s0s1 ð Þ=h1 n2þn3 aþn2þn3 in case C. If there exists t 0 that than s0 and assume that t 0 is larger than s1 when t 0 is closed to s0, the estimated value of h1 approximates 2 s0 t 0 =log a=a þ b 0 , where a is the number of gene Compared with IMa3 and Analyzed Real Data The model estimated by IMa3 is IM model, and we used a ﬁxed true species topology for IMa3. The real data are the genomic sequences of the human (H), chimpanzee (C), and gorilla (G) from Burgess and Yang (2008). The data set com- prises 14,663 autosomal loci, and the mean locus length is 508bp. Table 1 If we choose t 0 that is less The Accuracy of mstree They are as follows: h0 ¼ h1 ¼ 0:005, s0 ¼ 0:006, and s1 ¼ 0:004 (hominoids); h0 ¼ h1 ¼ 0:01, s0 ¼ 0:02, and s1 ¼ 0:01 (mangroves). For the three models, gene ﬂow is symmetrical and the migration rate (the expected number of migrants per generation) is 1. The number of loci is 10,000 and the number of replicates is 1,000. Analyzing the simulation data by using mstree, the results show that the parameter estimates are very close to the true values and are not sensitive to the model’s assump- tion about the relationship between isolation and migration (table 1). e in table 1 is the threshold value in mstree and trees, of which t1 is larger than s0 and b 0 is the number of gene trees, of which t1 belongs to t 0; s0 . Lastly, we estimate the value of s1 based on the values of s0 and h1 by using the formula 2 3 e2 s0s1 ð Þ=h1 n2þn3 aþn2þn3 in case C. If there exists t 0 that is less than s0 and satisﬁes 2 3 e2 s0t 0 ð Þ=h1 n2þn3 aþn2þn3, where a is is less than s0 and satisﬁes 2 3 e2 s0t 0 ð Þ=h1 n2þn3 aþn2þn3, where a is is less than s0 and satisﬁes 2 3 e the number of gene trees, of which t1 is larger than t 0 and the shape is ((k, l),m) (ﬁg. 2: G2–G3); n2 is the number of gene trees, of which the shape is ((k, m),l) (ﬁg. 2: G4); and n3 is the number of gene trees, of which the shape is ((l, m),k) (ﬁg. 2: G5), t 0 can be considered as the estimated value of s1. The number of loci is 10,000 and the number of replicates is 1,000. Analyzing the simulation data by using mstree, the results show that the parameter estimates are very close to the true values and are not sensitive to the model’s assump- tion about the relationship between isolation and migration (table 1). e in table 1 is the threshold value in mstree and Table 1 Table 1 The Estimated Species Divergence Time and Population Size with Different Threshold Value NOTE.—The hominoid set is h0¼ h1¼ 0.005, s0¼ 0.006, and s1¼ 0.004. The mangrove set is h0¼ h1¼ 0.01, s0¼ 0.02, and s1¼ 0.01. h and s estimates are scaled by 102. Gene ﬂow is symmetrical and the migration rate is 1. e is the threshold value in mstree. The number of loci is 10,000. The number of replicates is 1,000. IIM, isolation-with-initial- migration; SC, secondary contact; IM, isolation-with-migration. The best estimates are marked in bold. model by using seq-gen (Rambaut and Grassly 1997). The example of command is as follows: model by using seq-gen (Rambaut and Grassly 1997). The example of command is as follows: with equal probability when t1 is larger than s0. If there exists t 0 that satisﬁes 2a n2 þ n3, where a is the number of gene trees, of which t1 is larger than t 0 and the shape is ((k, l),m) (ﬁg. 2: G3); n2 is the number of gene trees, of which the shape is ((k, m),l) (ﬁg. 2: G4); and n3 is the number of gene trees, of which the shape is ((l, m),k) (ﬁg. 2: G5), t 0 can be considered as the estimated value of s0. Second, we can estimate the value of h0 based on the estimated value of s0 by using the formula with equal probability when t1 is larger than s0. If there exists t 0 that satisﬁes 2a n2 þ n3, where a is the number of gene trees, of which t1 is larger than t 0 and the shape is ((k, l),m) (ﬁg. 2: G3); n2 is the number of gene trees, of which the shape is ((k, m),l) (ﬁg. 2: G4); and n3 is the number of gene trees, of which the shape is ((l, m),k) (ﬁg. 2: G5), t 0 can be considered as the estimated value of s0. Second, we can estimate the value of h0 based on the estimated value of s0 by using the formula e2 s 00 0 s 0 0 ð Þ=h0 a aþb0 in case B. Genome Biol. Evol. 12(5):715–719 doi:10.1093/gbe/evaa087 Advance Access publication May 4, 2020 The Theoretical Model 12(5):715–719 doi:10.1093/gbe/evaa087 Advance Access publication May 4, 2020 GBE mstree: A Multispecies Coalescent Approach Table 1 The Estimated Species Divergence Time and Population Size with Different Threshold Value Threshold Hominoid Mangrove h0 h1 s0 s1 h0 h1 s0 s1 IIM model e ¼ 0.007 0.50 6 0.02 0.49 6 0.04 0.60 6 0.00 0.42 6 0.04 1.00 6 0.06 0.97 6 0.11 2.00 6 0.01 1.25 6 0.29 e ¼ 0.01 0.50 6 0.02 0.49 6 0.03 0.60 6 0.00 0.41 6 0.03 1.00 6 0.05 0.98 6 0.07 2.00 6 0.01 1.14 6 0.22 e ¼ 0.03 0.50 6 0.02 0.50 6 0.01 0.60 6 0.00 0.39 6 0.01 1.00 6 0.02 0.99 6 0.02 2.00 6 0.01 0.99 6 0.05 SC model e ¼ 0.007 0.50 6 0.02 0.48 6 0.05 0.60 6 0.00 0.43 6 0.05 1.00 6 0.06 0.95 6 0.14 2.00 6 0.01 1.31 6 0.33 e ¼ 0.01 0.50 6 0.01 0.49 6 0.04 0.60 6 0.00 0.42 6 0.03 1.00 6 0.04 0.97 6 0.09 2.00 6 0.01 1.21 6 0.27 e ¼ 0.03 0.50 6 0.01 0.50 6 0.02 0.60 6 0.00 0.39 6 0.01 1.00 6 0.02 0.99 6 0.03 2.00 6 0.01 1.01 6 0.07 IM model e ¼ 0.007 0.50 6 0.02 0.48 6 0.06 0.60 6 0.00 0.43 6 0.05 1.00 6 0.06 0.95 6 0.15 2.00 6 0.01 1.34 6 0.34 e ¼ 0.01 0.50 6 0.01 0.49 6 0.04 0.60 6 0.00 0.41 6 0.04 1.00 6 0.04 0.96 6 0.11 2.00 6 0.01 1.22 6 0.28 e ¼ 0.03 0.50 6 0.01 0.50 6 0.02 0.60 6 0.00 0.39 6 0.01 1.00 6 0.02 0.99 6 0.03 2.00 6 0.01 1.00 6 0.07 NOTE.—The hominoid set is h0¼ h1¼ 0.005, s0¼ 0.006, and s1¼ 0.004. The mangrove set is h0¼ h1¼ 0.01, s0¼ 0.02, and s1¼ 0.01. h and s estimates are scaled by 102. Gene ﬂow is symmetrical and the migration rate is 1. e is the threshold value in mstree. The number of loci is 10,000. The number of replicates is 1,000. IIM, isolation-with-initial- migration; SC, secondary contact; IM, isolation-with-migration. The best estimates are marked in bold. Table 1 The Estimated Species Divergence Time and Population Size with Different Threshold Value Table 1 Table 1 When we choose t 0 and t 00 that satisfy s0 t 0 < t 00, the estimated value of h0 approximates 2 t 00 t 0 =log a=a þ b 0 , where a is the number of gene trees, of which t0 is larger than t 00 and t1 is less than s0; b 0 is the number of gene trees, of which t0 belong to t 0; t 00 and t1 is less than s0. Similarly, we can also estimate the value of h1 based on the estimated value of s0 by using the formula e2 s0s 0 1 ð Þ=h1 a aþb0 in case A. If we choose t 0 that is less ./ms id="465" 3 50000 -T -I 3 1 1 1 -m 1 2 0 -m 2 1 0 - em 0.667 1 2 4 -em 0.667 2 1 4 -em 1 1 2 0 -em 1 2 1 0 - ej 1 2 1 -ej 2 3 1 j tail -nþ 4 j grep -v//> tree ./seq-gen -m HKY -l 500 -s 0.01 -t 2.0< tree > inﬁle ./ms id="465" 3 50000 -T -I 3 1 1 1 -m 1 2 0 -m 2 1 0 - em 0.667 1 2 4 -em 0.667 2 1 4 -em 1 1 2 0 -em 1 2 1 0 - ej 1 2 1 -ej 2 3 1 j tail -nþ 4 j grep -v//> tree ./seq-gen -m HKY -l 500 -s 0.01 -t 2.0< tree > inﬁle e2 s 00 0 s 0 0 ð Þ=h0 a aþb0 in case B. When we choose t 0 and t 00 that satisfy s0 t 0 < t 00, the estimated value of h0 approximates 2 t 00 t 0 =log a=a þ b 0 , where a is the number of gene trees, of which t0 is larger than t 00 and t1 is less than s0; b 0 is the number of gene trees, of which t0 belong to t 0; t 00 and t1 is less than s0. Similarly, we can also estimate the value of h1 based on the estimated value of s0 by using the formula e2 s0s 0 1 ð Þ=h1 a aþb0 in case A. The Accuracy of mstree We used the program ms (Hudson 2002) to simulate gene trees at multi loci under the IIM, secondary contact (SC), and IM model. For the IIM model, the gene ﬂow stopped at 2 3 s1 in the past; For the SC model, the time of SC began at 1 3 s1 in the past. Two sets of parameter values were used, roughly based on estimates from the hominoids (Burgess and Yang 2008) and the mangroves (Zhou et al. 2007). They are as follows: h0 ¼ h1 ¼ 0:005, s0 ¼ 0:006, and s1 ¼ 0:004 (hominoids); h0 ¼ h1 ¼ 0:01, s0 ¼ 0:02, and s1 ¼ 0:01 (mangroves). For th th d l ﬂ i t i l d th i ti We used the program ms (Hudson 2002) to simulate gene trees at multi loci under the IIM, secondary contact (SC), and IM model. For the IIM model, the gene ﬂow stopped at 2 3 s1 in the past; For the SC model, the time of SC began at 1 3 s1 in the past. Two sets of parameter values were used, roughly based on estimates from the hominoids (Burgess and Yang 2008) and the mangroves (Zhou et al. 2007). They are as follows: h0 ¼ h1 ¼ 0:005, s0 ¼ 0:006, and s1 ¼ 0:004 (hominoids); h0 ¼ h1 ¼ 0:01, s0 ¼ 0:02, and s1 ¼ 0:01 (mangroves). For the three models, gene ﬂow is symmetrical and the migration rate (the expected number of migrants per generation) is 1. The number of loci is 10,000 and the number of replicates is 1,000. Analyzing the simulation data by using mstree, the results show that the parameter estimates are very close to the true values and are not sensitive to the model’s assump- tion about the relationship between isolation and migration (table 1). e in table 1 is the threshold value in mstree and We used the program ms (Hudson 2002) to simulate gene trees at multi loci under the IIM, secondary contact (SC), and IM model. For the IIM model, the gene ﬂow stopped at 2 3 s1 in the past; For the SC model, the time of SC began at 1 3 s1 in the past. Two sets of parameter values were used, roughly based on estimates from the hominoids (Burgess and Yang 2008) and the mangroves (Zhou et al. 2007). Robustness of mstree and Analyzing Real Data Though our method is not affected by gene ﬂow between the sister species, our method assumes that there is no gene ﬂow between the ingroup and the outgroup. Therefore, we exam- ined the robustness of our method in the presence of gene ﬂow between the ingroup and the outgroup. The results are shown in supplementary table S8, Supplementary Material online. Our method is robust to the simulations based on IIM model between the ingroup and the outgroup. For the simulations based on SC and IM model between the ingroup and the outgroup, the accuracy of parameter estimates is on the decline. At last, we apply mstree to the genomic sequen- ces of the human (H), chimpanzee (C), and gorilla (G) (Burgess and Yang 2008). The estimates of parameters are similar to those of Burgess and Yang (2008), but the estimate of sHC is slightly higher (supplementary table S9, Supplementary Material online). In order to quantify uncertainty in the estimates obtained, we resort to bootstrapping with 100 replicates. The averages and the standard errors of estimates are as follows: bhHCG ¼ 0:0032 6 0:0000, bhHC ¼ 0:0068 6 0:0005, bsHCG ¼ 0:0059 6 0:0000, and bsHC ¼ 0:0038 6 0:0005. The Factors That Inﬂuence Parameter Estimates In addition, we performed more simulations to test how dif- ferent factors inﬂuence parameter estimates, such as the number of loci, migration rate, and the direction of migration. The numbers of loci are 5,000, 10,000, and 50,000; the mi- gration rates are 0.1, 1, and 10; and the directions of migra- tion are symmetrical and asymmetrical. The results are shown in supplementary tables S1–S6, Supplementary Material on- line. For the parameters h0, h1, and s0, the results show that larger number of loci makes the estimates more accurate and the estimates are not sensitive to the model’s assumption, migration rate, and the direction of migration. For the param- eter s1, we have the same conclusion except for the case that migration rate is 10. When migration rate is 10, asymmetrical gene ﬂow decreases the accuracy of s1 estimate. This indi- cates that the estimate of s1 is sensitive to the direction of migration with large migration rate (supplementary ﬁg. S1, Supplementary Material online). The examples of above sim- ulation commands are in the supplementary ﬁle S2, Supplementary Material online. The Simulation Although there has been some decline in the accuracy of parameter estimates because of the inferred error of gene trees, the estimates of mstree are still near to the true values and not sensitive to the model’s as- sumption. Comparing mstree with the program 3s (Dalquen et al. 2017) and IMa3 (Hey et al. 2018), mstree is faster than 3s and IMa3 (supplementary table S7, Supplementary Material online). Although 3s and IMa3 performed very well on some parameter estimates, the s1 estimates of 3s and s0 estimates of IMa3 were very poor. describes the degree of approximation between two sides of the formulas in cases A, B, and C. For example, in case A, e ¼ 0:03 means the value of e2 s0s 0 1 ð Þ=h1 a aþb0 = a aþb0 describes the degree of approximation between two sides of the formulas in cases A, B, and C. For example, in case A, e ¼ 0:03 means the value of e2 s0s 0 1 ð Þ=h1 a aþb0 = a aþb0 should be <0.03 when e2 s0s 0 1 ð Þ=h1 a aþb0. In mstree, the value of e must be <0.05. The results in table 1 show that the smaller e increased the standard deviations of the param- eter estimates and the estimate of s1. Therefore, we suggest that the value of e should be 0.03 when using mstree. Furthermore, we applied mstree to additional two parameter sets (Dalquen et al. 2017), which have larger parameter values and different values for two hs (table 2). The results show that mstree still performs well. The Simulation We simulated gene trees by using the program ms (Hudson 2002) and converted gene trees to sequence data under JC69 717 Genome Biol. Evol. 12(5):715–719 doi:10.1093/gbe/evaa087 Advance Access publication May 4, 2020 GBE Liu et al. Table 2 The Estimated Species Divergence Time and Population Size with Larger and Different Parameter Values Threshold h05 0.02, h15 0.03, s05 0.06, s15 0.04 h05 0.02, h15 0.01, s05 0.02, s15 0.01 h0 h1 s0 s1 h0 h1 s0 s1 IIM model e ¼ 0.03 2.00 6 0.08 2.97 6 0.10 5.99 6 0.03 3.90 6 0.17 2.00 6 0.06 1.00 6 0.02 2.00 6 0.01 1.00 6 0.03 SC model e ¼ 0.03 2.01 6 0.06 2.95 6 0.17 5.99 6 0.03 4.03 6 0.19 2.00 6 0.06 1.00 6 0.02 2.00 6 0.02 1.00 6 0.04 IM model e ¼ 0.03 2.00 6 0.05 2.94 6 0.29 5.98 6 0.05 3.89 6 0.46 2.00 6 0.05 1.00 6 0.02 2.00 6 0.02 1.00 6 0.04 NOTE.—h and s estimates are scaled by 102. Gene ﬂow is symmetrical and the migration rate is 1. e is the threshold value in mstree. The number of loci is 10,000. The number of replicates is 1,000. IIM, isolation-with-initial-migration; SC, secondary contact; IM, isolation-with-migration. NOTE.—h and s estimates are scaled by 102. Gene ﬂow is symmetrical and the migration rate is 1. e is the threshold value in mstree. The number of loci is 10,000. The number of replicates is 1,000. IIM, isolation-with-initial-migration; SC, secondary contact; IM, isolation-with-migration. NOTE.—h and s estimates are scaled by 102. Gene ﬂow is symmetrical and the migration rate is 1. e is the threshold value in mstree. The number of loci is 10,000. The number of replicates is 1,000. IIM, isolation-with-initial-migration; SC, secondary contact; IM, isolation-with-migration. Therefore, we need a program, such as PHYLIP, to infer gene trees when applying mstree to experimental data. Because inference of gene trees is associated with error and uncertainty, we did some simulations to investigate the effect of the gene tree uncertainty (supplementary table S7, Supplementary Material online). We used program ms and seq-gen (Rambaut and Grassly 1997) to generate sequence data under JC69 model and used program dnamlk in PHYLIP package to infer gene trees. Compared with 3s and IMa3 The input ﬁle of mstree is gene tree, which is in Newick for- mat, and the gene tree can be estimated from the observed sequence alignments where there must be three sequences, with one sequence from each species, at each locus. 718 Genome Biol. Evol. 12(5):715–719 doi:10.1093/gbe/evaa087 Advance Access publication May 4, 2020 8 Genome Biol. Evol. 12(5):715–719 doi:10.1093/gbe/evaa087 Advance Access publication May 4, 2020 GBE GBE mstree: A Multispecies Coalescent Approach Discussion Supplementary table S8, Supplementary Material online, shows the performance of mstree in the presence of gene ﬂow with species 3. Under the inﬂuence of gene ﬂow be- tween the ingroup and the outgroup, s0 was underestimated and was closed to the time that gene ﬂow stopped except for IIM model. When s0 was underestimated, the estimates of other parameters were far away from the true value. Burgess and Yang (2008) estimated divergence times under the as- sumption of no gene ﬂow. However, Zhu and Yang (2012) applied the test based on SIM3s model to a human–chimpan- zee–gorilla genomic data and the test results suggested gene ﬂow around the time of speciation of human and chimpan- zee. Compared with the estimated divergence times from Burgess and Yang (2008), the analysis from mstree suggested migrations between sister species. In addition, there are two signiﬁcant differences between mstree and COALGF (Tian and Kubatko 2016), which describes the distribution of coa- lescent histories under the coalescent model with gene ﬂow: 1) mstree uses the coalescent history distribution under coa- lescent model without gene ﬂow to infer model parameters based on summary statistics; however, COALGF computes probabilities of gene tree histories given species trees under the coalescent process with gene ﬂow and the results obtained from COALGF may be used to infer model param- eters based on a maximum likelihood framework. 2) mstree does not make any assumption about the mode of gene ﬂow between sister taxa; however, COALGF assumes that the mode of gene ﬂow between sister taxa is IM. We thank David Bryant and three anonymous reviewers for many critical and constructive comments, which have led to improvement of our article. This work was supported by the National Natural Science Foundation of China (No. 31501081 to Q.Y.). 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https://openalex.org/W2142040732	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/1471-2458-14-55	English	null	Validation of public health competencies and impact variables for low- and middle-income countries	BMC public health	2,014	cc-by	8,923	* Correspondence: P.Zwanikken@hetnet.nl 1Royal Tropical Institute, Amsterdam, The Netherlands Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Open Access Validation of public health competencies and impact variables for low- and middle-income countries Prisca AC Zwanikken1, Lucy Alexander2, Nguyen Thanh Huong3, Xu Qian4, Laura Magana Valladares5, Nazar A Mohamed6, Xiao Hua Ying4, Maria Cecilia Gonzalez-Robledo5, Le Cu Linh7, Marwa SE Abuzaid Wadidi8, Hanan Tahir9, Sunisha Neupane2 and Albert Scherpbier10 Zwanikken et al. BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 © 2014 Zwanikken 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. Royal Tropical Institute, Amsterdam, The Netherlands Full list of author information is available at the end of the article © 2014 Zwanikken 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. © 2014 Zwanikken et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: P.Zwanikken@hetnet.nl 1Royal Tropical Institute, Amsterdam, The Netherlands Full list of author information is available at the end of the article © 2014 Zwanikken 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. Background have defined their intended impact on the workplace and in society in general, by specifying outcome or im- pact indicators. Responding to the crisis in human resources for health and the need for a well-established public health work- force, the number of Master of Public Health (MPH) training programmes has increased, especially in low- and middle-income countries (LMICs) [1-4]. As the LMIC context differs deeply from High Income Countries, the question has been posed whether existing LMIC pro- grammes equip public health alumni to be effective, and whether the taught competencies from these programmes are relevant to their contexts [4-6]. Since the 1990s, in schooling and higher education globally, detailed descrip- tions of expected performance or competencies have been commonly used as drivers of curriculum development, programme evaluation, job function delineation and con- tinuous professional development assessments [7-9]. For some of these purposes, competencies are defined as the “effective application of available knowledge, skills, atti- tudes and values in complex situations” [7]. When six institutions offering MPH programmes came together in December 2011 to design a comparative im- pact evaluation across programmes, each brought to the discussion the set of key competencies which have gui- ded their programmes over the past decade. These were to serve as the basis for formulating the competencies and impact variables against which to evaluate the im- pact of the MPH programmes across all six institutions involved in this study. As part of the design, these compe- tencies and impact variables were to be validated using a Delphi process. All the institutions were engaged in training health and allied health professionals working in LMICs and included: School of Public Health, University of the Western Cape, South Africa; Hanoi School of Public Health, Vietnam; School of Public Health Fudan, China; National Institute of Public Health, Mexico; Uni- versity of Medical Science and Technology (UMST), Sudan (through the Ministry of Health), and the Royal Tropical Institute, the Netherlands. Background Over the past decade, public health competencies have received considerable attention, and have been developed and refined in a range of countries: in the United States of America (USA) they were formulated by the Council on Linkages Between Academia and Public Health Practice in 2001 and revisions adopted in 2010 [10]; the Public Health Agency of Canada published a list in 2007 [11], while in Europe, the Association of Schools of Public Health in the European Region (ASPHER) drafted a list in 2008, which were redefined in 2011 [12]. In the same year (2008), the United Kingdom (UK) Public Health Skills and Career Framework was endorsed [13], while in Australia, the Foundation Competencies for Master of Public Health alumni [14] were published in 2009. These public health competencies were, in many instances, developed through group discussions and a modified Delphi method, with varying degrees of input from academia and public health practitioners at different levels [8,12-14]. Abstract BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Zwanikken et al. BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Page 2 of 10 Abstract Background: The number of Master of Public Health (MPH) programmes in low- and middle-income countries (LMICs) is increasing, but questions have been raised regarding the relevance of their outcomes and impacts on context. Although processes for validating public health competencies have taken place in recent years in many high-income countries, validation in LMICs is needed. Furthermore, impact variables of MPH programmes in the workplace and in society have not been developed. Method: A set of public health competencies and impact variables in the workplace and in society was designed using the competencies and learning objectives of six participating institutions offering MPH programmes in or for LMICs, and the set of competencies of the Council on Linkages Between Academia and Public Health Practice as a reference. The resulting competencies and impact variables differ from those of the Council on Linkages in scope and emphasis on social determinants of health, context specificity and intersectoral competencies. A modified Delphi method was used in this study to validate the public health competencies and impact variables; experts and MPH alumni from China, Vietnam, South Africa, Sudan, Mexico and the Netherlands reviewed them and made recommendations. Results: The competencies and variables were validated across two Delphi rounds, first with public health experts (N = 31) from the six countries, then with MPH alumni (N = 30). After the first expert round, competencies and impact variables were refined based on the quantitative results and qualitative comments. Both rounds showed high consensus, more so for the competencies than the impact variables. The response rate was 100%. Conclusion: This is the first time that public health competencies have been validated in LMICs across continents. It is also the first time that impact variables of MPH programmes have been proposed and validated in LMICs across continents. The high degree of consensus between experts and alumni suggests that these public health competencies and impact variables can be used to design and evaluate MPH programmes, as well as for individual and team assessment and continuous professional development in LMICs. Keywords: Public health competencies, Impact, Low- and middle-income countries, Master of Public Health © 2014 Zwanikken 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. Zwanikken et al. Developing the public health competencies and impact variables The competencies and impact variables which were vali- dated in the Delphi process had been developed through the deliberations of the six LMIC schools of public health. At an early stage, the competencies were com- pared with those of the Council on Linkages Between Academia and Public Health Practice [10], as these com- petencies have been widely used elsewhere as a basis for curriculum design [24-26]. Having considered this frame- work, the team decided to structure the list of competen- cies into seven competency clusters. Taking into account the argument against ‘atomization’ or fragmentation into component parts of competencies/outcomes, and the rec- ognition that the overarching competence is often consid- ered the best expression thereof [27,28], the team used the clusters to condense and clarify the competencies. In the course of this clustering process, we decided to group ‘analytical/assessment skills’ with ‘public health sciences skills’, as we consider ‘analytical/assessment’ skills to be embedded in, and the active component of ‘public health skills’. The five public health experts from each country (N = 31) were asked to review and validate the public health competencies and impact variables using a Likert scale graded from 1 (signifying that the competence was of ‘poor’ relevance) to 5 (indicating ‘excellent’ relevance to the field of public health practice). The intention was that the decision to undertake further rounds of consultation with the same groups should be based on the degree of consensus found. Qualitative comments and suggestions were also invited. In making this comparison, additions to our compe- tencies are notable because we view them as important in the LMICs context. Gender issues were absent from the Council on Linkages framework. The ‘pro-poor and equity-based approach’ was specifically added in response to population needs of LMICs. Furthermore, the team added competencies related to the social determinants of health, as they are considered to be an important foun- dation for public health practice, as acknowledged in the work of the WHO Commission on the Social Determi- nants of Health, and others [29]. The conceptualization of cultural competencies was also expanded to encompass ‘context-sensitive competencies’, in recognition that health status is determined by far more than cultural or back- ground factors, including social, economic, political and gender factors. ‘Policy advocacy’ was added to the domain of policy, as well as the need for ‘context sensitivity’ of po- licies. Methods h BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Page 3 of 10 No agreement exists in the literature on how to mea- sure consensus; measures of central tendency and mea- sures of dispersion are often used [22]. According to Argyrous [23], the median can be used with ranked data (ordinal and interval/ratio), but this is not considered useful for scales with few values; in addition the mean can be used for data that are not skewed. Initially we determined the cut-off points as a mean < =3.9 or a vari- ation coefficient > =0.26. However, the data appeared to be skewed, so the median was chosen: a median of 4 or 5 was considered a good degree of consensus. Prior to embarking on the study, the ethics commit- tees of the six participating institutions, ie the University of Western Cape Senate Research and Ethics Commit- tee, Hanoi School of Public Health Ethic Committee, Fudan University School of Public Health Institutional Review Board, Sudan Medical and Scientific Research Ins- titute (SUMASRI) Ethical Clearance Committee, National Institute of Public Health Ethic Committee, Royal Tro- pical Institute Research Ethics Committee, granted ethical approval for the study. pp y The initial meeting was followed by a period of refine- ment and discussion of the validation design within the team, by email and Skype conferencing. Validation was undertaken using a modified Delphi process. A number of researchers have used the Delphi method to generate consensus on the public health competencies of different health professionals [18-21]. Though the original con- ceptualisation of the Delphi method includes at least two and sometimes three rounds of feedback by the same experts [21], a modified Delphi process was chosen: this involved consulting a group of experienced public health experts (1st round) invited by each convenor on the basis of maximum diversity, and if need be, a second round by the experts, followed by consultation of a simi- larly selected group of programme alumni (2nd or 3rd round), and possibly another round with the alumni. The rationale for including alumni was that their experience would enable them to critique the competencies and im- pact variables from the perspective of what they regard as relevant in their field of work. Maximum professional, gender and cohort diversity criteria were used in their selection. Methods h The team used a multistep process, starting with the December 2011 meeting of MPH programme convenors from six countries, to reach consensus on a set of public health competencies, develop a list of draft impact vari- ables and design the validation process. The process of developing the competencies and variables aimed to rep- resent the diversity amongst institutional competencies and learning objectives as well to harmonize and stream- line the competency statements sufficiently to establish a shared basis for the evaluation. Specific competencies ar- ticulated by particular schools were discussed until con- sensus was reached on whether and how to include them. The resulting set of competencies includes the common competencies of all schools and seeks to articulate key areas of public health performance. The process of modifi- cation was discussion, careful deliberation and consensus building during the face to face meeting, email communi- cations and two Skype meetings. In LMICs, the Public Health Foundation of India held a multi-country conference in 2008, attended by a wide range of local and international delegates and experts. Some delegates were commissioned to develop reports on the state of public health training in their own countries, with a view to informing the development of the public health curriculum in India [15]. Since then, public health competencies have also been developed in Latin America [16]. However public health competencies have not been accepted nor validated across LMICs. After the competencies were defined and agreed upon, impact variables were developed. The impact variables were divided into impact on the workplace, such as de- veloping improved working procedures within a work unit, and impact on the sector or society, such as im- proved quality of care for patients. The team formulated the impact variables through inductive logic while taking into consideration the public health competencies that had already been defined. Although these two levels of intended impact are linked, they were not constructed to be directly equivalent. Furthermore, although restructuring curricula in terms of competencies constitutes a statement of intent on be- half of the provider, it does not demonstrate whether these competencies have been acquired, nor whether the selected competencies had impact in the workplace or in society. A review by Zwanikken et al. [17] revealed that very few Masters programmes in health and health care Zwanikken et al. BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Zwanikken et al. Results Responses were received over two months from 31 experts (21 men and 10 women). Eighteen of the experts were from universities, seven from health system ma- nagement, three from service delivery institutions and three from NGOs, including international agencies such as the United Nations Family Planning Association. All public health experts had more than 10 years of experi- ence in public health. In the analysis of data, medians were calculated for all scores as well as for each country to identify cross-country variability (see Additional file 1). As described in the Methodology, the competencies and impact variables were validated using two Delphi rounds, yielding quantitative and qualitative results. Developing the public health competencies and impact variables ‘Intersectoral competencies’ were added to commu- nity competencies, because intersectoral engagement is regarded as a critical principle in furthering the impact Responses were entered and stored in Microsoft Office Excel® 2003 (Microsoft Corporation, USA), and calcula- tions were made using Excel. Since the results from the first round showed considerable consensus, a further round was not deemed to be required. The experts’ feedback, however, guided further refinement of the competencies and impact variables, which was then circulated to alumni (N = 30) across the six MPH pro- grammes for further validation. Once again, based on the level of consensus in the results of the graduate round, a further round with them was not deemed necessary. Page 4 of 10 Zwanikken et al. BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 of public health. A further contrast with the Council on Linkages framework is that where they assign an im- portant role to financial planning and management, this group’s competencies emphasize planning and manage- ment as a whole, including finances (Figure 1). ‘excellent relevance’ suggested that this effect on the grad- uate’s workplace or sector of society was very important, e.g. they were asked to rate the relevance of ‘Contributed to equity/pro-poor orientation towards health access at all levels’. Comments and additional suggestions were also re- ceived from the expert group. Validation by alumni Af h h After the researchers reached agreement on the revision, each school sent the competencies and impact variables out to five alumni for a second round of validation. The selection of alumni was based on maximum variation per school using criteria of gender, geographical location, year of graduation (between 2004 and 2010) and work- place type. Respondents were again asked to rate the relevance of each competency on a five-point Likert scale – i.e. whether each competency is expected of an MPH graduate working in public health; the key for sco- ring was revised for greater clarity, and ranged from 1 (‘Not a key competency’) to 5 (‘Highly relevant’) on the advice of one of the experts. Respondents were also asked to rate the drafted impact variables from 1 (‘Not a key variable’) to 5 (‘Highly relevant’). They were also asked for comments and additional suggestions. It took a further two months to gather feedback from these 14 men and 16 women. One of the alumni graduated in 2004; two in 2005; five in 2006; four in 2007; six in 2008; six in 2009; five in 2010; and one in 2011. Of them, ten alumni worked for higher education institutions, 12 in health system management, seven for service delivery institutions and one for an NGO. some too broad and some too ambitious, given certain contexts. All nine of the impact variables on society had a me- dian of 4 (Table 1) across the six countries. General feedback by some experts was that it was difficult to score these variables, as the scoring would depend on the context in which a graduate works. The importance of each variable would depend on the level and role of the graduate, as well as the specific field in which they work, for example as a policy maker, implementation manager, educator or researcher. One expert commen- ted that it would be difficult to attribute an indicator to one person, as so often public health workers operate in teams. Qualitative comments suggested that some soci- ety level impact variables were too broad, and too ambi- tious given certain contexts; in some cases, clarifications were suggested. Some cross-country variability was identified, with one school scoring overall lower regarding the competencies and variables. Validation by experts To ensure a maximum variation sample of experts in the public health field, the following criteria were used: reviewers should have a broad view on public health, at least 10 years’ work experience in public health, and work at different workplace types. The expert group was required to include both men and women, at least one person from a university (e.g. professor/programme trainer/policymaker of an educational department), one from health system management (i.e. at national/pro- vincial level), one from a service delivery institution (e.g. Centers for Disease Control and Prevention or other pub- lic health institutions) and one from a non-governmental organization (NGO). Respondents were recruited by the MPH convener of the respective schools by email or telephone. Quantitative analysis across all six countries revealed that 11 of the competencies had a median of 4, while 12 competencies had a median of 5, which shows a high de- gree of consensus. Qualitative comments focused mainly on the formulation of selected competencies, either point- ing out vagueness of expression, a dual focus question or adding to and improving formulations. Results for the workplace impact variables also showed a high level of consensus: 19 of them had a median of 4, two had a median of 4.5, and three had a median of 5 (Table 1). Two of the lowest variables (with a median of 3) were: the graduate had ‘published book chapters’, which was considered too high an expectation for an MPH graduate; and ‘Projects [were] rewarded and by what amount’; this was also considered too ambitious and concern was expressed that raising funds could often not be attributed to one person alone. Some ex- perts commented that several workplace variables re- quired a scale, that some were difficult to measure, Respondents were provided the competencies and im- pact variables and asked to rate the relevance of each competency and impact variable using a Likert scale from 1 (‘poor’ relevance) to 5 (‘excellent’ relevance’). The key to the competencies and impact variables noted: ‘Relevance in this study means that this particular competency is expected of a Public Health Masters graduate working in the field of Public Health’. As regards impact variables: Figure 1 Public health competencies. Figure 1 Public health competencies. Figure 1 Public health competencies. Page 5 of 10 Zwanikken et al. Validation by experts BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Table 1 Results of two Delphi validation rounds Round\median Median 3 Median 4 Median 5 Total Round 1 (experts) Number of competencies 11 12 23 Number of impact variables work 2 19 5 26 Number of impact variables society 9 9 Round 2 (alumni) Number of competencies 10 13 23 Number of impact variables work 19 7 26 Number of impact variables society 7 3 10 Table 1 Results of two Delphi validation rounds Round\median Median 3 Median 4 Median 5 Total partnership’, based on comments from experts. Eleven of the 26 impact variables in the workplace were reworded, based on qualitative feedback (Tables 1 and 3). Four of the nine impact variables on society were reworded based on the qualitative feedback, and one variable was added, based on comments from experts: ‘Influenced better un- derstanding of public health measures amongst the ge- neral population’ (Tables 1 and 4). Validation by alumni Af h h In this school’s survey, four out of 23 com- petencies had a median of 3, while other schools had only one competency with a median of 3, or none. For the im- pact variables at work, the same school scored three var- iables less than 3, while other schools scored one to three variables with a median of 3. Medians for each country were computed to identify cross country variability. Quantitative results revealed that ten competencies had a median of 4, and 13 competencies a median of 5 (Tables 1 and 2), showing a high degree of consensus (see Additional file 2). In relation to the impact variables in the workplace, 19 of the 26 variables had a median of 4, and seven had a median of 5 (Tables 1 and 3). Seven of the impact vari- ables in society had a median of 4, and three had a median of 5 (Tables 1 and 4). For impact variables on society, there were two schools which scored four variables with a median of 3. The com- petencies and variables which were scored lower in the one school were also not rated highly in other schools, and were changed. As regards to cross-country variability for the compe- tencies, alumni from one school rated two competencies at 3, alumni from two different schools scored five out of 23 work impact variables at 3, while alumni from other schools scored a median of 3 for zero, one or three variables. With regard to the impact variables in society, alumni from two different schools scored two and three different impact variables at 3. Based on the quantitative results and the qualitative comments from the experts, 14 of the 23 competencies were reworded to improve clarity (Tables 1 and 2). Two of the workplace impact variables were changed because the median of 3 was low: ‘Achievements which can be attributed to the leadership of the graduate, e.g. individ- ual or organisational awards’ was deleted as it was seen as overlapping with another variable, and the following variable was added: ‘Participated in building a successful General qualitative feedback suggested that the impact variables were dependent on the actual job or workplace of an MPH graduate, as well as the expectation of a Page 6 of 10 Zwanikken et al. Validation by alumni Af h h BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Table 2 Results of validation of competencies student at the start of a program, which concurred with the feedback from the experts. Qualitative feedback sug- and five of the 10 impact variables on society. For ex- ample, of the fourth competency, ‘Commissions research’, Table 2 Results of validation of competencies Competencies\validation Experts Alumni Cluster of competencies Detailed competencies (as sent to alumni) Median Median Public Health science skills including analytical assessment competencies 1. Applies the basic Public Health sciences (including but not limited to biostatistics, epidemiology, environmental health services, health services administration and social and behavioral health sciences) to Public Health policies and programs 5 5 2. Appraises scope, function and role of Public Health in relation to local context, health system and other social sectors 4 4 3. Assesses population health status and identifies population health problems, risk factors, related Social Determinants, and determines needs 5 5 4. Commissions and critically interprets research findings and/or develops protocol and collects, analyses and synthesizes reliable and valid data using qualitative and quantitative methods 5 4,75 Policy development competencies 5. Analyzes and evaluates policy options and determines feasibility for Public Health policies/programs in diverse community contexts, using appraisal of evidence 4 5 6. Participates in developing context sensitive policies and strategic plans and translates them into action 4 4 7. Understands and contributes to developing and using mechanisms to monitor and evaluate Public Health policies and regulations 4 4 8. Contributes to advocacy of new and existing health policies to the public health and other sectors 4 4 Communication competencies 9. Communicates concisely in writing and orally, in person and through electronic means with linguistic and cultural proficiency and appropriateness 5 5 10. Facilitates and integrates input to Public Health policy and programs from a wide range of individual and organizational stakeholders 4 4 11. Uses a variety of culturally appropriate approaches to disseminate Public Health information with consideration to ethical and confidential issues 5 5 Context sensitive competencies 12. Analyzes the role of gender, cultural, social, economic, political and behavioral factors in the accessibility, availability, acceptability and delivery of Public Health services and programs 4 5 13. Incorporates a Social Determinants of Health approach to Public Health needs 4 5 Community and inter-sectoral competencies 14. Validation by alumni Af h h Assesses and engages community actors and communities and their linkages and relationships that affect health in diverse social and cultural situations 4 4 15. Collaborates in community-based participatory efforts 5 4 16. Develops and maintains partnerships with key stakeholders, including from different sectors 4 4 Planning and management competencies 17. Uses evidence and good practice to address Public Health policy, planning and management issues 5 5 18. Plans, implements, monitors and evaluates Public Health interventions, programs, resources, services including input, process, outcome and impact 5 5 19. Prepares and contributes to manage and evaluate Public Health information systems, human, financial and logistic resources 4 4 Leadership and systems thinking competencies 20. Demonstrates leadership as a manager and in team efforts, and is able to lead in Public Health emergencies 5 5 21. Demonstrates professional judgment and ethical standards in data handling and addressing Public Health issues and diverse opinions 5 5 22. Leads with applying the understanding of the interconnectedness and dynamic interactions of the Public Health system 5 4,5 23. Continues life-long learning and professional development, and stimulates team to do so 5 4 Bold type indicates that the competency was changed after feedback from experts and discussion by the research group. Bold type indicates that the competency was changed after feedback from experts and discussion by the research group. and five of the 10 impact variables on society. For ex- ample, of the fourth competency, ‘Commissions research’, two alumni (A15 male, A20 male) commented: ‘Consider adding application of ethical principles’. For the second student at the start of a program, which concurred with the feedback from the experts. Qualitative feedback sug- gested that wording could be more specific in 12 of the 23 competencies, 11 of the 26 impact variables on work Zwanikken et al. BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Page 7 of 10 Page 7 of 10 Table 3 Results of validation of impact variables in workplace Experts Alumni Impact variables at workplace (as sent to alumni) Median Median 1. Created evidence (primary or secondary) for decision-making 5 5 2. Developed a study or a research proposal 4,5 5 3. Reported and made recommendations on population health status or needs 5 5 4. Contributed to change in policy at workplace where needed 4 4 5. Contributed to change in policy at one level higher than work institution 4 4 6. Validation by alumni Af h h Participated and influenced working committees for program design or policy formulation at provincial, national or international level 4 4 7. Published or posted in popular (including electronic) media 4 4 8. Made presentations at conferences 4 4,5 9. Published in peer reviewed publications 4 4 10. Contributed to writing a published chapter of a book 3 4 11. Tutored or taught Public Health professionals, trainees or students in the community 4 4 12. Developed, reviewed or commissioned educational or Health Promotion media and materials 4 4 13. Planned or implemented community health education courses and workshops 4 4,5 14. Intervened or worked with a Social Determinants of Health Framework in a way that promotes equity and/or is pro-poor 4 4 15. Collaborated/networked/developed partnerships successfully with other departments than health 4 4 16. Initiated, sustained and evaluated projects with community participation 4 4 17. Planned and implemented Public Health interventions, programs or policies based on consultation with stakeholders and using evidence and best practice 4,5 4 18. Implemented performance improvement strategies in response to monitoring and evaluation findings 5 5 19. Contributed to improvements in human resource management 4 4 20. Contributed to improving regular working procedures 4 4 21. Instrumental in initiating a change within the workplace, or at some level beyond 4 4 22. Contributed to addressing the determinants of health e.g. through planning processes, resource allocation or research 4 5 23. Raised a project grant 3 4 24. Contributed to reputation-building of workplace 4 4 25. Participated in national and international collaboration 4 4 26. Participated in building a successful partnership (added) 0 4 Achievements which can be attributed to the leadership of the graduate, e.g. individual or organisational awards (deleted) 4 0 Bold type indicates that the variable was changed after feedback from experts and discussion by the research group. Table 4 Results of validation of impact variables on society Experts Alumni Impact variables on society (as sent to alumni) Median Median 1. Contributed to changes in policy or strategy in general 4 5 2. Contributed to changed guidelines, regulations, ordinances beyond the workplace 4 4 3. Contributed to influencing communities, organisations, health sector and other sectors than health 4 4 4. Contributed to equity/pro-poor orientation towards health access at all levels 4 4 5. Contributed to changes in resource allocation for interventions, and research, orientated towards equity and addressing the determinants of health 4 4 6. Validation by alumni Af h h Contributed to equitable access to quality services 4 4,5 7. Contributed to improved Public Health in specific areas related to work context, e.g. improved utilization of services 4 4 8. Contributed to increased resource mobilization for Public Health 4 4 9. Contributed to increased resource mobilization for disadvantaged groups 4 4 10. Influenced better understanding of Public Health measures amongst general population (added) 0 5 Bold type indicates that the variable was changed after feedback from experts and discussion by the research group. Table 3 Results of validation of impact variables in workplace Table 3 Results of validation of impact variables in workplace Experts Alumni Impact variables at workplace (as sent to alumni) Median Median 1. Created evidence (primary or secondary) for decision-making 5 5 2. Developed a study or a research proposal 4,5 5 3. Reported and made recommendations on population health status or needs 5 5 4. Contributed to change in policy at workplace where needed 4 4 5. Contributed to change in policy at one level higher than work institution 4 4 6. Participated and influenced working committees for program design or policy formulation at provincial, national or international level 4 4 7. Published or posted in popular (including electronic) media 4 4 8. Made presentations at conferences 4 4,5 9. Published in peer reviewed publications 4 4 10. Contributed to writing a published chapter of a book 3 4 11. Tutored or taught Public Health professionals, trainees or students in the community 4 4 12. Developed, reviewed or commissioned educational or Health Promotion media and materials 4 4 13. Planned or implemented community health education courses and workshops 4 4,5 14. Intervened or worked with a Social Determinants of Health Framework in a way that promotes equity and/or is pro-poor 4 4 15. Collaborated/networked/developed partnerships successfully with other departments than health 4 4 16. Initiated, sustained and evaluated projects with community participation 4 4 17. Planned and implemented Public Health interventions, programs or policies based on consultation with stakeholders and using evidence and best practice 4,5 4 18. Implemented performance improvement strategies in response to monitoring and evaluation findings 5 5 19. Contributed to improvements in human resource management 4 4 20. Contributed to improving regular working procedures 4 4 21. Instrumental in initiating a change within the workplace, or at some level beyond 4 4 22. Contributed to addressing the determinants of health e.g. Validation by alumni Af h h through planning processes, resource allocation or research 4 5 23. Raised a project grant 3 4 24. Contributed to reputation-building of workplace 4 4 25. Participated in national and international collaboration 4 4 26. Participated in building a successful partnership (added) 0 4 Achievements which can be attributed to the leadership of the graduate, e.g. individual or organisational awards (deleted) 4 0 Bold type indicates that the variable was changed after feedback from experts and discussion by the research group. Table 4 Results of validation of impact variables on society Experts Alumni Impact variables on society (as sent to alumni) Median Median 1. Contributed to changes in policy or strategy in general 4 5 2. Contributed to changed guidelines, regulations, ordinances beyond the workplace 4 4 3. Contributed to influencing communities, organisations, health sector and other sectors than health 4 4 4. Contributed to equity/pro-poor orientation towards health access at all levels 4 4 5. Contributed to changes in resource allocation for interventions, and research, orientated towards equity and addressing the determinants of health 4 4 6. Contributed to equitable access to quality services 4 4,5 7. Contributed to improved Public Health in specific areas related to work context, e.g. improved utilization of services 4 4 8. Contributed to increased resource mobilization for Public Health 4 4 9. Contributed to increased resource mobilization for disadvantaged groups 4 4 10. Influenced better understanding of Public Health measures amongst general population (added) 0 5 Bold type indicates that the variable was changed after feedback from experts and discussion by the research group. Table 4 Results of validation of impact variables on society Experts Alumni Impact variables on society (as sent to alumni) Median Median 1. Contributed to changes in policy or strategy in general 4 5 2. Contributed to changed guidelines, regulations, ordinances beyond the workplace 4 4 3. Contributed to influencing communities, organisations, health sector and other sectors than health 4 4 4. Contributed to equity/pro-poor orientation towards health access at all levels 4 4 5. Contributed to changes in resource allocation for interventions, and research, orientated towards equity and addressing the determinants of health 4 4 6. Contributed to equitable access to quality services 4 4,5 7. Contributed to improved Public Health in specific areas related to work context, e.g. improved utilization of services 4 4 8. Contributed to increased resource mobilization for Public Health 4 4 9. Validation by alumni Af h h Contributed to increased resource mobilization for disadvantaged groups 4 4 10. Influenced better understanding of Public Health measures amongst general population (added) 0 5 *Bold type indicates that the variable was changed after feedback from experts and discussion by the research group. Zwanikken et al. BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Page 8 of 10 impact variable on society: ‘Contributed to changed guide- lines, regulations, ordinances beyond the workplace’, alumni commented: ‘It’s hard because of the old thought about what public health is, but we’re pushing for the change’ (A23, Female) and [it is] ‘not easy to demonstrate’ (A27, Female). (See Additional file 3). competencies’ was deemed important in the context of LMICs. Further high scoring competencies were ‘planning and management’, ‘communication’ as well as ‘leadership’ and ‘systems thinking’ competencies. Slightly lower ratings were assigned to ‘policy development’ and ‘community and intersectoral competencies’. Though still highly rated, the ‘policy development competencies’ and ‘community competencies’ might be less valued because of assumptions regarding the working level and roles played by MPH alumni. Discussion A f A set of competencies and impact variables of MPH programmes were formulated and validated with public health experts and alumni of the programmes. This is the first time, to our knowledge, that public health com- petencies have been validated for MPH programmes lo- cated in or intended for LMICs, across continents. It was also the first time that impact variables of MPH pro- grammes have been formulated and validated. Although there were some variations across countries, the results show an overall consensus of the 23 public health com- petencies, 26 impact variables on the workplace and 10 impact variables on society. The highest scoring impact variables on the workplace amongst experts and alumni were: ‘Created evidence for decision making’, ‘Developed a study or research pro- posal’, ‘Reported and made recommendations on po- pulations health status or needs’, as well as the variable ‘Implemented performance improvement strategies in response to monitoring and evaluation findings’. Inter- estingly the variable, ‘Contributed to addressing deter- minants of health’ was scored higher by alumni than experts, possibly reflecting the alumni’ current experi- ence in the field as well as recent public health deve- lopments emphasising the determinants. As for impact variables in society, there was not much difference be- tween the rating of experts and alumni, except for the first indicator: ‘Contributes to changes in policy or stra- tegy in general’: this was rated higher by the alumni. The highest rated competencies and variables were clearly strongly endorsed and indicated a high degree of consensus. The process of competency development has differed across the globe: in the USA as well as in the UK, a large number of experts were involved [8,13], but in both cases, the process was criticized for being too strongly directed by the higher ranks [12]. Another approach was taken by ASPHER in the European region, which in- volved local employer and workforce representatives [12]. Other recent studies reviewing public health competency formulation surveyed only specific stakeholders such as employers [29], experts [20], academic practitioners and employers, but not alumni [18,19]. Higher num- bers of people than were used in this study were some- times included in the panel [18,30], however, the response rate and the level of consensus was lower. None of the initiatives reviewed in the literature developed im- pact variables. References 1 H 1. Hongara C, McPake B: How to bridge the gap in human resources for health. Lancet 2004, 2004(364):1451–1458. 2. World Health Organization: The World Health Report 2006: Working together for health. Geneva: WHO press, World Health Organisation; 2006. 3. Petrakova A, Sadana R: Problems and progress in public health education. Bulletin of the World Health Organization 2007, 85(12):963–965. 4. Sadana R, Petrakova A: Shaping public health education around the world to address health challenges in the coming decades. Bull World Health Organ 2007, 85(12):902. 3. Petrakova A, Sadana R: Problems and progress in public health education. Bulletin of the World Health Organization 2007, 85(12):963–965. Inasmuch as this formulation and validation process of public health competencies is understood to be a first initiative and that impact variables for MPH alumni wor- king in LMICs have not previously been developed or at least publicised, the study can be said to have provided a foundation for further refinement, and suggested sur- prising consensus across countries. Although the social, cultural and political situation and the state of public health development differs considerably between the countries where the six MPH programmes are situated, clear consensus emerged as to what the public health competencies and impact variables should entail. 4. Sadana R, Petrakova A: Shaping public health education around the world to address health challenges in the coming decades. Bull World Health Organ 2007, 85(12):902. 4. Sadana R, Petrakova A: Shaping public health education around the world to address health challenges in the coming decades. Bull World Health Organ 2007, 85(12):902. 5. Sadana R, Chowdhury AMR, Petrakova A: Strengthening public health education and training to improve global health. Bull World Health Organ 2007, 85:163. 5. Sadana R, Chowdhury AMR, Petrakova A: Strengthening public health education and training to improve global health. Bull World Health Organ 2007, 85:163. 6. Plugge E, Cole D: Oxford graduates’ perceptions of a global health master’s degree: a case study. Human Res Health 2011, 9(1):26. 6. Plugge E, Cole D: Oxford graduates’ perceptions of a global health master’s degree: a case study. Human Res Health 2011, 9(1):26. 7. Calhoun JG, Davidson PL, Sinioris ME, Vincent ET, Griffith JR: Toward an understanding of competency identification and assessment in health care management. Qual Manag Health Care 2002, 11(1):14–38. 8. Calhoun J, Ramiah K, McGean Weist E, Shortell SM: Development of a core competency model for the Master of Public Health degree. Limitations Al h h Although the experts came from four different conti- nents and six countries, there was a high degree of con- sensus regarding the rating of competencies, though this applied to a lesser extent to the impact variables. It is suggested that consensus might have been promoted by social desirability: although the experts were anonymous to one another, they were selected by MPH programme convenors who had developed the competencies and variables. Intra-rater variability, however, showed scores from 1–5. In addition, no prioritization of competencies was requested which might have elicited even greater social desirability bias. By using selection criteria for experts to ensure maximum variation, this bias was reduced. Public health is in different stages of development in the different countries. However, in spite of this and other contextual diversity factors, the validation process yielded high consensus. It is possible, however, that if experts and alumni had been asked to prioritize or weight the compe- tencies and impact variables, differences would have be- come more pronounced [30]. The nature of the Delphi method is qualitative in de- sign and does not seek statistical representativeness in the number of experts invited, but rather attempts to achieve maximum variation of the characteristics of the experts, based on purposeful selection [21]. Future work could engage a larger number of experts or ensure a wider variety of experts. The validation by alumni yielded similar results, with increased congruency, i.e. no impact variable with a me- dian of 3, and more with a median of 5. For the graduate respondents, it is possible that social desirability may have influenced the results, as they were informed that the competencies and variables had already been re- viewed by experts. However given the fact that there was less consensus regarding the impact variables and intra-rater variability was mostly between 2–5, this was In this validation process, the ‘public health science skills’ as well as the ‘context sensitive competencies’ received the highest ratings from both experts and alumni: clearly the addition of the ‘context sensitive Zwanikken et al. BMC Public Health 2014, 14:55 http://www.biomedcentral.com/1471-2458/14/55 Page 9 of 10 Acknowledgements The contribution of the respondents to the Delphi survey – both experts and alumni – is greatly appreciated. The study for this article was funded by the respective institutions and partly funded by the Ministry of Foreign Affairs of the Netherlands. Author details 1 1Royal Tropical Institute, Amsterdam, The Netherlands. 2School of Public Health, University of the Western Cape, Capetown, South Africa. 3Hanoi School of Public Health, Hanoi, Vietnam. 4School of Public Health, Fudan University, Shanghai, China. 5National Institute of Public Health, Cuernavaca, Mexico. 6Ministry of Health, Khartoum, Sudan. 7Department of Demography, Hanoi School of Public Health, Hanoi, Vietnam. 8Human Resource 1Royal Tropical Institute, Amsterdam, The Netherlands. 2School of Public Health, University of the Western Cape, Capetown, South Africa. 3Hanoi School of Public Health, Hanoi, Vietnam. 4School of Public Health, Fudan University, Shanghai, China. 5National Institute of Public Health, Cuernavaca, Mexico. 6Ministry of Health, Khartoum, Sudan. 7Department of Demography, Hanoi School of Public Health, Hanoi, Vietnam. 8Human Resource Development, Federal Ministry of Health, Khartoum, Sudan. 9University of Medical Sciences and Technology, Khartoum, Sudan. 10Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands. Additional files probably not the case. In retrospect, it might have been better to engage the alumni in the first rather than the second round, to avoid this possible risk of bias. The ques- tion is, however, whether that would have yielded different results, given the already high consensus in the first round. The fact that the alumni were only invited to par- ticipate in the second round created the opportunity to improve the competencies and impact variables before they received them. The selection of alumni may also have been biased, as this was undertaken by MPH programme convenors; this bias was minimized through the use of ex- plicit selection criteria. Additional file 1: Responses experts quantitative. Additional file 2: Responses alumni quantitative. Additional file 3: Responses alumni qualitative and summarized quantitative. Additional file 1: Responses experts quantitative. Additional file 2: Responses alumni quantitative. Additional file 3: Responses alumni qualitative and summarized quantitative. Additional file 1: Responses experts quantitative. Additional file 2: Responses alumni quantitative. Additional file 3: Responses alumni qualitative and summarized quantitative. Author’s contributions PZ conceived the article. PZ, LA, NTH, QX, LMV, XHY, MCGR, MAW and SN were all involved in the data collection. All authors contributed to data analysis, writing and review. All authors read and approved the final manuscript. Competing interests The validated impact variables yielded relatively high consensus although less than the competencies. Both the experts and alumni commented that contextual factors, such as the position of the graduate or the level at which the graduate was working, influenced whether these var- iables could be measured; this was raised as a greater concern for the impact variables on society. Some cross- country variability was identified however, given the small number of experts and alumni per country and the fact that no specific school could be identified as differing markedly from others, and given the otherwise high con- sensus, these findings were thought not to be material to the study. The authors report no competing interests. Abbreviations l A: Alumnus; ASPHER: Association of schools of public health in the European region; MPH: Master of Public Health; LMIC: Low and middle income countries; NGO: Non-governmental organisation; UK: United Kingdom; USA: Unites States of America. Conclusion This study contributes to the debates and deliberations on appropriate selection of public health competencies in LMICs. The validation of the competencies and im- pact variables suggests that public health competencies in LMICs should differ from those in high income coun- tries by placing emphasis on factors which impact on the health of their populations, such as examining the social determinants of health, focusing on context speci- ficity and intersectoral competencies, with less emphasis on financial planning in management. Received: 16 February 2013 Accepted: 16 January 2014 Published: 20 January 2014 References 1 H Am J Public Health 2008, 98:1598–1607. 9. Frank JR, Mongroo R, Ahmad Y, Wang M, De Rossi S, Horsley T: Toward a definition of competency-based education in medicine: a systematic review of published definitions. Med Teach 2010, 32:631–637. 9. Frank JR, Mongroo R, Ahmad Y, Wang M, De Rossi S, Horsley T: Toward a definition of competency-based education in medicine: a systematic review of published definitions. Med Teach 2010, 32:631–637. 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References 1 H Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 30. Pfeiffer J, Beschta J, Hohl S, Wasserheit J: Competency-based curricula to transformglobal health: redesign with the end in mind. Acad Med 2013, 88. 10.1097/ACM.0b013e318276bdf4. Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission doi:10.1186/1471-2458-14-55 Cite this article as: Zwanikken et al.: Validation of public health competencies and impact variables for low- and middle-income countries. BMC Public Health 2014 14:55.
https://openalex.org/W2622554694	http://redc.revistas.csic.es/index.php/redc/article/download/973/1485	es		La accesibilidad de los portales web de las universidades públicas andaluzas	Revista española de documentación científica	2,017	cc-by	8,993	Revista Española de Documentación Científica 40(2), abril-junio 2017, e169 ISSN-L:0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 ESTUDIOS / RESEARCH STUDIES La accesibilidad de los portales web de las universidades públicas andaluzas Luis Alejandro Casasola Balsells, Juan Carlos Guerra González, María Araceli Casasola Balsells, Vicente Antonio Pérez Chamorro Universidad de Sevilla Correo-e: ale_casasola@hotmail.com \| ORCID iD: http://orcid.org/0000-0003-4976-5314 Correo-e: jcguegon@gmail.com \| ORCID iD: http://orcid.org/0000-0002-8943-8070 *Universidad Pablo de Olavide, Sevilla Correo-e: macasbal@upo.es \| ORCID iD: http://orcid.org/0000-0002-0539-6337 Correo-e: vapercha@upo.es \| ORCID iD: http://orcid.org/0000-0003-1047-4595 Recibido: 11-02-2016; 2ª versión: 01-07-2016; Aceptado: 05-09-2016. Cómo citar este artículo/Citation: Casasola Balsells, L. A.; Guerra González, J. C.; Casasola Balsells, M. A.; Pérez Chamorro, V. A. (2017). La accesibilidad de los portales web de las universidades públicas andaluzas. Revista Española de Documentación Científica, 40(2): e169. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 Resumen: En este trabajo se describe el análisis realizado en 2015 para la evaluación de la accesibilidad de los portales web de las universidades públicas andaluzas. Con el fin de determinar si las universidades ofrecen información accesible, se ha comprobado el cumplimiento de las Pautas de Accesibilidad de Contenidos Web (WCAG 2.0) establecidas por el Consorcio World Wide Web (W3C). Para ello, se ha diseñado una metodología de análisis que combina tres herramientas automáticas (eXaminator, accesibilidad web del MINHAP, y TAW) y un análisis manual con objeto de dotar de mayor fiabilidad y validez a los resultados. Aunque en términos generales los resultados son aceptables, un análisis detallado muestra que aún queda camino por recorrer hasta conseguir una accesibilidad completa para toda la comunidad universitaria. A este respecto, en el trabajo se sugieren mejoras en los errores comunes encontrados sobre accesibilidad que puedan servir de ayuda al diseño web de las Universidades. Palabras clave: Accesibilidad de contenidos web; diseño web; discapacidad; eXaminator; herramientas de evaluación de accesibilidad web; universidades andaluzas; TAW; W3C; WAI; WCAG 2.0. Web accessibility of public universities in Andalusia Abstract: This paper describes an analysis conducted in 2015 to evaluate the accessibility of content on Andalusian public university websites. In order to determinate whether these websites are accessible, an assessment has been carried out to check conformance with the latest Web Content Accessibility Guidelines (WCAG 2.0) established by the World Wide Web Consortium (W3C). For this purpose, we have designed a methodology for analysis that combines the use of three automatic tools (eXaminator, MINHAP web accessibility tool, and TAW) with a manual analysis to provide a greater reliability and validity of the results. Although the results are acceptable overall, a detailed analysis shows that more is still needed for achieving full accessibility for the entire university community. In this respect, we suggest several corrections to common accessibility errors for facilitating the design of university web portals. Keywords: Web content accessibility; web design; disability; eXaminator; web accessibility evaluation tools; Andalusian Universities; Web Accessibility Observatory; TAW; W3C; WAI; WCAG 2.0. Copyright: © 2017 CSIC. Este es un artículo de acceso abierto distribuido bajo los términos de la licencia Creative Commons Attribution (CC BY) España 3.0. 1 Luis Alejandro Casasola Balsells, Juan Carlos Guerra González, María Araceli Casasola Balsells, Vicente Antonio Pérez Chamorro 1. INTRODUCCIÓN El acceso equitativo de las Tecnologías de la Información y Comunicación (TIC) es un derecho para toda la ciudadanía, pero para las personas con discapacidad debería ser, además, una oportunidad para poder integrarlas, favoreciendo su autonomía. Las innovaciones y avances tecnológicos plantean oportunidades y también amenazas, dentro de las cuales se identifica la denominada “brecha digital”, esto es, el espacio social, técnico y político que distancia a sectores privilegiados de los más desfavorecidos (con limitaciones técnicas o físicas) en cuanto al acceso a la información de dominio público (González, 2006). La “accesibilidad web” implica que todos los usuarios, independientemente de su discapacidad o de su contexto de navegación, puedan disponer de los servicios y contenidos de un sitio web (Hassan y Martín, 2003). Algunos países han reconocido esta necesidad y en el caso de España se han desarrollado diversas iniciativas legislativas para que las Administraciones Públicas impulsen, desarrollen y apliquen los estándares de accesibilidad web para las personas con discapacidad y, en particular, en las páginas web de centros públicos universitarios. 2. OBJETIVOS El objetivo principal de este estudio es obtener una panorámica general de la accesibilidad que presentan los portales de las diez universidades públicas andaluzas, analizando la situación actual sobre el cumplimiento de los niveles mínimos exigidos al respecto. Con carácter específico, se reflexiona en el trabajo sobre los problemas de accesibilidad más comunes y relevantes de los portales universitarios analizados, y se proponen mejoras con objeto de contribuir a un mejor desempeño y satisfacción de los usuarios. La literatura empírica (Chacón-Medina y otros, 2013; Discapnet, 2004; 2006; 2010; Hilera y otros, 2013; Ribera y otros, 2009; Rocha y otros, 2011; Rodríguez y Viera, 2006; Román y otros, 2013, Térmens y otros, 2003; Toledo, 2001; Toledo y otros, 2013; Torres, 2013) ha puesto de manifiesto deficiencias en la accesibilidad de los portales web de universidades españolas, fundamentalmente originadas por la falta de aplicación de pautas de accesibilidad de los contenidos publicados en la web. El presente trabajo contribuye a la literatura previa en dos sentidos. En primer lugar, con relación a la metodología utilizada, ya que no se han encontrado estudios que analicen de forma exhaustiva la accesibilidad de portales utilizan- 2 do una combinación de herramientas manuales y automáticas, tomando como base la versión más reciente, la 2.0, de las Pautas de Accesibilidad de Contenidos Web (Web Content Accessibility Guidelines, WCAG). Y en segundo lugar, hemos estimado conveniente utilizar la herramienta dispuesta por el Observatorio de Accesibilidad del Ministerio de Hacienda y Administraciones Públicas (en adelante Observatorio de Accesibilidad del MINHAP) debido a su carácter comprehensivo en la evaluación de accesibilidad web para Administraciones Públicas y a que no se han encontrado estudios que hayan utilizado este software. El trabajo que se presenta a continuación se organiza de la siguiente forma. En el siguiente apartado se muestran los conceptos y estándares relacionados con la accesibilidad web y su regulación legal en España. Igualmente se revisan las conclusiones principales de la literatura previa sobre accesibilidad web en portales universitarios. En el apartado cuarto se exponen las métricas y herramientas utilizadas, así como las entidades analizadas. En el apartado quinto se presentan los resultados obtenidos del análisis de accesibilidad web, así como un conjunto de recomendaciones, indicando los errores que más frecuentemente se han encontrado y proponiendo algunas posibles soluciones para solventarlos. Finalmente, se exponen las conclusiones del estudio, limitaciones y futuras líneas de investigación. 3. MARCO TEÓRICO 3.1. La accesibilidad web La sociedad actual exige una mejora en la accesibilidad de los servicios para evitar la exclusión social, y una de las áreas a mejorar es el acceso a la información disponible en la web. Por “accesibilidad web” se puede entender el conjunto de tecnologías, normas de aplicación y diseño, que facilitan la utilización de los sitios web siguiendo el principio del diseño universal o “diseño para todos” (Hassan y Martín, 2004), siendo una estrategia cuyo fin es hacer los diferentes entornos y productos accesibles y comprensibles, así como utilizables por todo el mundo, de la forma más independiente y natural posible, sin la necesidad de adaptaciones. La accesibilidad web implicaría que todas las personas independientemente de la discapacidad que presenten (física, intelectual o técnica) o de la que se derive de los contextos de uso, como tecnológicos o ambientales, van a poder hacer uso de la web (Hassan y Martín, 2003). Esto significa no sólo poder navegar, sino también interactuar y poder aportar contenidos, ya que la accesibilidad intenta Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 La accesibilidad de los portales web de las universidades públicas andaluzas alcanzar que las interfaces de usuario se adapten y acomoden a esas diferencias, de manera que cualquiera pueda utilizarlas y acceder a la información1. La aplicación de los requisitos de accesibilidad web, además de permitir y mejorar el acceso de todas las personas a los contenidos de la web, conlleva también otras ventajas adicionales como la independencia de tecnología y dispositivos, la mejora de la eficiencia y tiempo de respuesta, la reducción en costes y simplificación del desarrollo, o la mejora de la imagen de responsabilidad social (González, 2006). 3.2. Pautas, normas y estándares de accesibilidad web La Ley 34/2002, de 11 de julio, de Servicios de la Sociedad de la Información y de Comercio Electrónico (BOE, 2002) introdujo por primera vez la obligación legal de que la información proporcionada vía web por las Administraciones Públicas españolas fuera accesible para las personas con discapacidad y edad avanzada, fijándose como plazo el 31 de diciembre de 2005 (Poncela, 2006). Sin embargo, no se especificaba un nivel mínimo de accesibilidad, sino que se siguieran los criterios de accesibilidad al contenido generalmente reconocidos. En años posteriores, fue aprobada la normativa legal que hacía igualmente referencia a la necesidad de que la información a través de Internet fuera accesible a personas discapacitadas (BOE, 2003; 2007a; 2007b). El Real Decreto 1494/2007, de 12 de noviembre (BOE, 2007c), abrió una nueva etapa al tener las Administraciones Públicas que cumplir con las prioridades 1 y 2 de la norma UNE 139803:2004 (AENOR, 2004) a partir del 31 de diciembre de 2008. Esta norma UNE equivale a las Pautas de Accesibilidad para el Contenido Web (Web Content Accessibility Guidelines, WCAG) 1.0 elaboradas por el Consorcio de la Web (World Wide Web Consortium, W3C) mediante uno de sus grupos de trabajo, la Iniciativa de Accesibilidad Web (Web Accessibility Initiative, WAI) (W3C, 1999). Adicionalmente, mediante la Ley 56/2007, de 28 de diciembre, de Medidas de Impulso de la Sociedad de la Información (BOE, 2007d) se revisó, actualizó y amplió el contenido de la Ley 34/2002, de 11 de julio, referida a la accesibilidad de las páginas de Internet. A raíz de la actualización de WCAG a la versión 2.0 (W3C, 2008), la norma AENOR (2004) fue sustituida por la UNE 139803:2012 (AENOR, 2012; BOE, 2012). Por lo tanto, actualmente las Administraciones Públicas para alcanzar el nivel medio de accesibilidad web deberían cumplir completamente con un nivel de conformidad AA según la WCAG 2.0. Cuando se cumple con las WCAG 2.0, se debe incluir en dichas páginas una Declaración del Nivel de Conformidad, a través de una Sección de Accesibilidad y/o logotipo en su portal, que informe a los usuarios de este cumplimiento. En este caso, las WCAG 2.0 especifican las condiciones que debe cumplir dicha declaración, que deberá incluir al menos los siguientes elementos: • Fecha en que se realiza la declaración de nivel de conformidad. • Título, versión e identificador de recursos uniforme (Uniform Resource Identifier, URI) de las WCAG 2.0. • Nivel de conformidad alcanzado (nivel A, AA o AAA). • Alcance, es decir, la enumeración precisa de las páginas que cumplen con las WCAG 2.0, a través de una lista o expresión regular de los localizadores de recursos uniformes (Uniform Resource Locators, URLs). • Listado de las tecnologías usadas compatibles con la accesibilidad de las que se depende para acceder al sitio web, como el Lenguaje de Marcado de Hipertexto Extendido (Extensible Hypertext Markup Language, XHTML) o las Hojas de Estilo en Cascada (Cascading Style Sheets, CSS). En la iniciativa WCAG 2.0 se plantean cuatro principios fundamentales (perceptibilidad, operatividad, comprensibilidad y robustez) para la accesibilidad al contenido, que se desarrollan en un total de doce pautas. Estas pautas tienen en cuenta los diferentes elementos de accesibilidad que deben cumplirse y que abarcan, desde la concepción de la web, hasta su puesta a disposición a los diferentes usuarios. En la tabla I se muestran los objetivos de los principios y sus correspondientes pautas. 3.3. Literatura empírica sobre accesibilidad web en portales Universitarios La literatura empírica ha analizado en diferentes países la accesibilidad de sitios web universitarios, como Argentina (Laitano, 2015), Australia (Alexander, 2004; Alexander y Rippon, 2007), Estados Unidos (Thompson y otros, 2010), Finlandia (Karhu y otros, 2012), Perú (Córdova, 2012) o Turquía (Kurt, 2011). De forma general, se observa que la mayoría de los portales universitarios analizados incumplen pautas de accesibilidad web, por lo que se hace necesario que los diseñadores y desarrolladores verifiquen y revisen los resultados de validación para asegurar el acceso universal a la información. Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 3 Luis Alejandro Casasola Balsells, Juan Carlos Guerra González, María Araceli Casasola Balsells, Vicente Antonio Pérez Chamorro Tabla I. Principios y Pautas de las WCAG 2.0 Principios Pautas 1.1) Proporcionar alternativas textuales para todo contenido no textual de forma que se pueda convertir a otros formatos que las personas necesiten, tales como textos ampliados, braille, voz, símbolos o en un lenguaje más simple. 1.- Perceptibilidad: La información y los componentes de la interfaz de usuario deben ser presentados de modo que puedan ser percibidos. 1.2) Referidos a medios tempodependientes, es decir, proporcionar alternativas, como transcripciones textuales, donde se incluya una secuencia correcta de descripciones de la información visual y auditiva mostrada en las páginas. 1.3) Crear contenido que pueda presentarse de diferentes formas (por ejemplo, con una disposición más simple) sin perder información o estructura. 1.4) Facilitar a los usuarios ver y oír el contenido, incluyendo la separación entre el primer plano y el fondo. 2.1) Proporcionar acceso a toda la funcionalidad mediante el teclado. 2.- Operatividad: Los componentes de la interfaz de usuario y la navegación deben ser operables. 2.2) Ofrecer a los usuarios el tiempo suficiente para leer y usar el contenido. 2.3) No diseñar contenido de un modo que se sepa podría provocar ataques, espasmos o convulsiones. 2.4) Proporcionar medios para ayudar a los usuarios a navegar, encontrar contenido y determinar dónde se encuentran. 3.- Comprensibilidad: La información y el manejo de la interfaz de usuario deben ser comprensibles. 4.- Robustez: El contenido debe ser suficientemente robusto como para ser interpretado de forma fiable por una amplia variedad de aplicaciones de usuario, incluyendo las ayudas técnicas. 3.1) Hacer que los contenidos textuales resulten legibles y comprensibles. 3.2) Hacer que las páginas web aparezcan y operen de manera predecible. 3.3) Ayudar a los usuarios a evitar y corregir los errores. 4.1) Maximizar la compatibilidad con las aplicaciones de usuario actuales y futuras, incluyendo las ayudas técnicas. Por ejemplo, Laitano (2015) indicaba que las barreras de accesibilidad web en el espacio universitario público argentino eran mayoritariamente graves. Asimismo, Alexander (2004) advertía que casi la totalidad de los portales universitarios australianos incumplían las pautas básicas de accesibilidad. En una investigación posterior, Alexander y Rippon (2007) encontraron que la accesibilidad no había mejorado, sino que incluso había empeorado ligeramente. El estudio sobre universidades estadounidenses (Thompson y otros, 2010) mostraba que la mayoría de sus páginas no eran completamente accesibles. Sólo el estudio sobre universidades finlandesas (Karhu y otros, 2012) concluyó que sus resultados no eran tan deficientes en comparación con los de universidades de otros países. Es interesante observar que, en general, es reducido el número de universidades que obtienen un nivel de adecuación notable, y que gran parte de los problemas en los portales universitarios podrían ser fácilmente corregidos, implementando las técnicas más básicas de accesibilidad web. 4 La evaluación de la accesibilidad de sitios web universitarios ha sido igualmente un campo activo de investigación académica en España (Hilera y otros, 2013; Rocha y otros, 2011; Rodríguez y Viera, 2006; Térmens y otros, 2003; Toledo, 2001; Toledo y otros, 2013; Torres, 2013). Asimismo, el Observatorio de la Accesibilidad TIC de Discapnet ha elaborado varias oleadas de informes específicos (Discapnet, 2004; 2006; 2010) con el fin de evaluar el grado de avance en la accesibilidad web por las Universidades. Generalmente, los trabajos empíricos se caracterizan por la aplicación de herramientas automáticas para la evaluación de la accesibilidad, siendo TAW (Test de Accesibilidad Web) una de las más utilizadas, y en menor medida, otras herramientas como Bobby (Toledo, 2001), WebXACT (Rodríguez y Viera, 2006) o SortSite Professional (Román y otros, 2013). Por otra parte, son limitados los estudios (ChacónMedina y otros, 2013; Hilera y otros, 2013; Román y otros, 2013; Torres, 2013) que se han basado en la actualización más reciente de las WCAG 2.0. Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 La accesibilidad de los portales web de las universidades públicas andaluzas Algunos trabajos realizan también, además, una evaluación de la usabilidad (Rocha y otros, 2011; Román y otros, 2013), de la legibilidad (Hilera y otros, 2013); o incluyen en su valoración el cumplimiento de la sección 508 (Ribera y otros, 2009), de uso obligatorio en todas las agencias federales de Estados Unidos y con similares recomendaciones a las descritas en las WCAG. Los primeros trabajos sobre portales universitarios españoles concluían que el nivel de accesibilidad era bastante bajo. Toledo (2001) analizó ocho universidades andaluzas y demuestra que la mayor parte de las páginas web de las universidades andaluzas analizadas no respetaban las pautas básicas de un diseño web accesible según las WCAG 1.0. Estos malos resultados son consistentes con otros trabajos que han analizado posteriormente el cumplimiento de las WCAG 1.0 por las universidades españolas (Discapnet 2004; 2006; Ribera y otros 2009; Rodríguez y Viera, 2006; Térmens y otros, 2003), siendo también limitados los avances y mejoras observados. A raíz de la entrada en vigor de la normativa introducida en 2007, la literatura identifica un mayor nivel de cumplimiento de los criterios de accesibilidad web por las universidades españolas (ChacónMedina y otros, 2013: Discapnet, 2010; Hilera y otros, 2013; Ribera y otros, 2009; Román y otros, 2013; Toledo y otros, 2013). No obstante, también evidencia que sigue siendo bajo el nivel de accesibilidad en general, así como el incumplimiento de las pautas básicas de diseño accesible. 4. METODOLOGÍA Son numerosas las herramientas que permiten evaluar automáticamente la accesibilidad de una página o sitio web, y generar un informe de cumplimiento de estándares y pautas de accesibilidad (Serrano, 2009; MINHAP, 2014). Estas herramientas funcionan bien y son consideradas un buen punto de partida que ahorra trabajo posterior, pero no son suficientes, ya que a veces muestran como error algo que no lo es o, por el contrario, no detectan algunos errores existentes. Además, entre las distintas herramientas de evaluación automática existen diferencias en los resultados, debido a la implementación que cada una hace de la verificación. Por eso se recomienda emplear varias herramientas automáticas para obtener resultados más fiables así como completarlos con un análisis manual (MINHAP, 2014; W3C, 2015). El trabajo plantea un estudio empírico de la accesibilidad web de las diez Universidades públicas de Andalucía (Almería, Cádiz, Córdoba, Granada, Huelva, Internacional de Andalucía, Jaén, Málaga, Pablo de Olavide, y Sevilla). El análisis y evaluación de los portales va a permitir averiguar si actualmente cumplen con los requerimientos normativos en cuanto su nivel de accesibilidad, cómo se están aplicando las recomendaciones de acceso al contenido en la web, así como identificar los errores que más frecuentemente se cometen. El análisis ha sido llevado a cabo entre los meses de julio y septiembre de 2015. Se ha diseñado y desarrollado un proceso que combina las siguientes siete herramientas automáticas y manuales de evaluación de accesibilidad web para determinar el nivel de conformidad de los portales con la norma WCAG 2.0: Herramienta 1.- Comprobación de la existencia de la Sección de Accesibilidad. Inicialmente se ha comprobado el nivel de accesibilidad que las universidades exponen en sus páginas webs. Según la normativa vigente, las páginas deben ofrecer al usuario información sobre su nivel de accesibilidad y facilitar un sistema de contacto para que pueda transmitir las dificultades de acceso al contenido o formular cualquier queja, consulta o sugerencia de mejora. Herramienta 2.- Validador X(HTML) del W3C2. Se ha realizado una comprobación, análisis y validación previa de la página principal, verificando las gramáticas y otros estándares del código X(HTML). Herramienta 3.- Validador CSS del W3C3. Se ha utilizado el validador de las hojas de estilo CSS para comprobar el cumplimiento de la página principal de cada portal con los estándares del W3C. Herramienta 4.- Herramienta del Observatorio de Accesibilidad del MINHAP4. Se ha seleccionado la herramienta online que ofrece el Observatorio de Accesibilidad del MINHAP a través de su Comunidad de Accesibilidad5 por su elevada calidad y claridad en los resultados. Este servicio de diagnóstico permite, a partir de una URL indicada, la selección aleatoria de un número de páginas indicado. La herramienta analiza estas páginas conforme al tipo de estudio seleccionado y remite un informe, donde se realiza una estimación del nivel de accesibilidad de las mismas asignándoles una puntuación de 0 a 10. Agregando todos los resultados se ofrece también una estimación de la situación global del portal. Para la exploración automática del portal, se ha comenzado por la página principal de cada universidad. A partir de esta página, y de la profundidad6 y amplitud7 seleccionadas por el usuario, se realiza la exploración. En este trabajo se ha escogido una muestra de profundidad y amplitud 2x2, por lo que la herramienta selecciona un número variable de Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 5 Luis Alejandro Casasola Balsells, Juan Carlos Guerra González, María Araceli Casasola Balsells, Vicente Antonio Pérez Chamorro páginas de forma aleatoria (hasta un máximo de 5, incluyendo la página principal de cada universidad). Para su análisis, el Observatorio de Accesibilidad del MINHAP evalúa un total de 20 verificaciones distribuidas en dos grupos de análisis y adecuación: Análisis I (características genéricas de accesibilidad y estándares de una página) y Análisis II (características concretas de accesibilidad). Estas verificaciones contemplan los aspectos más importantes y relevantes que una página web accesible debe cumplir. En las tablas IIa y IIb, se muestran las verificaciones referidas, agrupadas conforme el nivel de análisis. Tabla IIa. Definición de puntos de verificación. Nivel de Análisis I Id Nombre Pregunta 1 Existencia de alternativas textuales ¿Los elementos no textuales tienen texto alternativo adecuado? 2 Uso de encabezados ¿Se usan encabezados para reflejar la estructura del documento de forma aceptable? 3 Uso de listas ¿Las listas están correctamente marcadas? 4 Tablas de datos ¿Las tablas de datos tienen encabezados, información de resumen adecuada y asociación de celdas en caso de ser complejas? 5 Agrupación estructural ¿Se realiza un marcado adecuado de los párrafos de texto? 6 Separación de contenido y presentación ¿Se separa adecuadamente el contenido de la presentación, sin usar estilos para transmitir información ni elementos estructurales únicamente con fines para la presentación? 7 Identificación del idioma principal ¿Se identifica el idioma principal correctamente? 8 Identificación de los cambios de idioma ¿Se identifican los cambios de idioma correctamente? 9 Contraste suficiente ¿El contraste entre el color del texto y el color de fondo es el suficiente? 10 Sección de Accesibilidad ¿La página tiene un enlace a la Sección de Accesibilidad y ésta tiene dirección de contacto y fecha de revisión? Tabla IIb. Definición de puntos de verificación. Nivel de Análisis II Id Nombre Pregunta 1 Navegación con Javascript accesible ¿Se usa Javascript de forma independiente de dispositivo? 2 Control del usuario ¿Puede el usuario controlar los parpadeos, las redirecciones y actualizaciones correctamente? 3 Formularios ¿Todos los controles tienen etiquetas asociadas, se agrupan los controles relacionados y se identifican los campos obligatorios en los formularios? 4 Título de página y de marcos ¿La página y los marcos tienen un título significativo que identifica su contenido? 5 Enlaces descriptivos ¿Los enlaces tienen un texto adecuado? 6 Cambios de contexto ¿Los cambios de contexto se realizan adecuadamente? 7 Compatibilidad ¿El código es procesable? 8 Múltiples vías de navegación ¿Existe un mapa web o un buscador? 9 Foco del teclado ¿Se respeta la visibilidad y el orden del foco del teclado? 10 Navegación consistente ¿El uso de los enlaces es consistente y el esperado por los usuarios? 6 Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 La accesibilidad de los portales web de las universidades públicas andaluzas El grado de adecuación del nivel de análisis I y II se obtiene a partir de la conformidad de las verificaciones de dicho nivel. Hay que tener en cuenta que el grado de adecuación es independiente de la puntuación obtenida, ya que se trata de una métrica para clasificar ese portal en uno de los siguientes tipos: • Parcial. Es una adecuación inferior a las dos siguientes. Se obtiene cuando el grupo de adecuación Prioridad 1 tiene al menos dos verificaciones con modalidad Falla. • Prioridad 1. Se obtiene cuando el grupo de adecuación Prioridad 1 tiene un máximo de una verificación con modalidad Falla, y el grupo de adecuación Prioridad 2 tiene al menos dos verificaciones con modalidad Falla. • Prioridad 1 y 2. Se trata de la mejor adecuación posible. Se obtiene cuando los grupos de adecuación Prioridad 1 y Prioridad 2 tienen un máximo de una verificación, con modalidad Falla. Herramienta 5.- eXaminator . Esta herramienta ha sido utilizada debido a que ofrece como resultado global, una puntuación de 1 (muy mal) a 10 (excelente), basándose en algunas de las técnicas más representativas de la UNE 139803:2012 y nivel de adecuación AA. Por otro lado, proporciona puntuaciones concretas para cada tipo de discapacidad (limitaciones visuales totales, visuales graves parciales, en los miembros superiores, de comprensión y derivadas de la edad). Se ha analizado con esta herramienta la página principal de cada portal. 8 Herramienta 6.- TAW WCAG 2.0 Online9. Se ha escogido el Test de Accesibilidad Web, TAW (García, 2006), por la completitud de resultados que proporciona y ser la más utilizada en investigaciones recientes sobre la materia (Chacón-Medina y otros, 2013; Hilera y otros, 2013; Toledo y otros, 2013). Este software analiza la página web basándose en las WCAG 2.0 y proporciona un informe agrupando los resultados en tres tipos: problemas (son necesarias las correcciones); advertencias (es necesario revisar manualmente) y no verificados (requieren una comprobación completamente manual). Mediante la misma se obtiene un listado de todos los elementos problemáticos para que puedan comprobarse manualmente y los organiza según cada principio. Al igual que eXaminator, se ha analizado la página principal de cada portal. Herramienta 7.- Verificación manual. Para el análisis manual se ha utilizado la metodología de evaluación de conformidad según WCAG 2.0 (W3C, 2015) y los puntos de verificación (Quevedo, 2009) elaborados por la W3C. Igualmente, se ha analizado la página principal de cada portal. En el proceso se han incluido las pautas que se tienen que comprobar de forma manual, y coincidentes con los errores obtenidos por el test TAW WCAG 2.0 Online. Posteriormente, se ha cumplimentado la plantilla, indicando en el campo “Resultado de la Revisión Manual” si la página cumple cada una de las verificaciones exigidas. Para ello, se han utilizado algunas herramientas manuales, como los navegadores gráficos más populares (Google Chrome, Internet Explorer y Mozilla Firefox con los complementos Accessibility Evaluation Toolbar10 y Web Developer11). Para la evaluación de color y contraste se han utilizado las herramientas GrayBit12 y WCAG Contrast Checker13. Una vez realizadas la comprobación manual y cumplimentada la plantilla, se ha calculado una “Puntuación Media Manual” (PMM). En este caso, se ha decidido obtenerla tomando como base otras herramientas, ofreciendo un resultado entre 0 y 10. PMM = (SRV / VP) x 10 Siendo: PMM: Puntuación Media Manual. SRV: Suma de Resultados de todas las Verificaciones de la página. VP: Número de Verificaciones Puntuables de la página. Una vez recopilados los resultados obtenidos con las distintas herramientas, se ha calculado una puntuación global de accesibilidad web para cada Universidad, conforme a la siguiente fórmula: PMT = (PMP + PME + PMM) / 3 Siendo: PMT: Puntuación Media Total. PMP: Puntuación Media obtenida por el Observatorio de Accesibilidad del MINHAP. PME: Puntuación Media obtenida con eXaminator. PMM: Puntuación Media obtenida en el test manual. Para interpretar el grado de accesibilidad de las puntuaciones obtenidas se ha tenido en cuenta las siguientes tres escalas (Discapnet, 2010): • “Cumplimiento”: una puntuación entre siete y diez puntos indicaría un sitio web con pocas barreras a los contenidos para personas con discapacidad y que, por lo tanto, cumpliría con la legislación vigente. • “Cumplimiento moderado”: una puntuación igual o superior a cinco e inferior a siete puntos indicaría la existencia de un mayor número de barreras a los contenidos y, en consecuencia, tendría un cumplimiento moderado. Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 7 Luis Alejandro Casasola Balsells, Juan Carlos Guerra González, María Araceli Casasola Balsells, Vicente Antonio Pérez Chamorro • “Incumplimiento”: para aquellos sitios web con una puntuación menor de cinco puntos los cuales tendría importantes barreras de acceso a los contenidos y que, por consiguiente, no cumplirían con la legislación. han tomado para conseguir los niveles de accesibilidad actual y las intenciones futuras de aumentar la accesibilidad de sus páginas webs. El acceso a esta información en estos portales ha sido sencillo y cómodo. En la tabla III se presentan los resultados relativos al análisis de la presencia de la Sección de Accesibilidad en los portales web. Sin embargo, el acceso a esta información en los portales de las Universidades de Córdoba, Huelva, Internacional de Andalucía y Málaga, no ha sido fácil ya que, en su página principal, no cuentan con los logotipos de declaración de conformidad con las WCAG ni con un enlace visible en la mencionada página que informe sobre la accesibilidad del sitio web. Con respecto a la información sobre accesibilidad se observa que las Universidades de Almería, Cádiz, Granada, Jaén, Pablo de Olavide y Sevilla, informan claramente sobre el grado de accesibilidad de sus páginas, incluyendo en su página principal, los logotipos de declaración de conformidad con las pautas de accesibilidad a los contenidos web, y/o cuentan con el enlace “Accesibilidad” en el que se detalla, además del nivel de accesibilidad alcanzado, información sobre legislación, medidas que se Para estos casos se ha tenido que recurrir a la búsqueda de la información a través de los mapas web y en los buscadores para su localización. Todas declaran tener un nivel de conformidad AA, sin embargo, solamente la Universidad de Granada y la Universidad Internacional de Andalucía dicen basarse en las WCAG 2.0. Se observa que ninguna cumple completamente con los requisitos de la W3C, y no se ha encontrado en ninguna de ellas la fecha de revisión de la conformidad. 5. RESULTADOS 5.1. Análisis automático y manual Tabla III. Comprobación de la existencia de la Sección de Accesibilidad en los portales Universidad Portal Web Logo y enlace “Accesibilidad” en página principal Declaración del Nivel de Conformidad (fecha de revisión) Tecnologías compatibles Sistema de Contacto Almería http://www.ual.es Sí, tanto logo como enlace. Nivel AA de WCAG 1.0 (no consta fecha) XHTML 1.0 CSS 2.1 Sí Cádiz http://www.uca.es/es Sí, tanto logo como enlace. Nivel AA de WCAG 1.0 (no consta fecha) XHTML 1.0 CSS 2.1 Sí Córdoba http://www.uco.es No. Ambos constan en su Sede Electrónica. Nivel AA de WCAG 1.0 (no consta fecha). No consta No consta Granada http://www.ugr.es Dispone de enlace. No tiene logo. Nivel AA de WCAG 2.0 (no consta fecha) XHTML 1.0 CSS 2.1 Sí Huelva http://www.uhu.es No. Ambos constan en su Campus Virtual. Nivel AA de WCAG 1.0 (no consta fecha) XHTML 1.0 CSS 2.1 Sí Internacional de Andalucía http://www.unia.es No. Ambos constan en su Servicio de Publicaciones. Nivel AA de WCAG 2.0 (no consta fecha) No consta Sí Jaén http://www.ujaen.es Sí, tanto logo como enlace. Nivel AA de WCAG 1.0 (no consta fecha) No consta Sí Málaga http://www.uma.es No. Ambos constan en un enlace externo. Nivel AA de WCAG 1.0 (no consta fecha) No consta No consta Pablo de Olavide http://www.upo.es Sí, tanto logo como enlace. Nivel AA de WCAG 1.0 (no consta fecha) XHTML 1.0 CSS 2.1 Sí Sevilla http://www.us.es Sí, tanto logo como enlace. Nivel AA de WCAG 1.0 (no consta fecha) XHTML 1.0 CSS 2.1 Sí 8 Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 La accesibilidad de los portales web de las universidades públicas andaluzas En las tablas IVa y IVb se presentan los resultados relativos al análisis de la accesibilidad web de los portales de las universidades públicas andaluzas con las herramientas automáticas y manuales. Los valores medios obtenidos indican que el nivel de accesibilidad web de las Universidades analizadas es, en general, intermedio ya que todas las herramientas utilizadas muestran unos valores en torno a seis puntos de diez, siendo inferiores las calificaciones obtenidas por la herramienta manual que las obtenidas por las herramientas automáticas del Observatorio de Accesibilidad del MINHAP y eXaminator. Con respecto a los valores medios de las distintas limitaciones obtenidos por la herramienta eXaminator, no se aprecia una gran diferencia, siendo el valor mínimo 5,91 para las relacionadas con la comprensión y un valor máximo de 6,59 para las visuales graves parciales. Según la Puntuación Media Total (PMT), se distinguen tres grupos diferenciados. El primero correspondería a las Universidades de Granada, Jaén, Almería y Cádiz. Estas son las que mejor cumplen con la accesibilidad al tener una puntuación igual o superior a 7. Un segundo grupo lo formarían las universidades Pablo de Olavide, Sevilla, Córdoba e Internacional de Andalucía, que se encuentran en un nivel intermedio, al tener puntuaciones iguales o superiores a 5 e inferiores a 7. El último grupo con un nivel bajo estaría formado por la Huelva y Málaga al tener puntuaciones inferiores a 5. De acuerdo con la PMT, entre la más accesible (Universidad de Granada) y la menos accesible (Universidad de Málaga) hay una diferencia superior a 3 puntos. Tabla IVa. Resultados del análisis de accesibilidad web de las Universidades públicas de Andalucía con herramientas automáticas y manuales Herramienta Observatorio de Accesibilidad MINHAP eXaminator Limitaciones: Universidades Almería Cádiz Córdoba Granada Huelva Internacional de Andalucía Jaén Málaga Pablo de Olavide Sevilla Valor Medio Puntuación Media (PMP) Nivel de Adecuación Puntuación Media (PME) 7,67 Parcial 8,0 8,0 8,4 7,4 6,8 4,9 7,14 Parcial 5,64 Parcial 7,78 Prioridad 1 6,00 Parcial 6,30 Parcial 7,20 Parcial 4,70 Parcial 5,94 Parcial 5,94 Parcial 6,43 Visuales totales Visuales Miembros graves superiores parciales Comprensión Derivadas de la edad 7,4 7,8 8,4 8,1 7,7 7,5 7,6 5,0 5,2 4,6 4,2 5,1 8,3 7,7 8,2 10 7,7 8,4 4,1 4,0 4,5 4,5 3,4 4,4 4,2 4,4 4,3 3,9 3,4 4,7 8,3 7,7 8,6 9,2 7,9 8,3 5,2 4,6 5,8 5,1 5,1 5,8 7,0 7,4 7,1 6,4 6,8 7,3 5,8 5,9 5,7 6,4 5,3 5,8 6,32 6,15 6,59 6,52 5,91 6,58 Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 9 Luis Alejandro Casasola Balsells, Juan Carlos Guerra González, María Araceli Casasola Balsells, Vicente Antonio Pérez Chamorro Tabla IVb. Resultados del análisis de accesibilidad web de las Universidades públicas de Andalucía con herramientas automáticas y manuales TAW WCAG 2.0 Online Herramienta Universidades Almería Manual Problemas Advertencias No verificados Puntuación Media Manual (PMM) 0 133 17 7,50 PUNTUACIÓN MEDIA TOTAL (PMT) 7,72 Cádiz 15 159 17 7,18 7,24 Córdoba 15 90 14 4,75 5,10 Granada 4 84 18 7,25 7,78 Huelva 3 2 18 4,00 4,70 Internacional de Andalucía 13 175 15 4,62 5,04 Jaén 13 206 16 7,75 7,75 Málaga 54 188 16 3,75 4,55 Pablo de Olavide 4 86 16 7,25 6,73 Sevilla 27 135 16 4,62 5,49 14,80 125,80 16,30 5,87 6,21 Valor Medio Según las diferentes herramientas empleadas, destaca positivamente la Universidad de Granada con una puntuación de 7,78 según la herramienta del Observatorio de Accesibilidad del MINHAP. Conforme a la herramienta eXaminator, destacan las Universidades de Jaén y Granada con una puntuación de 8,3. Asimismo, según la herramienta TAW WCAG 2.0 Online, las universidades con menos incidencias son Almería con cero “problemas”, Huelva con dos “advertencias” y Córdoba con catorce “no verificados”. Según el test manual, la Universidad de Jaén con un 7,75 cuenta con la mayor puntuación. Por último, con respecto a los resultados desagregados por limitaciones ofrecidos por la herramienta eXaminator, observamos que las universidades de Almería, Jaén y Granada son las que obtienen mejores puntuaciones. Por el contrario, la Universidad que ofrece la puntuación más baja según la herramienta del Observatorio de Accesibilidad del MINHAP sería la Universidad de Málaga con 4,70. Asimismo, la Universidad de Huelva muestra resultados más bajos según la herramienta eXaminator, ofreciendo una puntuación de 4,1 y según el Test Manual con un valor de 4,0. Según la herramienta TAW WCAG 2.0 Online, la Universidad de Málaga es la que muestra mayor número de problemas encontrados, con un total de 54. Por último, distinguiendo por las limitaciones ofrecidas por la herramienta eXaminator, las universidades que obtienen puntuaciones inferiores son las de Huelva e Internacional de Andalucía. Al comparar nuestros resultados con trabajos previos donde se han evaluado universidades pú- 10 blicas andaluzas, se comprueba que, en general, la accesibilidad ha ido evolucionando positivamente, por lo que se deduce que los responsables de los portales han tenido en cuenta la accesibilidad en sus desarrollos web. La Universidad de Granada obtiene los mejores resultados en nuestro estudio y son consistentes con los de investigaciones previas (Chacón-Medina y otros, 2013; Román y otros, 2013; Torres, 2013). Las Universidades de Almería, Cádiz, Córdoba, Internacional de Andalucía y Sevilla alcanzan buenas valoraciones en los estudios previos, habitualmente por encima de la media. Contrastándolos con nuestros resultados, sólo las dos primeras obtienen unas puntuaciones notables, mientras que las restantes no logran los resultados esperados. Por otra parte, se observa en las investigaciones previas que las Universidades de Jaén, Málaga y Pablo de Olavide generalmente obtienen puntuaciones cercanas a la media o algo inferiores. A este respecto, la Universidad de Jaén alcanza una de las mejores puntuaciones en nuestros análisis, mientras que la Universidad de Málaga obtiene la peor valoración. También llama la atención, en uno de los estudios más recientes (Toledo y otros, 2013), el excelente resultado obtenido por la Universidad Pablo de Olavide, aunque en nuestro estudio sólo muestra un nivel intermedio de adecuación. Por último, la Universidad de Huelva habitualmente no suele lograr buenos resultados, situándose en puestos por debajo de la media; y de forma similar ocurre en nuestra investigación, al obtener la segunda peor puntuación. Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 La accesibilidad de los portales web de las universidades públicas andaluzas 5.2. Errores comunes y posibles soluciones En la tabla V se exponen, de forma ya resumida, los valores medios del cumplimiento de los criterios de conformidad agrupados según los cuatro principios correspondientes, y que han sido obtenidos a partir del análisis manual realizado a los portales web. A este respecto, una gran parte de estos errores suelen ser ocasionados por el uso inadecuado de las gramáticas empleadas en el diseño web, es decir, XHTML y CSS. A continuación, se discuten los errores más comunes en el cumplimiento del nivel de conformidad A y se proponen posibles correcciones para solventarlos. Con relación al criterio de conformidad “1.1.1.Contenido no textual” del principio de perceptibilidad, el 60% de los portales web presentan páginas con imágenes sin textos alternativos, inadecuados, o bien texto vacío. En otros casos algunos de los botones de los formularios no tienen etiquetas textuales <label> asociadas. Las páginas deberían incluir textos alternativos añadiendo, dentro de las etiquetas de HTML, atributos tales como <title>, ofreciendo una explicación adicional al texto que aparezca, o bien añadiendo el atributo <alt>. Las imágenes que no transmitan contenidos o sean decorativas se ofrecerán con el texto alternativo vacío (alt=””) o aplicadas como fondos de imagen CSS. Por ello, es necesario que todas las imágenes cuenten con un texto descriptivo alternativo. Asimismo, el 90% de los portales web analizados presentan diferentes errores en cuanto al cumplimiento del criterio de conformidad “1.3.1.- Información y relaciones” del principio de perceptibilidad. Por ejemplo, se ha detectado la presencia de encabezados (títulos) seguidos del mismo nivel (o superior) sin contenido entre ellos o que no tienen texto. No se usa correctamente el anidamiento de encabezados, existe una inadecuada agrupación estructural, no separando entre el contenido y su presentación. A este respecto, las páginas web no deberían tener niveles vacíos cuando se declaren, y deberían establecerse de forma secuencial, agrupando de forma más consistente siguiendo los estándares. Igualmente, se deben emplear hojas de estilo separadas del código del programa. En todos los portales webs analizados se han identificado errores relacionados con el criterio de conformidad “2.4.4.- Propósito de los enlaces (en contexto)” del principio de operatividad ya que existen enlaces sin contenido textual, sobre todo por ser imágenes. Debería añadirse dicho contenido textual mediante la propiedad <alt>. Cuando se generan nuevas ventanas en determinados enlaces, dentro del mismo bloque, se observa en el 60% de los portales web analizados que no se avisa al usuario de que ese enlace se va a abrir en una nueva ventana, conforme al criterio de conformidad “3.2.1.- Al recibir el foco” del principio de comprensibilidad. A este respecto deberían incorporarse descripciones y elementos que ayuden al usuario a elegir entre diversos destinos. Un 60% de los portales web no cumple con el criterio de conformidad “3.3.2.- Etiquetas o instrucciones. Formularios” del principio de comprensibilidad, debido a que el elemento <input> no tiene una etiqueta asociada explícitamente de forma correcta. Como corrección se deberían proporcionar las suficientes etiquetas, avisos e instrucciones necesarias para los elementos interactivos. Por ejemplo, escribiendo la etiqueta <label for <nombre id>> y agrupando los campos relacionados con etiqueta <fieldset> y <legend>. Por último, con respecto al criterio de conformidad “4.4.1.- Compatibilidad. Análisis” del principio de robustez, un 90% de los portales analizados presentan errores de sintaxis en las gramáticas existentes empleadas (XHTML). A este respecto debería comprobarse el código y validarlo mediante las herramientas online que dispone el W3C. Tabla V. Resultados del análisis manual de los principios accesibilidad WCAG 2.0 Principios (criterios de conformidad) Cumplimiento Incumplimiento No Aplica 1. Perceptibilidad (22) 31,8% 21,8% 46,4% 2. Operatividad (20) 41,5% 24,0% 34,5% 3. Comprensibilidad (17) 42,4% 30,0% 27,6% 4. Robustez (2) 30,0% 70,0% - 37,9% 26,4% 35,7% Total Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 11 Luis Alejandro Casasola Balsells, Juan Carlos Guerra González, María Araceli Casasola Balsells, Vicente Antonio Pérez Chamorro 6. DISCUSIÓN Y CONCLUSIONES La web se creó como una red de conocimiento que ha supuesto un gran avance en cuanto al acceso universal a la información. Actualmente, debido a diferentes razones, existen barreras en la web, que pueden dificultar o impedir el acceso a los contenidos. Aunque es frecuente que se piense que la accesibilidad es algo que atañe únicamente a personas discapacitadas, se trata de una idea equivocada, ya que cualquiera puede tener dificultades de acceso a la web en un determinado momento. Por ello, la accesibilidad web es el recurso que permitirá eliminar, o al menos minimizar, estas barreras que impiden a los usuarios acceder a la información, de ahí la importancia de su cumplimiento. En la última década, en España se han realizado importantes avances legislativos en materia de accesibilidad web en Administraciones Públicas. No obstante, aún queda mucho margen de mejora en cuanto al cumplimiento y aplicación de la misma. La aplicación de la accesibilidad web supone un ahorro en términos de costes en su futura optimización, redunda en el corto plazo en beneficio de los usuarios y refrenda el carácter social de la institución. Los resultados del trabajo son consistentes con los obtenidos en estudios previos sobre la accesibilidad web de universidades y permiten conocer la situación actual de la accesibilidad web de las mismas. En términos generales, se comprueba que los portales han ido incorporando la accesibilidad en sus desarrollos cumpliendo con la legislación vigente en cuanto al nivel de conformidad AA. No obstante, ninguna de las Universidades públicas andaluzas se adecúan completamente al nivel de conformidad AA de la norma WCAG 2.0. Se ha limitado este trabajo al análisis de las universidades públicas andaluzas. Futuros trabajos podrían continuar con la línea de Chacón-Medina y otros (2013) y de Hilera y otros (2013) ampliando la muestra de portales web a las más representati- vas en el ámbito nacional e internacional, incluyendo asimismo a las universidades privadas. Igualmente, podrían existir limitaciones por la metodología utilizada en el trabajo. Por ejemplo, se podría considerar que la muestra de páginas analizadas no ha sido suficiente. No obstante, se ha analizado al menos la página principal con todas las herramientas automáticas y manuales, así como un número aleatorio de cinco páginas con la herramienta del Observatorio de Accesibilidad del MINHAP. A este respecto, también podría haber sido seleccionada una muestra de las páginas con más visitas del portal y que contuvieran elementos problemáticos en cuanto a accesibilidad, como documentos descargables, formularios, tablas o archivos de audio o vídeo. En cuanto a futuras líneas de análisis, deberían plantearse trabajos empíricos con una metodología experimental que evalúen el impacto de los distintos tipos de discapacidades de los usuarios en la accesibilidad web y que complementen los resultados obtenidos mediante herramientas automáticas y manuales de evaluación. Por otra parte, también podría analizarse el impacto de las innovaciones en cuanto al hardware, como lectores de pantalla, dispositivos móviles y otras interfaces hombre/máquina, y mejoras en las herramientas software, por ejemplo HTML5 o CSS3 Speech Module, que faciliten la proliferación de contenidos accesibles y el progreso de la web semántica. 7. AGRADECIMIENTOS A D. Fernando Díaz del Río, profesor del Departamento de Arquitectura y Tecnología de Computadores de la ETSII de la Universidad de Sevilla, tutor del Trabajo Fin de Grado titulado “Análisis de Accesibilidad Web de las Universidades Públicas Andaluzas”. 8. NOTAS [1] La accesibilidad y usabilidad web son conceptos estrechamente relacionados pero a su vez diferentes. Mientras que la accesibilidad se basa en la facilidad de acceso al aplicar un diseño universal a un producto o servicio para que no excluya a ningún usuario, la usabilidad se preocupa por mejorar la facilidad de uso de las páginas web, por ejemplo, en relación a la rapidez de aprendizaje, la eficiencia de uso y la satisfacción del usuario (Nielsen, 1993). [5] Cualquier gestor de las Administraciones Públicas responsable de los portales web correspondientes puede solicitar formar parte de esta comunidad y hacer uso de los servicios ofrecidos. Se trata de un punto de encuentro para los gestores de portales web de las Administraciones Públicas de cualquier ámbito administrativo donde se puede compartir experiencias y resolver dudas. [2] https://validator.w3.org/ [6] [3] https://jigsaw.w3.org/css-validator/ [4] http://administracionelectronica.gob.es/comunidades/accesibilidad La profundidad hace referencia al número de clics necesarios para llegar hasta una página específica. De esta forma, una página de profundidad cuatro será aquella para la que ha sido necesario seguir cuatro enlaces desde la página principal para llegar a ella. 12 Rev. Esp. Doc. Cient., 40(2), abril-junio 2017, e169. ISSN-L: 0210-0614. doi: http://dx.doi.org/10.3989/redc.2017.2.1372 La accesibilidad de los portales web de las universidades públicas andaluzas [7] La amplitud hace referencia al número de páginas analizadas en cada nivel de profundidad, es decir, por cada nivel que el rastreador se adentre en el portal web, se seleccionarán tantas páginas como estén definidas en la amplitud. 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https://openalex.org/W2170123786	https://bmcgastroenterol.biomedcentral.com/counter/pdf/10.1186/1471-230X-12-121	English	null	Increased proton pump inhibitor and NSAID exposure in irritable bowel syndrome: results from a case-control study	BMC gastroenterology	2,012	cc-by	5,384	Abstract Background: Patients with irritable bowel syndrome (IBS) seen by a gastroenterologist often utilize medications that may alter intestinal homeostasis. The question arises whether exposure to these drugs is associated with the development of IBS symptoms. Aim of this study was therefore to assess the use of PPIs and NSAIDs in patients with IBS versus controls. Methods: Cases of IBS from the last 5 years were reviewed. All patients having had at least one prescription for a particular drug (PPIs, NSAIDs, SSRIs, diuretics, ACE inhibitors) in the 6 months prior to the time of initial symptom onset were considered exposed. The control group consisted of individuals randomly selected from the general population. Results: 287 cases of IBS were retrieved for analysis together with 287 age and sex-matched controls. Exposure to PPIs and NSAIDs was significantly higher in IBS patients, whereas no association between ACE inhibitor use and IBS was found. PPIs were not significantly associated when excluding patients with gastrointestinal reflux disease or functional dyspepsia. Exposure to SSRIs was also positively associated with IBS, but only when patients with psychiatric comorbidity were included in the analyses. Conclusions: Medications that may alter intestinal homeostasis such as NSAIDs and PPIs were more frequently used in IBS patients compared to controls. This association might be relevant for everyday clinical practice, but it is remains to be elucidated whether this association is of etiological nature. Conclusions: Medications that may alter intestinal homeostasis such as NSAIDs and PPIs were more frequently used in IBS patients compared to controls. This association might be relevant for everyday clinical practice, but it is remains to be elucidated whether this association is of etiological nature. Keywords: Irritable bowel syndrome, Proton pump inhibitors, NSAIDs, Small intestinal bacterial overgrowth, Intestinal permeability Keywords: Irritable bowel syndrome, Proton pump inhibitors, NSAIDs, Small intestinal bacterial overgrowth, Intestinal permeability Since the pathogenesis of IBS is still poorly unders- tood, the question arises whether the intake of medica- tion can contribute to or trigger onset of symptoms in IBS patients, as these patients often utilize medications that may potentially alter intestinal homeostasis. One postulated hypothesis points to increased intestinal per- meability in IBS patients induced by exposure to certain medication, such as non-steroidal anti-inflammatory drugs (NSAIDs), thereby allowing luminal antigens to enter the lamina propria and eliciting an immune and inflammatory reaction [3]. Abstract In particular, proton pump inhibitors (PPIs) have also been suggested to be involved in the patho- genesis of IBS, primarily through alteration of intestinal microbiota composition [4]. PPIs are one of the most frequently prescribed classes of medication worldwide Increased proton pump inhibitor and NSAID exposure in irritable bowel syndrome: results from a case-control study aniel Keszthelyi1,2, Gwen H Dackus2, Gwen M Masclee2, Joanna W Kruimel2 and Ad AM Masclee © 2012 Keszthelyi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: daniel.keszthelyi@maastrichtuniversity.nl 1Top Institute Food and Nutrition, Wageningen, The Netherlands 2Division of Gastroenterology-Hepatology, Department of Internal Medicine, Maastricht University Medical Centre+, Maastricht, The Netherlands Keszthelyi et al. BMC Gastroenterology 2012, 12:121 http://www.biomedcentral.com/1471-230X/12/121 Keszthelyi et al. BMC Gastroenterology 2012, 12:121 http://www.biomedcentral.com/1471-230X/12/121 Open Access Background Irritable bowel syndrome (IBS) is a condition characte- rized by recurrent abdominal pain or discomfort accom- panied by abnormalities in bowel habits [1]. It leads to considerable morbidity as it is associated with tripling of missed work-days and doubling of illness-related costs [1]. National Health Service costs in the UK are 60% higher in IBS patients than in non-IBS control patients [2]. As care-seeking behavior can be characteristic for IBS patients, these patients are also prone to suffer from polyphragmasia and polypharmacia. Keszthelyi et al. BMC Gastroenterology 2012, 12:121 http://www.biomedcentral.com/1471-230X/12/121 Page 2 of 6 Page 2 of 6 practitioners or other medical specialists for diagnostic work up and therapy were identified by reviewing medical records in the period from May 2006 to November 2010. Symptoms included abdominal pain and discomfort as- sociated with diarrhea or constipation, bloating and ab- dominal distension. At the time of the index visit to our outpatient clinic, the duration of the symptoms was assessed. In case of patients having had symptoms for at least 6 months, the diagnosis of IBS was made based on Rome III criteria at the time of the index visit. In case of patients having IBS symptoms for less than 6 months, diagnosis according to Rome III criteria was confirmed using questionnaires sent to patients to assess symp- tom duration following initial symptom onset. Only patients fulfilling Rome III criteria were included in the investigation. because they combine a high level of efficacy with low to- xicity. In 2009, expenses for omeprazole and esomeprazole only reached €150 million in the Netherlands [5]. In the five years since the introduction of esomeprazole in 2001, prescriptions for PPIs have doubled [6]. With 5 million prescriptions, omeprazole became the second most fre- quently prescribed medication in the Netherlands in 2009 [5]. A recent socio-demographic study from the Netherlands found that 11.8% of the general population had at least one prescription for a PPI in the year 2006 [7]. It has also been demonstrated that in IBS patients, the use of PPIs generates an excess health cost of €80 per case per year [8]. We have recently demonstrated that exposure to PPIs was associated with increased risk for developing micro- scopic colitis [9], a condition known to share symptoms and clinical features with IBS [10] and to be associated with changes in intestinal permeability [11]. Background PPIs have not only been demonstrated to be able to alter intestinal barrier function [12], but also to induce profound changes in intestinal microbiota composition [13], which in turn may also lead to secondary changes in epithelial integrity [14]. It has therefore also been hypothesized that PPIs use is associated with IBS through causing small intestinal bacterial overgrowth (SIBO) [4]. Due to these presumed alterations in intestinal physiology induced by PPI and/or NSAID use, we postulate that PPIs and NSAIDs may play an etiological role in the development of IBS. More speci- fically, we hypothesize that patients with IBS will be more often exposed to drugs affecting intestinal homeostasis (PPIs and NSAIDs) at time of presentation with symp- toms. In this study, we aimed to establish a relationship between PPI or NSAID use and IBS by comparing expos- ure these drugs in comparison to control group from the general population. Written informed consent was obtained from patients to obtain detailed information of their pharmacy records. Their medical records were reviewed to assess medical history and comorbidities. Information on prescribed me- dication for the period of two years prior to the index visit was obtained from the pharmacy database. All patients having had at least one prescription for a period of four weeks for a PPI (esomeprazole, omeprazole, pantoprazole, lansoprazole or rabeprazole) in a dose of 20 mg (for lanso- prazole 15 mg) or more in the 180 days prior to the time of the initial onset of symptoms were considered exposed to PPIs. Patients exposed to PPIs only in the period after initial onset of symptoms were not included in the analyses. Similarly, we investigated exposure to NSAIDs (as drugs known to alter intestinal barrier function). The following doses were considered as minimal drug expos- ure: diclofenac 12.5 mg, ibuprofen 200 mg, ketoprofen 100 mg, indomethacin 25 mg, aceclofenac 100 mg, nabu- metone 500 mg, naproxen 250 mg, aspirin 500 mg. COX- 2 inhibitors were not included in the analyses. Also, ex- posure to selective serotonin reuptake inhibitors (SSRIs, drugs frequently prescribed to IBS patients) and as control medication exposure to diuretics and angiotensin convert- ing enzyme (ACE) inhibitors, drugs that have not been associated with IBS, was investigated. Background The rationale for selecting a time window of 180 days between drug expos- ure and diagnosis of IBS was the minimum of six months duration of symptoms needed for IBS diagnosis (Rome III criteria) and the considerable delay that may occur bet- ween initial symptom onset and referral with eventual diagnosis by a gastroenterologist. Psychiatric comorbidity was defined as evidence of depression or anxiety disorder in medical history as diagnosed according to the DSM IV. Patients We performed a retrospective, observational, case–con- trol study at the Division of Gastroenterology-Hepato- logy at the Maastricht University Medical Center, the Netherlands, a tertiary and regionally also secondary re- ferral medical center. Patients presenting with symp- toms characteristic for IBS who had been referred to a gastroenterologist at our outpatient clinic by general Methods h d The study was approved by the Medical Ethics Committee of the Maastricht University Medical Centre+ (MUMC), Maastricht, the Netherlands (reference number 0904015), and performed in full accordance with the European directive 91/507/EEG, and the Declaration of Helsinki (as amended in Tokyo, Venice, Hong Kong, Somerset West and Edinburgh. Note of clarification added in Washington and Tokyo). Controls The control group consisted of 408 individuals randomly selected from the general population in Maastricht, the Netherlands. Residents with a permanent address in Page 3 of 6 Page 3 of 6 Keszthelyi et al. BMC Gastroenterology 2012, 12:121 http://www.biomedcentral.com/1471-230X/12/121 Table 1 Demographic characteristics of irritable bowel syndrome (IBS) patients and controls IBS patients N = 287 Controls N = 287 P value Age (years) 36.7 ± 16 37.4 ± 16 0.64 Gender 75% female 75% female 1.00 BMI (kg/m2) 25.8 ± 3.4 24.7 ± 5.1 0.06 Table 1 Demographic characteristics of irritable bowel syndrome (IBS) patients and controls was as follows: 38% diarrhea-predominant (IBS-D), 30% constipation-predominant (IBS-C) and 32% mixed sub- type (IBS-M). A significantly higher numer of IBS had comorbid conditions (FD or GERD, psychiatric condition, fibromyalgia, rheumatoid arthritis), compared to controls (see Table 2, all p < 0.001). Exposure to PPIs and NSAIDs was significantly higher in IBS patients (see Table 2). Co-exposure to PPIs and NSAIDs was also significantly higher in IBS (7.6% vs 0%, χ2 = 17.4, p < 0.001). Of the patients using NSAIDs (59/287), 37% also used PPIs at the same time (22/287). See Table 3 for a summary of the type of PPIs and NSAIDs patients were exposed to. Maastricht were eligible for selection. Potential controls were selected by a random computerized selection from the municipality residential register. Controls received questionnaires regarding their current medical status and drug exposure. Individuals who self-admitted to being diagnosed with IBS were excluded. Controls for analyses (n = 287) were selected from this group and matched to IBS cases by age (within 1 year) and gender. IBS patients more frequently used SSRIs (Table 2). However, no association with SSRI use was found when correcting for psychiatric comorbidity. When patients with psychiatric co-morbidity were excluded from analy- sis, ORs for PPIs, NSAIDs diuretics and ACE inhibitors remained unchanged, whereas SSRI exposure proved not to be associated with IBS (Table 2). Statistical analysis Statistical analyses were performed using χ2 test to com- pare gender and comorbidities. Independent Student’s t test was used to compare age and BMI. Generalized linear model for binomial regression also adjusted for comorbi- dities (psychiatric, gastrointestinal reflux disease [GERD], functional dyspepsia [FD], rheumatoid disorders, fibro- myalgia) was used to calculate ORs and 95% CIs using SPSS, version 20.0 (Chicago, IL). Statistical significance was predetermined as p < 0.05. No association between ACE inhibitor or diuretic use and IBS was found (Table 2). No significant association was found with regards to IBS-subtype (diarrhea, consti- pation or mixed) and drug exposure. As PPIs are most frequently prescribed for upper GI tract complaints originating primarily from FD or GERD, analyses were repeated with exclusion of patients and controls with documented comorbid GERD or dyspepsia. These analyses showed similar ORs for NSAIDs, SSRIs, diuretics and ACE inhibitors. However, PPI exposure proved not be associated with the disorder in the group of IBS patients without comorbid GERD or dyspepsia (OR 2.1 [0.8-5.4]). Discussion In this study, we found that a significantly higher num- ber of IBS patients use certain medications that can po- tentially alter intestinal homeostasis, such as PPIs and NSAIDs, in comparison to controls. Medication use of patients was examined in the period preceding the time of presentation at our clinic with their IBS symptoms, ai- ming to establish a temporal relationship between medica- tion use and onset of symptoms. PPIs and NSAIDs are often used simultaneously, with the former frequently co-prescribed to reduce gastrointes- tinal injury due to the latter. In our study, over a third of the patients using NSAIDs also used PPIs – presumably for gastroprotection. Recent video capsule studies suggest [22,23] a very high incidence (55-70%) of intestinal dam- age in healthy humans taking both NSAIDs and PPIs for 2 weeks. A more recent study performed in rats demon- strated that PPIs lead to a marked exacerbation of small intestinal ulceration induced by NSAIDs, which was trans- ferable to germ-free mice via microbiota isolated from the PPI-treated rats. This observation points to an important role for microbial alterations. When PPIs were adminis- tered alone, significant changes in intestinal microbiota were observed, with 80% reduction in the levels of the beneficial Bifidobacteria spp, whereas little effect was de- tected on the morphology of the intestinal mucosa [24]. We here demonstrate in our study population of IBS patients a positive association with IBS and exposure to PPIs and NSAIDs, but also to SSRIs. The former two are associated with alterations of intestinal physiology, where- as the latter is often prescribed in IBS due to concomitant psychiatric comorbidity. In order to delineate between a potential causal effect and mere therapeutic utilization of these drugs, we conducted analysis with the exclusion of IBS patients with certain comorbid conditions. For ins- tance, IBS patients often suffer from functional dyspepsia; the degree of overlap varies between 15 and 42% [15,16]. Because patients with IBS are more likely to have GERD and dyspepsia in comparison to controls, they are also more likely to receive PPI therapy. Therefore, following initial analyses, we conducted additional analyses exclud- ing the cases and controls exposed to PPIs due to therapy for concomitant upper GI complaints based on functional dyspepsia or GERD. In this case, PPI exposure was not associated with IBS. Results Table 3 Types of drugs used by irritable bowel syndrome patients known to alter intestinal physiology [19] and in particular barrier function, previous studies suggest that the associa- tion with NSAIDs is due to the tendency of patients with IBS having pain complaints rather than analgesics being a causative factor [17]. We therefore conducted analyses with the exclusion of subjects with rheumatoid disorders, as a common source of extragastrointestinal pain. Results of these analyses still showed a significant association bet- ween NSAIDs use and IBS. This could be explained by the fact that patients were taking NSAIDs due to pain symp- toms related to conditions other than rheumatoid disor- ders. On the other hand, recent findings have shown that NSAIDs can compromise intestinal permeability in IBS patients to a greater extent than in healthy subjects [3]. Furthermore, another epidemiological study demonstrated that IBS patients using NSAIDs were also more likely to have persistent irritable bowel syndrome [20]. This ap- pears to be in line with the hypothesis that NSAID therapy affects intestinal permeability resulting in sustained low- grade mucosal inflammation [21]. This implies that even if NSAIDs do not necessarily trigger IBS symptoms, they may be able to sustain the condition by altering intes- tinal physiology and in particular by impairing intestinal permeability. known to alter intestinal physiology [19] and in particular barrier function, previous studies suggest that the associa- tion with NSAIDs is due to the tendency of patients with IBS having pain complaints rather than analgesics being a causative factor [17]. We therefore conducted analyses with the exclusion of subjects with rheumatoid disorders, as a common source of extragastrointestinal pain. Results of these analyses still showed a significant association bet- ween NSAIDs use and IBS. This could be explained by the fact that patients were taking NSAIDs due to pain symp- toms related to conditions other than rheumatoid disor- ders. On the other hand, recent findings have shown that NSAIDs can compromise intestinal permeability in IBS patients to a greater extent than in healthy subjects [3]. Furthermore, another epidemiological study demonstrated that IBS patients using NSAIDs were also more likely to have persistent irritable bowel syndrome [20]. This ap- pears to be in line with the hypothesis that NSAID therapy affects intestinal permeability resulting in sustained low- grade mucosal inflammation [21]. Results This implies that even if NSAIDs do not necessarily trigger IBS symptoms, they may be able to sustain the condition by altering intes- tinal physiology and in particular by impairing intestinal permeability. arthritis. These analyses showed similar ORs to the ana- lysis with the inclusion of all subjects. Results During the investigated period, 521 cases were identified as having IBS according the Rome III criteria. From these, a total of 287 cases gave informed consent to assess their pharmacy records. These cases of IBS along with 287 ran- domly selected age and sex-matched controls were identi- fied and retrieved for detailed analysis. For demographic characteristics, see Table 1. Distribution of IBS subtypes Similarly, as NSAIDs are often prescribed for rheumatic disorders, analyses were also conducted with the exclusion of patients and controls with documented rheumatoid Table 2 Results for binary logistic regression analysis using comorbidities and exposure to drugs in IBS IBS patients N = 287 Controls N = 287 OR [95% CI] uncorrected for comorbidities OR [95% CI] corrected for comorbidities OR [95% CI] excluding patients with psychiatric comorbidity OR [95%] excluding patients with RA or arthritis OR [95% CI] excluding patients with GERD/FD FD or GERD 25.2% 9.8% NA 2.0 [1.1-3.5] 2.8 [1.5-5.2] 2.1 [1.8-3.8] NA Psychiatric comorbidity 40.4% 3.5% NA 16.6 [7.9-34.8] NA 17.0 [8.0-36.2] 33.3 [11.6-95.5] Fibromyalgia 4.4% 0% NA NA NA NA NA Rheumatoid arthritis 6.1% 0% NA NA NA NA NA PPIs 21.2% 5.2% 2.2 [1.1-5.0] 2.1 [1.1-4.1] 3.0 [1.3-7.0] 3.1 [1.5-6.2] 2.1 [0.8-5.4] NSAIDs 20.55% 3.8% 3.8 [1.7-8.6] 5.2 [2.5-11.0] 4.1 [1.7-9.6] 5.7 [2.65-12.2] 5.5 [2.3-13.3] SSRIs 10.84% 2.1% 3.7 [1.3-10.9] 0.9 [0.3-2.7] 6.3 [0.7-56.8] 0.75 [0.25-2.3] 2.8 [0.5-14.7] Diuretics 5.2% 2.8% 1.0 [0.3-3.6] 1.1 [0.3-3.4] 2.4 [0.7-7.9] 1.4 [0.5-3.7] 2.6 [0.8-9.6] ACE-I 2.8% 1.4% 1.2 [0.2-4.0] 1.4 [0.3-6.0] 0.9 [0.2-4.4] 1.3 [0.3-5.2] 0.8 [0.1-5.0] PPIs = proton pump inhibitors PPIs, NSAIDs = non-steroidal anti-inflammatory drugs, SSRIs = selective serotonin reuptake inhibitors, ACE-I = angiotensin- converting enzyme inhibitors, RA = rheumatoid arthritis, GERD = gastroesophageal reflux disease, FD = functional dyspepsia. NA = not applicable. 2 Results for binary logistic regression analysis using comorbidities and exposure to drugs in IBS Table 2 Results for binary logistic regression analysis using comorbidities and Keszthelyi et al. BMC Gastroenterology 2012, 12:121 http://www.biomedcentral.com/1471-230X/12/121 Page 4 of 6 Page 4 of 6 Table 3 Types of drugs used by irritable bowel syndrome patients Type of PPI1 Type of NSAID2 Pantoprazole 44.0% Diclofenac 47.0% Omeprazole 28.8% Ibuprofen 41.2% Esomeprazole 27.1% Naproxen 21.6% Rabeprazole 6.7% Aceclofenac 3.9% 1 6 patients used multiple PPIs. 2 7 patients used multiple NSAIDs. PPI = proton pump inhibitor. NSAID = non-steroidal anti-inflammatory drug. Discussion We therefore presume that the expo- sure to PPIs can be explained by the therapeutic use of PPIs for upper GI complaints. It is generally accepted that PPI therapy can alter in- testinal microbial profiles by inducing hypochlorhydria resulting in a diminished host defense against certain bac- teria [25-28]. A recent study by Lombardo et al. indeed suggested that PPI therapy in humans may potentially re- sult in small intestinal bacterial overgrowth (SIBO) [13]. It is not known whether the changes in intestinal microbiota induced by PPI therapy contribute to the development of symptoms and clinical conditions such as IBS [29]. In ano- ther recent study using duodenal aspirates, no clear asso- ciation was found between SIBO with IBS or PPI use [30]. It therefore still remains unclear whether SIBO, if at all present in IBS, is a cause or merely an epiphenomenon of We also found a positive association with the exposure to NSAIDs, reproducing previous literature findings [17]. Pain-related symptoms of extragastrointestinal origin fre- quently observed in IBS could be the explanation for the increased use of NSAIDs in this population [18]. More- over, as IBS patients tend to suffer from recurring and therapy-refractory abdominal pain, overuse of NSAIDs in IBS patients can be extremely common in everyday clini- cal practice. Therefore, despite the fact that NSAIDs are Keszthelyi et al. BMC Gastroenterology 2012, 12:121 http://www.biomedcentral.com/1471-230X/12/121 Page 5 of 6 are prone to over-utilize drugs. Therefore, one should be aware that prescribing PPIs for upper GI complaints or NSAIDs for pain relief may potentially trigger mecha- nisms resulting in symptom generation representative for IBS. Animal data suggest that the combination of PPIs and NSAIDs, in particular, is able to induce profound al- terations in intestinal homeostasis. As case–control stu- dies generally do not allow interpretation of a cause-effect relationship, further research should include prospective evaluation of PPI users and NSAIDs users monitoring the development of IBS-symptoms in relation to drug expo- sure to ascertain whether this increased exposure to PPIs and NSAIDs should be considered as legitimate etiological factors in IBS. IBS, as microbial alterations most probably are not the sole explanation for symptom development in IBS [29]. Overall, it is tempting to assume that PPIs can potentially induce alterations in intestinal microbiota, albeit not to a clinically significant degree, which can in turn impair the capacity of the intestine to respond to noxious agents, such as NSAIDs. References 1. Drossman DA, Camilleri M, Mayer EA, Whitehead WE: AGA technical review on irritable bowel syndrome. Gastroenterology 2002, 123(6):2108–2131. 2. Akehurst RL, Brazier JE, Mathers N, O'Keefe C, Kaltenthaler E, Morgan A, Platts M, Walters SJ: Health-related quality of life and cost impact of irritable bowel syndrome in a UK primary care setting. Pharmacoeconomics 2002, 20(7):455–462. 3. Kerckhoffs AP, Akkermans LM, de Smet MB, Besselink MG, Hietbrink F, Bartelink IH, Busschers WB, Samsom M, Renooij W: Intestinal permeability in irritable bowel syndrome patients: effects of NSAIDs. Dig Dis Sci 2010, 55(3):716–723. 4. Spiegel BM, Chey WD, Chang L: Bacterial overgrowth and irritable bowel syndrome: unifying hypothesis or a spurious consequence of proton pump inhibitors? Am J Gastroenterol 2008, 103(12):2972–2976. 5. Stichting Farmaceutische Kerngetallen (SKF): Facts and figures 2009; 2010. Available from: http://www.sfk.nl/pdf-documenten/data-en-feiten/data-en- feiten-2009, accessed 15 August. 6. Forgacs I, Loganayagam A: Overprescribing proton pump inhibitors. BMJ 2008, 336(7634):2–3. 7. van Boxel OS, Hagenaars MP, Smout AJ, Siersema PD: Socio-demographic factors influence chronic proton pump inhibitor use by a large population in the Netherlands. Aliment Pharmacol Ther 2009, 29(5):571–579. 7. van Boxel OS, Hagenaars MP, Smout AJ, Siersema PD: Socio-demographic factors influence chronic proton pump inhibitor use by a large population in the Netherlands. Aliment Pharmacol Ther 2009, 29(5):571–579. 8. Goettsch WG, van den Boom G, Breekveldt-Postma NS, Smout AJ, Herings RM: Treatment patterns and health care costs of mebeverine-treated IBS patients: a case–control study. Pharmacoepidemiol Drug Saf 2004, 13(11):803–810. Competing interests h h d l h The authors declare that they have no competing interests. Abbreviations BS bl b IBS: Irritable bowel syndrome; NSAID: Non-steroidal anti-inflammatory drug; PPI: Proton pump inhibitor; SSRI: Selective serotonin reuptake inhibitor; ACE: Angiotensin converting enzyme; SIBO: Small intestinal bacterial overgrowth. Discussion Such ‘two-hit’ theory could provide an explanation for the relevance of co-exposure to PPIs and NSAIDs in the onset of IBS symptoms [31]. Besides PPIs and NSAIDs, the use of SSRIs was also associated with IBS in our study, but only when patients with psychiatric comorbidity were included in the ana- lyses. Excluding IBS patients with psychiatric comorbid- ities from the analyses, accounting for 38% of our study population, resulted in a loss of positive association with exposure to SSRIs. This may be due to the high preva- lence of psychiatric comorbidities in this population for which IBS patients receive SSRI therapy. This observa- tion therefore suggests that the association of IBS and SSRI use is probably due to the therapeutic application of SSRIs for preexistent psychiatric conditions. Although no data were available on the duration of psychiatric co- morbid conditions, we postulate that these may have been present prior to the onset of the gastrointestinal symp- toms and are therefore less likely to be involved in trigge- ring IBS symptoms. Authors’ contribution DK l d d d DK analyzed data and wrote manuscript, GD, GM and JWK collected data, AAM supervised manuscript preparation. All authors read and approved the final manuscript. Although we were able to demonstrate a positive asso- ciation with IBS and the use of drugs known to alter in- testinal physiology, our study is hypothesis generating rather than proving an etiological relationship due to a number of limitations. While it is apparent that IBS pa- tients utilize medication more often than controls, case– control studies generally do not allow interpretation with respect to a causal relation. In an attempt to establish a temporal relationship, we aimed to assess exposure to medication prior to onset of symptoms, implicating that this exposure can potentially trigger symptoms characte- ristic for IBS. However, we were not able to report on exact symptom history in the 180-day period investigated for drug exposure in relation to drug intake, which hin- ders the establishment of a true cause-effect relationship. We were also unable to report on a potential relationship between drug use and the severity of IBS-related symp- toms. Furthermore, we could not take into account the use of over-the-counter medication. Also, our patient po- pulation is possibly a selected population consisting of pa- tient presenting to our secondary/tertiary referral, which may not represent the IBS population as a whole. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 27. Thorens J, Froehlich F, Schwizer W, Saraga E, Bille J, Gyr K, Duroux P, Nicolet M, Pignatelli B, Blum AL, et al: Bacterial overgrowth during treatment with omeprazole compared with cimetidine: a prospective randomised double blind study. 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https://openalex.org/W3021348595	https://mfr.edp-open.org/10.1051/mfreview/2020013/pdf	English	null	Multi objective optimization of wear behaviour of Aluminum MMCs using Grey-Taguchi method	Manufacturing review	2,020	cc-by	5,633	Received: 15 December 2019 / Accepted: 18 March 2020 Received: 15 December 2019 / Accepted: 18 March 2020 Abstract. In recent years, metal matrix composite (MMCs) have been receiving worldwide attention on account of their superior strength-to-weight ratio and stiffness. Among the several classes of composite materials, Aluminium matrix ceramic reinforcement composites have attracted increasing attention due to their unique properties such as better speciﬁc strength, speciﬁc stiffness, wear resistance, excellent corrosion resistance, high elastic modulus and light weight. The aim of the present investigation is to optimize the dry sliding wear parameters of Aluminum LM25 matrix reinforced with silicon carbide (SiC) (5 wt.%) and Copper (Cu) (3 wt.%) using Taguchi based grey relational analysis. In this work, the composite is prepared using stir casting method. The specimens are prepared according to ASTM standard. Using pin-on-disc apparatus, wear tests are conducted as per Taguchi’s L9 orthogonal array and optimum wear parameters are identiﬁed with an objective to minimise the wear rate and coefﬁcient of friction based on the grey relational grade. The effect of parameters on the wear rate and coefﬁcient of friction was determined using Analysis of variance (ANOVA). Finally, the experimental results were veriﬁed using conﬁrmation tests and the SEM analysis was carried out to study the wear mechanism. Keywords: Aluminium–metal matrix composite / optimization / grey relational analysis / ANOVA * e-mail: vpmuthu2001@yahoo.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Introduction wear rate and the increase in applied load results in increase of the wear rate of the MMCs. However, it was decreased with increase in speed. Gaitonde et al. [7], Ravi Kumar et al. [8], and Zhang et al. [9] reported that the wear resistance of MMCs depends upon shape of the particle, distribution of reinforcement material and volume fraction. Shanawaz Patil et al. [10] concluded that the wear rate for the metal matrix composite increases with increase in speed for a given constant load and time. Ravi et al. [11] conducted the tribological tests on Al6061 reinforced with redmud and reported that red mud particles in Al-6061 increase the hardness and coefﬁcient of friction decrease with increase in weight percent of red mud reinforcement. Amuthakkannan et al. [12] investigated the wear rate of the composites with parameters such as percent of reinforce- ment, Sliding velocity and Normal load using Grey relation analysis. They found that all the parameters taken for the wear study are signiﬁcant and the most signiﬁcant parameter was on Normal load and the optimized parameters are Sliding velocity (m/sec) of 2, Normal load (N) of 20, Reinforcement of basalt ﬁber (percent) of 10. Arulraj et al. [13] investigated the tribological behaviour of hybrid Metal Matrix (Al-Al2O3-B4C) Com- posite. They concluded that the applied load and sliding wear rate and the increase in applied load results in increase of the wear rate of the MMCs. However, it was decreased with increase in speed. Gaitonde et al. [7], Ravi Kumar et al. [8], and Zhang et al. [9] reported that the wear resistance of MMCs depends upon shape of the particle, distribution of reinforcement material and volume fraction. Shanawaz Patil et al. [10] concluded that the wear rate for the metal matrix composite increases with increase in speed for a given constant load and time. Ravi et al. [11] conducted the tribological tests on Al6061 reinforced with redmud and reported that red mud particles in Al-6061 increase the hardness and coefﬁcient of friction decrease with increase in weight percent of red mud reinforcement. Amuthakkannan et al. [12] investigated the wear rate of the composites with parameters such as percent of reinforce- ment, Sliding velocity and Normal load using Grey relation analysis. Multi objective optimization of wear behaviour of Aluminum MMCs using Grey-Taguchi method Mechanical Engineering Department, University College of Engineering, Anna University, Ramanathapuram 623 513, Tamilnadu, India Manufacturing Rev. 7, 16 (2020) © P. Muthu, Published by EDP Sciences 2020 https://doi.org/10.1051/mfreview/2020013 Available online at: https://mfr edp open org Manufacturing Rev. 7, 16 (2020) © P. Muthu, Published by EDP Sciences 2020 https://doi.org/10.1051/mfreview/2020013 Manufacturing Rev. 7, 16 (2020) © P. Muthu, Published by EDP Sciences 2020 https://doi.org/10.1051/mfreview/2020013 Available online at: https://mfr.edp-open.org Available online at: https://mfr.edp-open.org RESEARCH ARTICLE 2 Experimental procedures The signal-to-noise ratio is a logarithmic function used to optimize the process or product design, minimizing the variability. In general, signal to noise (S/N) ratio (h, dB) represents quality characteristics for the observed data in the Taguchi design of experiments. There are 3 signal-to- noise ratios of common interest for optimization of Static Problems. They are lower-the-better, higher-the-better and nominal-the better. The S/N ratio with lower-the- better characteristics (wear rate, coefﬁcient of friction etc.,) can be calculated using equation (1) 2.2 Plan of experiment The Taguchi design method was developed by Dr. Genichi Taguchi. It is a simple and robust technique for optimizing the process parameters. In this method, load, sliding velocity and sliding distance are taken as main process parameters input parameters and the experiment is performed as per L9 orthogonal array. The levels of various input process parameters according to the L9 orthogonal array for the experiments are shown in Table 2. 1 Introduction They concluded that the wear rate of LM6-M alloy increases with increase in percentage of silicon than LM4-T6 alloy. Akhlaghi et al. [17,18] studied the tribological behaviour of SiC and Gr reinforced Al6061. They ﬁnd that the wear rate of the prepared composite increases with increase of SiC. Sanjeev Das et al. [19] studied the abrasive wear of Al-Cu alloy with alumina and Zircon sand particles and concluded that wear resistance of the composite increases with the addition of alumina and zircon particles. Ranganath et al. [20] investigated that the wear resistances of Al6061 reinforced with garnet particles. They reported that the wear rate of Garnet particle reinforced composites is more than the unreinforced alloy specimens and the wear rate decreased with increasing garnet content. Also they found that the increase in applied load increases the wear rate of the composites as well as the matrix alloy. Umanath et al. [21] investigated the wear behaviour of Al6061reinforced with SiC and Al2O3 hybrid metal matrix composites. They reported that the wear resistance of the 5 percent hybrid composite is poorer than that of the 15 percent composite. In the present investigation, an attempt has been made to determine best parameters such as load, sliding velocity and sliding distance to optimize the wear rate and coefﬁcient of friction (COF) simultaneously using Grey Taguchi method. The optimized parameters were then veriﬁed through a conﬁrmation experiment. Finally, the worn out surfaces and wear mechanism of the matrix composites were studied using SEM. Table 2. Input process parameters with their values at three levels. Table 2. Input process parameters with their values at three levels. Factors Level 1 Level 2 Level 3 Load (N) 10 20 30 Sliding velocity (m/s) 2.0 2.4 2.7 Sliding distance (m) 1000 1150 1300 Factors Level 1 Level 2 Level 3 Load (N) 10 20 30 Sliding velocity (m/s) 2.0 2.4 2.7 Sliding distance (m) 1000 1150 1300 the cast samples were taken away from the mould and machined to a dimensions of 10 mm in diameter and 30 mm in length as per ASTM G99-95 (2010). 1 Introduction They found that all the parameters taken for the wear study are signiﬁcant and the most signiﬁcant parameter was on Normal load and the optimized parameters are Sliding velocity (m/sec) of 2, Normal load (N) of 20, Reinforcement of basalt ﬁber (percent) of 10. Arulraj et al. [13] investigated the tribological behaviour of hybrid Metal Matrix (Al-Al2O3-B4C) Com- posite. They concluded that the applied load and sliding Due to the advancement in science and technology, there is a demand of advanced engineering materials in the ﬁeld of automotive and aerospace areas. These area demands materials having better mechanical and tribological properties than the conventional materials. In recent years, metal matrix composite (MMCs) are playing an important role in various applications due to their high temperature resistance and strength [1]. The MMC is the combination of two or more constituents in which one is matrix and other is reinforcements. At present, aluminium based metal matrix composites are widely used in many engineering applications because of their improved wear resistance, low density, enhanced strength and stiffness. The tribological parameters such as applied load (Prasad et al. [2], Bai et al. [3] sliding speed (Lee et al. [4], Sannino et al. [5]), and percent of ﬂy ash control the friction and wear performance. Sreenivasan et al. [6] reported the wear behaviour aluminium reinforced with TiB2 that the increase in TiB2 results in decrease of the P. Muthu: Manufacturing Rev. 7, 16 (2020) 2 Table 1. Chemical composition of LM25 (in weight %). Composition Percentage (%) Si 6.5–7.5 Fe 0.5 Cu 0.1 Mn 0.3 Mg 0.2–0.6 Pb 0.1 Ni 0.1 Zn 0.1 Sn 0.05 Ti 0.2 Al balance velocity were important parameters followed by time duration on wear rate. Bhaskar et al. [14] investigated the wear behaviour of Al2024 reinforced with beryl particulate MMC’s. They ﬁnd that wear rate and coefﬁcient of friction of Al2024 and the composites decreases with increase in sliding speed up to 3 m/s for a load of 40 N, after that wear rate and coefﬁcient of friction slightly increases as the sliding speed increases. Ahemad et al. [15] studied the tribological behaviour of LM25 reinforcement with SiC that the wear rate varies linearly with normal load. Also, they Narayan et al. [16] studied the wear behaviour of LM4 (Al-Si5Cu3)-T6/LM6 (Al-Si12)-M using Taguchi Ap- proach. 2.1 Materials and methods No Load (N) Sliding velocity (m/s) Sliding distance (m) Density g/mm3 Initial mass (g) Final mass (g) 1 10 2 1000 0.002688 6.360864 6.351364 2 10 2.4 1150 0.002688 6.370356 6.358111 3 10 2.7 1300 0.002688 6.430784 6.41875 4 20 2 1150 0.002688 6.440568 6.431701 5 20 2.4 1300 0.002688 6.150237 6.138626 6 20 2.7 1000 0.002688 6.400665 6.382087 7 30 2 1300 0.002688 6.090467 6.050778 8 30 2.4 1000 0.002688 6.265047 6.217758 9 30 2.7 1150 0.002688 6.390743 6.373643 P. Muthu: Manufacturing Rev. 7, 16 (2020) 3 Table 3. Experimental plan and results. Ex. No Load (N) Sliding velocity (m/s) Sliding distance (m) Density g/mm3 Initial mass (g) Final mass (g) 1 10 2 1000 0.002688 6.360864 6.351364 2 10 2.4 1150 0.002688 6.370356 6.358111 3 10 2.7 1300 0.002688 6.430784 6.41875 4 20 2 1150 0.002688 6.440568 6.431701 5 20 2.4 1300 0.002688 6.150237 6.138626 6 20 2.7 1000 0.002688 6.400665 6.382087 7 30 2 1300 0.002688 6.090467 6.050778 8 30 2.4 1000 0.002688 6.265047 6.217758 9 30 2.7 1150 0.002688 6.390743 6.373643 Table 3. Experimental plan and results. Fig. 1. Pin on disc apparatus. n in Table 3. Initial volume of the specimen ¼ Initial weight of the specimen Density of the specimen ð2Þ Final volume of the specimen ¼ Final weight of the specimen Density of the specimen ð3Þ Initial volume of the specimen ¼ Initial weight of the specimen Density of the specimen ð2Þ ð2Þ Final volume of the specimen ¼ Final weight of the specimen Density of the specimen ð3Þ ð3Þ Then ﬁnal volume loss of the specimen, wear rate and coefﬁcient of friction are calculated using equations (4)–(6) and is shown in Table 4. Volume loss of the specimen ¼ Final volume of the specimen Initial volume of the specimen ð4Þ Wear rate ¼ Volume loss Sliding distance mm3 m ð5Þ ð4Þ Fig. 1. Pin on disc apparatus. ð5Þ 2.3 Wear test ð6Þ The pin on disc apparatus (Ducom, model No: ED-201, Bangalore, India) (see Fig. 1) is used to evaluate the wear rate and coefﬁcient of friction of the specimens. 2.4 Grey relational analysis (GRA) Tests were conducted as per L9 orthogonal array under dry sliding conditions as per ASTM G99-95 (2010) at a room temperature. The counter-face disc is made of EN-31 steel with the hardness of 62 HRC. Initially, the test samples were polished metallographically. The initial weight of the specimen was measured using a single pan electronic weighing machine with least count of 0.0001 g. Then the test the pin was pressed against the counterpart rotating against EN-31 steel with the hardness of 62 HRC. After the test, the specimens were removed, cleaned with acetone, dried and the specimen was weighed to ﬁnd the ﬁnal weight of the specimen. From the initial and ﬁnal weight of the specimen the initial and ﬁnal volume of the specimen are calculated using the equations (2) and (3) and Single performance characteristics can be easily optimized by Taguchi method. However, it is a challenging task for analyzing and solving multi performance characteristics. GRA is one of the techniques used for optimization of control factors having multi-performances. The various steps of GRA technique are: 2.1 Materials and methods In this investigation, LM25 was used as a matrix material. Silicon carbide and copper were used as reinforcements. The chemical composition of the LM25 in weight percent is given in Table 1. The silicon carbide particle size ranges from 30 to 40 microns. The composite with 5 wt.% of SiC particles and 3 percent Cu was fabricated by the mechanical stir casting technique. Weighed amount of LM25 was melted at 850 °C in an electric resistance furnace using a graphite crucible. Before adding SiC and Cu particulates to LM25, they were preheated to a tempera- ture of about 350 °C. Then they were added to the molten metal at 850 °C and stirred continuously for uniform distribution. The stirring was done at 750 rpm for 7–9 min. Then the molten material was poured into a permanent metallic mould and allowed to solidify. After solidiﬁcation, hij ¼ 10log 1 n X n j¼1 y2 ij ð1Þ ð1Þ where hij is the j th S/N ratio of the i th experiment, yij is the i th experiment at the j th test, n is the total number of the tests. P. Muthu: Manufacturing Rev. 7, 16 (2020) 3 2.3 Wear test The pin on disc apparatus (Ducom, model No: ED-201, Bangalore, India) (see Fig. 1) is used to evaluate the wear rate and coefﬁcient of friction of the specimens. Tests were conducted as per L9 orthogonal array under dry sliding conditions as per ASTM G99 95 (2010) at a is shown in Table 3. Initial volume of the specimen ¼ Initial weight of the specimen Density of the specimen ð2Þ Final volume of the specimen ¼ Final weight of the specimen Density of the specimen ð3Þ Then ﬁnal volume loss of the specimen, wear rate and coefﬁcient of friction are calculated using equations (4)–(6) and is shown in Table 4. Volume loss of the specimen ¼ Final volume of the specimen Initial volume of the specimen ð4Þ Wear rate ¼ Volume loss Sliding distance mm3 m ð5Þ Coefficient of friction ðCOFÞ¼ Frictional force Normal force ð6Þ 2.4 Grey relational analysis (GRA) Fig. 1. Pin on disc apparatus. Table 3. Experimental plan and results. Ex. Step 1: Normalization f In this step, measured features of quality characteristics i.e., wear rate and coefﬁcient of friction are ﬁrst normalized ranging from zero to one. This process is known as Grey relational generation. Three approaches are used for normalization. If the target value of original sequence is P. Muthu: Manufacturing Rev. 7, 16 (2020) 4 Table 4. Wear rate and coefﬁcient of friction. Ex. No Load (N) Sliding velocity (m/s) Sliding distance (m) Initial volume (mm3) Final volume (mm3) Volume loss (mm3) Frictional force (N) Wear rate (mm3/m) Coefﬁcient of friction 1 10 2 1000 2366.393 2362.859 3.534226 3.52 0.003534 0.3521 2 10 2.4 1150 2369.924 2365.369 4.555432 4.03 0.003961 0.4032 3 10 2.7 1300 2392.405 2387.928 4.476935 3.78 0.003444 0.3781 4 20 2 1150 2396.045 2392.746 3.298735 2.21 0.002868 0.1105 5 20 2.4 1300 2288.035 2283.715 4.319568 13.52 0.003323 0.6760 6 20 2.7 1000 2381.2 2374.288 6.911458 7.21 0.006911 0.3605 7 30 2 1300 2265.799 2251.033 14.76525 8.47 0.011358 0.2823 8 30 2.4 1000 2330.747 2313.154 17.59263 11.75 0.017593 0.3916 9 30 2.7 1150 2377.509 2371.147 6.361607 11.44 0.005532 0.3813 Table 4. Wear rate and coefﬁcient of friction. inﬁnite, then it has a characteristic of “the-larger-the- better.” The original sequence can be normalized using equation (7) After obtaining the grey relational coefﬁcient, the Grey relational grade corresponding to each performance characteristic is computed. The grey relational grade can be obtained using equation (10) X i k ð Þ ¼ X0 i k ð Þ minX0 i k ð Þ maxX0 i k ð Þ minX0 i k ð Þ ð7Þ gi ¼ 1 n Xn k¼1 ji k ð Þ ð10Þ ð7Þ ð10Þ when the lower-the-better characteristic of the original sequence is considered, then the original sequence should be normalized using equation (8) where gi is the grey relational grade for the ith experiment and n is the number of performance characteristics. X i k ð Þ ¼ maxX0 i k ð Þ X0 i k ð Þ maxX0 i k ð Þ minX0 i k ð Þ ð8Þ ð8Þ 3 Results and discussion where X0 i k ð Þ is the value after the grey relational generation (data preprocessing), max X0 i k ð Þ is the largest value of X0 i k ð Þ, minX0 i k ð Þ is the smallest value of X0 i k ð Þ, and X0 is the desired value. As per grey relational analysis, the experimental results are normalized using equation (4) ranging from zero and then using equation (5), the normalized results are converted into grey relational coefﬁcient to represent the correlation between the desired and actual data. Finally, by averaging the grey relational coefﬁcient, the grey relational grades and their rank are calculated. The S/N ratio, Normalized S/N ratio of wear rate and COF along with Grey relational coefﬁcient, Grey relational Grade and Rank are shown in Table 5. Step 2 Calculation of Grey relational coefﬁcient and grey relational grade Step 2 Calculation of Grey relational coefﬁcient and grey relational grade S/N ratio Normalized S/N ratio Grey relational coefﬁcient Grey relational grade Rank Wear rate COF Wear rate COF Wear rate COF 1 49.034 9.069146 0.1151204 0.6397029 0.36104 0.58119 0.47111 8 2 48.043 7.893899 0.1780054 0.7144087 0.37821 0.63646 0.50734 6 3 49.258 8.450164 0.1008986 0.6790492 0.35737 0.60905 0.48321 7 4 50.848 19.13275 0.0000000 0.0000000 0.33333 0.33333 0.33333 9 5 49.569 3.401066 0.0811809 1.0000000 0.35240 1 0.67620 2 6 43.209 8.861894 0.4848702 0.6528771 0.49254 0.59023 0.54139 4 7 38.894 10.98476 0.7587617 0.5179347 0.67454 0.50913 0.59183 3 8 35.093 8.141660 1.0000000 0.6986595 1.00000 0.62395 0.81197 1 9 45.142 8.373912 0.3621696 0.6838962 0.43943 0.61266 0.52605 5 Table 5. S/N ratio, Normalized S/N ratio of wear rate and COF along with Grey relational coefﬁcient, Grey relational Grade and Rank. Table 6. Main effects on grey grades. Level Load Sliding velocity Sliding distance 1 0.4872 0.4654 0.6082* 2 0.5170 0.6652* 0.4556 3 0.6433* 0.5169 0.5838 Delta 0.1561 0.1997 0.1526 Rank 2 1 3 * Optimum values. Fig. 2. Effect of grey relational grade. Fig. 2. Effect of grey relational grade. Table 7. Results of ANOVA on grey grade. Source DF Adj. SS Adj. MS F-Value P-Value %C Load 2 0.041197 0.020598 114.70 0.009 28.14 Sliding velocity 2 0.064535 0.032267 179.67 0.006 44.08 Sliding distance 2 0.040308 0.020154 112.22 0.009 27.53 Residual error 2 0.000359 0.000180 0.25 Total 8 0.146399 100 Table 7. Results of ANOVA on grey grade. gives the largest average response is selected. From Table 6 and Figure 2, the optimum factors for both the wear rate and the coefﬁcient of friction obtained for the hybrid composite are 30 N load (level 3), 2.4 m/s sliding velocity (level 2), and 1000 m sliding distance (level 1) combination. signiﬁcance of 5 percent, i:e., the level of conﬁdence 95 percent. Table 7 shows the results of the ANOVA for the overall Grey relational grade of MMCs. It is observed from the table that the signiﬁcant factor is sliding velocity. The sliding velocity (44.08 percent) inﬂuences more on the multi-performance characteristics of LM25/SiC/Cu hybrid composite followed by load (28.14 percent) and sliding distance (27.53 percent). 3.1 Analysis of variance (ANOVA) ANOVA is a statistical method used to interpret the experimental results. In this work, ANOVA is used to analyze the effects of load, sliding velocity and sliding distance on wear. This analysis was carried out for a level of Step 2 Calculation of Grey relational coefﬁcient and grey relational grade S/N ratio Normalized S/N ratio Grey relational coefﬁcient Grey relational grade Rank Wear rate COF Wear rate COF Wear rate COF 1 49.034 9.069146 0.1151204 0.6397029 0.36104 0.58119 0.47111 8 2 48.043 7.893899 0.1780054 0.7144087 0.37821 0.63646 0.50734 6 3 49.258 8.450164 0.1008986 0.6790492 0.35737 0.60905 0.48321 7 4 50.848 19.13275 0.0000000 0.0000000 0.33333 0.33333 0.33333 9 5 49.569 3.401066 0.0811809 1.0000000 0.35240 1 0.67620 2 6 43.209 8.861894 0.4848702 0.6528771 0.49254 0.59023 0.54139 4 7 38.894 10.98476 0.7587617 0.5179347 0.67454 0.50913 0.59183 3 8 35.093 8.141660 1.0000000 0.6986595 1.00000 0.62395 0.81197 1 9 45.142 8.373912 0.3621696 0.6838962 0.43943 0.61266 0.52605 5 Table 6. Main effects on grey grades. Level Load Sliding velocity Sliding distance 1 0.4872 0.4654 0.6082* 2 0.5170 0.6652* 0.4556 3 0.6433* 0.5169 0.5838 Delta 0.1561 0.1997 0.1526 Rank 2 1 3 * Optimum values. Fig. 2. Effect of grey relational grade. Table 5. S/N ratio, Normalized S/N ratio of wear rate and COF along with Grey relational coefﬁcient, Grey relational Grade and Rank. Ex. No. S/N ratio Normalized S/N ratio Grey relational coefﬁcient Grey relational grade Rank Wear rate COF Wear rate COF Wear rate COF 1 49.034 9.069146 0.1151204 0.6397029 0.36104 0.58119 0.47111 8 2 48.043 7.893899 0.1780054 0.7144087 0.37821 0.63646 0.50734 6 3 49.258 8.450164 0.1008986 0.6790492 0.35737 0.60905 0.48321 7 4 50.848 19.13275 0.0000000 0.0000000 0.33333 0.33333 0.33333 9 5 49.569 3.401066 0.0811809 1.0000000 0.35240 1 0.67620 2 6 43.209 8.861894 0.4848702 0.6528771 0.49254 0.59023 0.54139 4 7 38.894 10.98476 0.7587617 0.5179347 0.67454 0.50913 0.59183 3 8 35.093 8.141660 1.0000000 0.6986595 1.00000 0.62395 0.81197 1 9 45.142 8.373912 0.3621696 0.6838962 0.43943 0.61266 0.52605 5 Table 6. Main effects on grey grades. Level Load Sliding velocity Sliding distance 1 0.4872 0.4654 0.6082* 2 0.5170 0.6652* 0.4556 3 0.6433* 0.5169 0.5838 Delta 0.1561 0.1997 0.1526 Rank 2 1 3 * Optimum values. Fig. 2. Effect of grey relational grade. Table 5. S/N ratio, Normalized S/N ratio of wear rate and COF along with Grey relational coefﬁcient, Grey relational Grade and Rank. Ex. No. Step 2 Calculation of Grey relational coefﬁcient and grey relational grade In this step, the Grey relational coefﬁcient is calculated to express the relationship between the ideal and the actual experimental results. The grey relational coefﬁcient can be found using equation (9). From the value of Grey relational grade in Table 5, the main effects are tabulated in Table 6 and the factor effects are plotted in Figure 2. Since the grey relational grade represents the level of correlation between the reference sequence and the comparability sequence, the greater value of the grey relational grade means that the comparability sequence has a stronger correlation to the reference sequence [22]. In other words, regardless of category of the performance characteristics, a greater grey relational grade value corresponds to better performance [23]. Therefore, the optimal level of the machining parameters is the level with the greatest grey relational grade value. The reference sequence that was selected here had the “smaller-the-better” characteristic. Therefore, the comparability sequence with the larger value of the grey relational grade would induce smaller wear rate and coefﬁcient of friction. Based on this premise, the level that ji k ð Þ ¼ Dmin þ &:Dmax DoiðkÞ þ &:Dmax ð9Þ ð9Þ where z(∊ 0,1) = distinguished coefﬁcient, ς = 0.5 is generally used. ji (k) is grey relational coefﬁcient, Dmin is the smallest value of Doi(k), Dmax is the largest value of Doi(k), Doi (k) is the deviation sequence of the reference sequence X o k ð Þ, and the comparability sequence X i k ð Þ, namely Doi k ð Þ ¼ jXo k ð Þ Xi k ð Þj Dmax ¼ max ∀jei max ∀k jX o k ð Þ X j k ð Þj Dmin ¼ min ∀jei min ∀k jX o k ð Þ X j k ð Þj P. Muthu: Manufacturing Rev. 7, 16 (2020) 5 Table 5. S/N ratio, Normalized S/N ratio of wear rate and COF along with Grey relational coefﬁcient, Grey relational Grade and Rank. Ex. No. 3.2 Conﬁrmation test After the optimal level of parameters is identiﬁed, a conﬁrmation experiment is carried out to predict and P. Muthu: Manufacturing Rev. 7, 16 (2020) 6 Table 8. Results of conﬁrmation experiments. Initial testing parameters Optimum testing parameters Prediction Experiment Combination of testing A2B1C2 A3B2C1 A3B2C1 Wear rate 0.002868 0.017593 COF 0.1105 0.3916 Grey relational grade 0.333333 0.818299111 0.811977 Table 8. Results of conﬁrmation experiments. Fig 3. SEM micrographs of LM25/SiC/Cu hybrid Metal Matrix Composites. verify the improvement of the quality characteristic using the optimal level of design parameters. The predicated gray relational grades of using the optimum level of the dry sliding parameters are calculated using equation (11). verify the improvement of the quality characteristic using the optimal level of design parameters. The predicated gray relational grades of using the optimum level of the dry sliding parameters are calculated using equation (11). ^g ¼ gm þ X q i¼1 gi gm ð Þ ð11Þ ð11Þ where gm is the total mean gray relational grade, gi is the mean gray relational grade at the optimum level, and ‘q’ is the number of main parameters that signiﬁcantly affect the coefﬁcient of friction and wear rate. Table 8 shows the comparison of the predicted grey relational grade with the actual grey relational grade obtained in the experiment using the optimal process parameters. Fig 3. SEM micrographs of LM25/SiC/Cu hybrid Metal Matrix Composites. This indicate the simultaneous occurrence of formation and delamination of MML. This is due to the properties of SiC and Cu. From this, it is concluded that the less worn surface was identiﬁed in LM25 hybrid composite than the unreinforced LM25. This analysis proves that aluminium alloy’s wear resistance can be improved with the addition of SiC and Cu reinforcements. From Table 8, it is clear that the optimal data obtained from the conﬁrmation test, which was conducted with the level settings parameters of load (30 kN), Sliding velocity (2.4 m/s) and sliding distance (1000 m) had good agree- ment with the predicted model. Hence the grey relational analysis based on Taguchi method for the optimization of the multi response problems is a very useful tool for predicting the wear rate and coefﬁcient of friction of LM25/ SiC/Cu hybrid metal matrix composites. 4 Conclusions In the present work, the use of Taguchi’s orthogonal array with grey relational analysis to optimize the multiple performance characteristics of LM25 hybrid composites such as wear rate and coefﬁcient of friction has been reported. 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Arumaikkannu, R. 3.3 Surface morphology SEM micrographs of the worn surfaces of (a) LM25 (b) LM25+5% SiC (c) LM25+ 3% Cu (d) LM25+5%SiC+3%Cu at Load 30 N, Sliding speed 2.4 m/s and Sliding distance 1000 m. 3.3 Surface morphology The SEM image of the prepared LM25/SiC/Cu hybrid metal matrix composites is shown in Figure 3. It indicates the nearly uniform distribution of the reinforcement particles in the composite. – The LM25 hybrid composites have been successfully produced by the stir casting route. The microstructural study clearly reveals a nearly uniform distribution of SiC and Cu in the LM25 matrix alloy. After conducting of wear test at optimum conditions (Load 30 N, Sliding speed 2.4 m/s and Sliding distance 1000 m), the morphological changes on the worn surface LM25, LM25 + 5 wt. percent SiC, LM25 + 3 wt. percent Cu and LM25 + 5 wt. percent SiC + 3 wt. percent Cu were examined through scanning electron microscope (SEM). The SEM image of LM25 (Fig. 4a) shows scratches and deep grooves along the sliding direction. The large amount of plastic deformation was also observed. The SEM image of LM 25 + 5 wt. percent SiC (Fig. 4b) shows scratches but grooves becomes smaller than the LM25 alone. In the SEM images of LM 25 + 3 percent Cu (Fig. 4c) scratches become smaller than the previous compositions. In the SEM image of LM 25 + 5 percent SiC + 3 percent Cu (Fig. 4d), the degree of formation of cracks on the wear surface is not much. It exhibits relatively smoother MML and grooves due to delamination of mechanically mixed layer (MML). – Grey relational analysis in the Taguchi method for the optimization of the multi response problems is a very useful tool for predicting the wear rate and coefﬁcient of friction of LM25/SiC/Cu hybrid Metal Matrix Composites. – From, ANOVA, it is revealed that the sliding velocity (44.08 percent) inﬂuences more on the multi-perfor- mance characteristics of LM25/SiC/Cu hybrid compos- ite followed by load (28.14percent) and sliding distance (27.53percent). ( ) – The wear rate is increased with load. The wear resistance of the alloy is improved signiﬁcantly due to particle addition. The composite exhibited lower wear rate than that of the matrix alloy. P. Muthu: Manufacturing Rev. 7, 16 (2020) 7 Fig. 4. SEM micrographs of the worn surfaces of (a) LM25 (b) LM25+5% SiC (c) LM25+ 3% Cu (d) LM25+5%SiC+3%Cu at Load 30 N, Sliding speed 2.4 m/s and Sliding distance 1000 m. Fig. 4. Cite this article as: Poovalingam Muthu, Multi objective optimization of wear behaviour of Aluminum MMCs using Grey- Taguchi method, Manufacturing Rev. 7, 16 (2020) References Subramanian, Analysis of parameters inﬂuencing wear and frictional behaviour of aluminium–ﬂy ash composites, Tribology Trans. 55 (2012) 723–729 17. S. Mahdavi, F. Akhlaghi, Effect of SiC content on the processing, compaction behavior and properties of Al6061/ SiC/Gr hybrid composites, J. Mater. Sci. 46 (2011) 1502–1511 18. F. Akhlaghi, S. Mahdavi, Effect of the SiC content on the tribological properties of hybrid Al/Gr/SiC composites processed by in situ powder metallurgy (IPM) method, Adv. Mater. Res. 264–265 (2011) 1878–1886 9. L. Zhang, X.B. He, X.H. Qu, B.H. Duan, X. Lu, M.L. Qin, Dry sliding wear properties of high volume fraction SiCp/Cu composites produced by pressureless inﬁltration, Wear 265 (2008) 1848–1856 P. Muthu: Manufacturing Rev. 7, 16 (2020) 8 hybrid metal matrix composites, Composites: Part B 53 (2013) 159–168. 19. S. Das, S. Das, K. Das, Abrasive wear of zircon sand and alumina reinforced Al-4.5 wt. percent Cu alloy matrix composites A comparative study, Compos. Sci. Technol. 67 (2007) 746–751 22. C.P. Fung, Manufacturing process optimization for wear property of ﬁbre-reinforced polybutylene terephthalate composites with grey relational analysis, Wear 254 (2003) 298–306 ( ) 20. G. Ranganath, S.C. Sharma, M. Krishna, Dry sliding wear of garnet reinforced zinc/aluminium metal matrix composites, Wear 251 (2001) 1408–1413 23. N. Tosun, C. Cogun, H. Pihtili, The effect of cutting parameters on wire crater sizes in wire EDM, Int. J. Adv. Manuf. Technol. 21 (2003) 857–865 21. K. Umanath, K. Palanikumar, S.T. Selvamani, Analysis of dry sliding wear behaviour of Al6061/SiC/Al2O3 Cite this article as: Poovalingam Muthu, Multi objective optimization of wear behaviour of Aluminum MMCs using Grey- Taguchi method, Manufacturing Rev. 7, 16 (2020)
https://openalex.org/W2340594345	https://europepmc.org/articles/pmc4839738?pdf=render	English	null	Ultra-Sensitive Humidity Sensor Based on Optical Properties of Graphene Oxide and Nano-Anatase TiO2	PloS one	2,016	cc-by	6,479	Ultra-Sensitive Humidity Sensor Based on Optical Properties of Graphene Oxide and Nano-Anatase TiO2 Mahdiar Ghadiry1, Mehrdad Gholami2, C. K. Lai1, Harith Ahmad1, W. Y. Chong1 Mahdiar Ghadiry1, Mehrdad Gholami2, C. K. Lai1, Harith Ahmad1, W. Y. Chong1 1 Photonics Research Center, University of Malaya, Kuala Lumpur, 50603, Malaysia, 2 Department o Chemistry, Marvdasht Branch, Islamic Azad University, P.O. Box 465, Marvdasht, Iran Mahdiar Ghadiry1, Mehrdad Gholami2, C. K. Lai1, Harith Ahmad1, W. Y. Chong1 mahdiar@um.edu.my * mahdiar@um.edu.my * mahdiar@um.edu.my Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Abstract a1111 Generally, in a waveguide-based humidity sensors, increasing the relative humidity (RH) causes the cladding refractive index (RI) to increase due to cladding water absorption. How- ever, if graphene oxide (GO) is used, a reverse phenomenon is seen due to a gap increase in graphene layers. In this paper, this interesting property is applied in order to fabricate dif- ferential humidity sensor using the difference between RI of reduced GO (rGO) and nano- anatase TiO2 in a chip. First, a new approach is proposed to prepare high quality nano-ana- tase TiO2 in solution form making the fabrication process simple and straightforward. Then, the resulted solutions (TiO2 and GO) are effortlessly drop casted and reduced on SU8 two channels waveguide and extensively examined against several humid conditions. Investi- gating the sensitivity and performance (response time) of the device, reveals a great linear- ity in a wide range of RH (35% to 98%) and a variation of more than 30 dB in transmitted optical power with a response time of only ~0.7 sec. The effect of coating concentration and UV treatment are studied on the performance and repeatability of the sensor and the attrib- uted mechanisms explained. In addition, we report that using the current approach, devices with high sensitivity and very low response time of only 0.3 sec can be fabricated. Also, the proposed device was comprehensively compared with other state of the art proposed sen- sors in the literature and the results were promising. Since high sensitivity ~0.47dB/%RH and high dynamic performances were demonstrated, this sensor is a proper choice for bio- medical applications. OPEN ACCESS OPEN ACCESS Citation: Ghadiry M, Gholami M, Lai CK, Ahmad H, Chong WY (2016) Ultra-Sensitive Humidity Sensor Based on Optical Properties of Graphene Oxide and Nano-Anatase TiO2. PLoS ONE 11(4): e0153949. doi:10.1371/journal.pone.0153949 Editor: Amitava Mukherjee, VIT University, INDIA Received: November 12, 2015 Accepted: April 6, 2016 Published: April 21, 2016 Copyright: © 2016 Ghadiry 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. Citation: Ghadiry M, Gholami M, Lai CK, Ahmad H, Chong WY (2016) Ultra-Sensitive Humidity Sensor Based on Optical Properties of Graphene Oxide and Nano-Anatase TiO2. PLoS ONE 11(4): e0153949. doi:10.1371/journal.pone.0153949 Editor: Amitava Mukherjee, VIT University, INDIA Copyright: © 2016 Ghadiry et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. RESEARCH ARTICLE Introduction Funding: The authors would like to acknowledge the full support from University of Malaya through their funding LRGS (2015), NGOD/um/KPT, GA010-2014, UM.C/625/1/HIR/MOHE/SCI/29. Monitoring humidity is essential is many industries and applications [1]. It is vital in medical and chemical industries to monitor patients breathing profile and control the chemical pro- cesses [2, 3]. In addition, it is essential in aerospace and civil engineering. As an example, it is checked regularly in huge structures like bridges and planes to assess the risk of leakage due to corrosion [4]. Besides, it is influential parameter in health of workers and product quality in Competing Interests: The authors have declared that no competing interests exist. 1 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Ultra-Sensitive Humidity Sensor food engineering [5]. Consequently, it has been a hot research topic for last decades and differ- ent types of humidity sensors including optical [6–8], resistive [9], thermal conductivity and capacitive [10] sensors have been designed and reported in the literature [11]. Depending on the applications, a humidity sensor requires high sensitivity, high speed (low response time) and noise immunity [10, 12]. One way to simply meet these requirements is to utilize optical devices. Geometry simplicity and small dimension of optical sensors bring about implementation of light-weight devices in a way that it is possible to simply insert them in medical probes and construction material [2]. Additionally, the sensitivity and detection range of optical devices can be significantly higher than that of the other conventional devices such as electrochemical sensors. These promising characteristics, make the optical devices pertinent alternatives for applications where high speed, low noise and high accuracy are appealed [13]. Furthermore, in situations, where humidity changes rapidly, sensors with low response and recovery time are needed. Such applications includes process control in industry, meteorology and variety of other medical applications such as human breath monitoring [14, 15]. This shows the need for fast, highly sensitive and low cost humidity sensors. Therefore, in this research, an optical method is proposed to implement a humidity sensor using nano-anatase TiO2 particles and reduced graphene oxide (rGO). These two material have been employed in a variety of sensing applications such as gas and humidity sensing and promising results have been reported [16–19]. The basic idea is to measure the relative humid- ity using the difference between refractive indices (power loss) of rGO and TiO2. Nano-Anatase TiO2 powder A new approach is presented here to prepare TiO2 NPs. Initially 2 ml of aetylacetone solution was mixed with 2 ml titanium isopropoxide in a 10 ml measuring cylinder, then the prepared solution was mixed with 40 ml of absolute ethanol in a beaker under vigorous stirring at room temperature. Later, it was added to a solution containing 10 mL of 5% (W/V) of urea dropwise in a beaker under stirring at room temperature. The final solution was pale yellow in color with a pH value of 5.6. After vigorous stirring of this solution for one hour, the whole mixture was trans- ferred into a 120 ml Teflon-lined stainless steel autoclave for 18 h at 150°C and cooled to room temperature naturally. The produced solution was centrifuged with DI water and absolute ethanol for several times. This residue was dried for 3 h at 80°C. The resulted powder was grinded to obtain fine powder which was used for preparation of nano-anatase TiO2 solutions of different concentrations. In this experiment, solutions of 0.01% (Sample 1), 0.02% (Sample 2), 0.04% (Sam- ple 3) and 0.08% (Sample 4) (W/V) of nano-anatase TiO2 were prepared from the solid powder. Graphene Oxide GO solution was prepared by an improved version of Hummer’s method according to [20]. The GO solution in de-ionized water as the solvent has a concentration of about 1 μg/μL. Raman spectra of the produced GO displayed in Fig 1, clearly shows D and G peaks indicating the quality of the material according to argument reported in [21]. Introduction In material preparation, a new approach is engaged to prepare TiO2 nanoparticles with very small particle size around 5 nm in solution form making the fabrication low-cost, effective and smooth. X-ray diffraction (XRD) An x-ray diffraction (XRD) instrument (Siemens D5000) with Cu Kα radiation was employed to analyze the synthesized powder. The XRD pattern of the synthesized nano TiO2 is repre- sented in Fig 4. The sharp picks are corresponded to the diffraction pattern of anatase with ref- erence number: 01-073-1764, which confirms the phase of the prepared material. Field emission scanning electron microscopy (FESEM) Field emission scanning electron microscopy (FESEM) was used in order to perform structural and morphological analysis of the prepared TiO2 powder and rGO. Fig 2(a) and 2(b) illustrate PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 2 / 14 Ultra-Sensitive Humidity Sensor Fig 1. The Raman spectra of GO indicating D and G peaks. doi:10.1371/journal.pone.0153949.g001 Fig 1. The Raman spectra of GO indicating D and G peaks. doi:10.1371/journal.pone.0153949.g001 Fig 1. The Raman spectra of GO indicating D and G peaks. doi:10.1371/journal.pone.0153949.g001 the FESEM image of TiO2 nanoparticles and TiO2 powder (subset) and layered structure of the synthesized GO respectively. In addition, in Fig 2(c), TiO2 (silver-color solution) and rGO (dark brown) are seen in solution form. the FESEM image of TiO2 nanoparticles and TiO2 powder (subset) and layered structure of the synthesized GO respectively. In addition, in Fig 2(c), TiO2 (silver-color solution) and rGO (dark brown) are seen in solution form. Result of size distribution analysis of the TiO2 sample is depicted in Fig 3. Accordingly, it can be recognized that most of particle size have a diameter less than 10 nm and maximum concentration is seen on 5 nm while in commercial TiO2, the average particle size is around 20 nm. PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Device fabrication A SiO2 wafer is selected as substrate and SU8 polymer waveguide is fabricated on top of that. The waveguide consists of two layers being undercladding and core layers. SU-8 2010 polymer Fig 2. The FESEM images of titanium dioxide nano particles (a); same material in form of powder (subset); layered structure of GO (b); prepared TiO2 and GO solution to be drop casted on the waveguide (c). d i 10 1371/j l 0153949 002 Fig 2. The FESEM images of titanium dioxide nano particles (a); same material in form of powder (subset); layered structure of GO (b); prepared TiO2 and GO solution to be drop casted on the waveguide (c). doi:10.1371/journal.pone.0153949.g002 doi:10.1371/journal.pone.0153949.g002 3 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Ultra-Sensitive Humidity Sensor Fig 3. Normalized size distribution of the prepared TiO2 nano particles. doi:10.1371/journal.pone.0153949.g003 Fig 3. Normalized size distribution of the prepared TiO2 nano particles. doi:10.1371/journal.pone.0153949.g003 doi:10.1371/journal.pone.0153949.g003 is used for both layers with different concentration of 100% and 70% respectively. The under- clad with a thickness of around 12 μm is formed by spin coating and then it is cured at 95°C by soft baking for 2 minutes and post baking for 40 seconds respectively. To form the core layer, another SU-8 layer with refractive index of 1.569 (at 1550 nm wave- length) and thickness of 8 μm is spin-coated. The undercladding is patterned by using UV exposure and contact photolithography. Once more, a curing process is carried out at 95°C for 30 minutes. Then the rGO and TiO2 dispersed in DI water are drop-casted on two waveguides as illustrated in Fig 5 and treated with UV for 1 hour. Later, fiber arrays are aligned to the chan- nels and the device is ready to use. The waveguide structure, thickness of the layers and Fig 4. XRD pattern of synthesized TiO2 NPs in anatase form. doi:10.1371/journal.pone.0153949.g004 Fig 4. XRD pattern of synthesized TiO2 NPs in anatase form. doi:10.1371/journal.pone.0153949.g004 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 4 / 14 Ultra-Sensitive Humidity Sensor Fig 5. Device structure and dimensions of layer and coatings. doi:10.1371/journal.pone.0153949.g005 Fig 5. Device structure and dimensions of layer and coatings. doi:10.1371/journal.pone.0153949.g005 evice structure and dimensions of layer and coatings. doi:10.1371/journal.pone.0153949.g005 doi:10.1371/journal.pone.0153949.g005 dimension of the coatings are stated in this figure as well. Device fabrication Based on our thickness analysis of our 25 samples using drop-casting techniques, we calculated the accuracy of the method to be lower than 50 nm while using other techniques such as plasma deposition, accuracy of ±10 is achievable. However, those methods need expensive machines making the fabrication process challenging. dimension of the coatings are stated in this figure as well. Based on our thickness analysis of our 25 samples using drop-casting techniques, we calculated the accuracy of the method to be lower than 50 nm while using other techniques such as plasma deposition, accuracy of ±10 is achievable. However, those methods need expensive machines making the fabrication process challenging. PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Results and Discussion Mechanism if sensing is straightforward. The electromagnetic field propagation inside the core is not fully confined inside that. A part of the field termed evanescent field can escape the core and propagate inside the cladding. These two fields are related by condition of electromagnetic field continuity on the border of the coating and core the layers. Accordingly, the field power loss inside the core will be affected by any change in the evanescent field [22]. Therefore, if the RH changes, the RI of the cladding will change and thus the power loss in the guided waveguide is affected. As a result, it is concluded that this field’s strength depends on the refractive index of the core and coating, input field strength and the coating thickness. As a design parameter, there- fore, the thickness plays an vital role in sensitivity and performance of the sensor [23]. Thus, different concentrations of the coating have been prepared and used on the sensor and the cor- responding performance (response and recovery times) and sensitivity are stated in Tables 1 and 2 for individual and differential sensors respectively. The response and recovery time are calculated based on the maximum recorded values of response and recovery time for both coatings. It can be seen that the sensitivity increases with the concentration while the performance decreases. When the coating thickness is too low (Sample 1), it is much less than that of the penetration depth of the evanescent wave. Correspondingly, a lesser number of particles are accessible to interact with the evanescent wave. By increasing the humidity, water molecules that are diffused into the coating layer could interact with a limited number of particles only resulting in a limited absorption of the optical power carried by the evanescent field in the sens- ing region. Thus, low sensitivity and response time are observed. On the other hand, in a thick cladding layer, numerous water molecules are diffused and the variation in the refractive index is much which results in high sensitivity. However, the associa- tion and dissociation processes of water molecules are timely which causes the recovery and response time to rise. Moreover, the hysteresis of the system increases because of the non-dis- sociated molecules which are trapped inside the coating layer. According to Table 2, it is seen that response time of 220 ms is achievable with acceptable sensitivity (Sample 1). Humidity sensing measurement Fig 6 illustrates the experimental configuration of the humidity sensing. A gas tube which pro- vides controlled humidity is placed above the sensing region. Humid nitrogen gas is made by bubbling the dry nitrogen through water. The fellow is controlled by flow meters. The response and recovery times of the proposed device to humid air is measured using a photodiode and a mechanical shutter which is located between the gas source and the sensor. A commercial HI 5862 hydrometer provided by Hanna instruments is used to measure the relative humidity. A polarizer controller (PC) is employed to adjust polarization and to obtain highest extinction ratio. In addition, an optical attenuator is used for power matching between rGO and TiO2 coatings. At the output, two USB power meter are used in order to measure the loss of rGO and TiO2 channels at the same time during the experiments in different humid conditions. Then, the outputs are processed with analyzer program installed on computer and the charts are plotted. Fig 6. Experimental configuration to control relative humidity inside the chamber and measure the power loss variation at different RHs. doi:10 1371/journal pone 0153949 g006 Fig 6. Experimental configuration to control relative humidity inside the chamber and measure the power loss variation at different RHs. Fig 6. Experimental configuration to control relative humidity inside the chamber and measure the power loss variation at different RHs. doi:10.1371/journal.pone.0153949.g006 doi:10.1371/journal.pone.0153949.g006 5 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Ultra-Sensitive Humidity Sensor PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Ultra-Sensitive Humidity Sensor Table 1. The effect of coating thickness on the sensitivity and response time of the rGO and TiO2 sensors. Sample Thickness (μm) Sensitivity (dB/RH%) Response Time (s) Recovery time (s) TiO2 GO TiO2 GO TiO2 GO TiO2 GO Sample 1 0.2 0.5 0.09 0.11 0.23 0.17 0.33 0.23 Sample 2 0.7 1.1 0.23 0.23 0.74 0.63 0.91 0.76 Sample 3 0.9 2.3 0.39 0.33 1.83 1.71 2.07 1.93 Sample 4 1.1 3.9 0.505 0.43 2.44 2.33 2.81 2.64 doi:10.1371/journal.pone.0153949.t001 Table 1. The effect of coating thickness on the sensitivity and response time of the rGO and TiO2 sensors. Table 1. The effect of coating thickness on the sensitivity and response time of the with ω, tg, ε0, σi, σr being radian frequency, interlayer distance of graphene layers, permittivity of graphene, and imaginary and real part of graphene’s conductivity respectively, as the inter- layer distance increases, the RI decreases and then the loss decreases too. In contrast, when the rGO sheet is exposed to dry air, water desorption process decreases the interlayer distance (i.e. of rGO films shrinking), and therefore the RI and the conductivity increases and decreases respectively. At low range of RH between 10 to 35%, water molecules can be absorbed only at the surface of the coating, which causes only slight absorption of light at the coating-air border. Thus, low sensitivity is observed. However, for higher RH range between 35 to 90%, water molecules can penetrate and be adsorbed into the holes and cracks of the coating and form monolayers of water there, which can cause the real part of the coating RI to change. This results in substantial radiation and influences the coating’s transverse guiding. Although primarily, the core’s real part of RI is dominant in the first region, with the increase in RH, the imaginary part may also contribute. Stewart et al. in [26] have reported comparable mechanism in D-fiber-based gas sensor. In our case, the gas can be replaced by water molecules and therefore, the same phe- nomena could be expected. In the third region above 90%, multilayers of water molecules are formed which results in absorption saturation and sensitivity decrease in comparison with the second region. The influence of UV exposure duration on sensitivity of TiO2 sensor is studied and the results are shown in Fig 8. Apparently, the sensitivity increases as the UV exposure time increases. Results and Discussion However, since high sensitivity and performance are in focus of this study, we further examine the 2th sensor (Sample 2) in the rest of this paper. The power loss profile in respect with RH is illustrated in Fig 7. Moreover, the proposed sensor is compared with other samples with different coating materials. As can be seen, high linearity is observed for wide range of RH between 35 to 98%. Additionally, power loss variation is also very large from 20 to 51 dB for RH in range of 30% to 98%. In general, if the RH increases, the coating RI increases as well due to water absorption in the coating [11]. This phenomenon substantially changes the boundary condition at guide- coating interface reducing the beam confinement in the core (guide). Therefore, the power loss at the output decreases. Although this is valid for TiO2, the scenario is different for rGO. As the RH increases, the rGO’s refractive index and power loss decreases as can be seen in Fig 7. Water molecules pene- trate into the layered structure of the rGO and clusters are formed between their h-bondings and rGO’s epoxy and hydroxyl groups. This process, which is called water swelling, increases the interlayer distance of GO layers. According to reported equation for graphene refracted index ng [24, 25], ng ¼ 1 2otgε0 !1=2½ si þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 4s2 r þ s2 i p ð1Þ ð1Þ 6 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 doi:10.1371/journal.pone.0153949.t001 However, there is a saturation point around 0.47 dB/% RH for exposure more than 90 min. The effect of UV treatment can be explained by contact angle (CA) concept. The water mol- ecule CA, which is the angle between the tangent line of the liquid phase at the interface of the solid surface and the solid-liquid-gas phases [27] is commonly used to estimate the surface wet- tability. As shown in Fig 9, the TiO2 CA is originally around few tenth of degrees. Due to exposing UV to the surface, the CA can be reduced to almost 0° which means that the surface is highly hydrophilic. The CA has been measured employing a CA-X, Kyowakaimenkagaku CA meter and the UV has been illuminated by a fluorescent black light bulb and an Hg-Xe lamp with intensities of 0.59 and 40 mW/cm2 respectively. Table 2. The effect of the coating thickness on the response time and sensitivity of the differential device. Sample Response time (s) Recovery time (s) Sensitivity (dB/RH%) Sample 1 0.23 0.33 0.20 Sample 2 0.74 0.91 0.46 Sample 3 1.83 2.07 0.72 Sample 4 2.44 2.81 0.93 doi:10.1371/journal.pone.0153949.t002 Table 2. The effect of the coating thickness on the response time and sensitivity of the differential device. PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 7 / 14 Ultra-Sensitive Humidity Sensor Fig 7. The proposed sensor power loss profile compared to other methods using various coating materials. For detail about other materials preparation please refer [11]. doi:10.1371/journal.pone.0153949.g007 Fig 7. The proposed sensor power loss profile compared to other methods using various coating materials. For detail about other materials preparation please refer [11]. doi:10.1371/journal.pone.0153949.g007 Fig 7. The proposed sensor power loss profile compared to other methods using various coating materials. For detail about other materials preparation please refer [11]. doi:10.1371/journal.pone.0153949.g007 doi:10.1371/journal.pone.0153949.g007 These findings indicate that the hydrophilic properties of the surface is a function of com- pressive stress which is caused by the surface volume expansion. When TiO2 is exposed to UV light, the electron-hole pairs (ehp) are produced and the these photo-generated holes are trapped in the lattice oxygen sites according to [28, 29]. Most of these trapped holes react with the water molecules, which produce OH radicals as illustrated in Fig 10. Nonetheless, a portion of these trapped holes react with TiO2 molecules at the titanium site and the bonds between the oxygen ions and the lattice titanium are broken. PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 For the charge compensation, new OH Fig 8. Results of applying UV in order to increase the sensitivity of the proposed device. doi:10.1371/journal.pone.0153949.g008 Fig 8. Results of applying UV in order to increase the sensitivity of the proposed device. doi:10.1371/journal.pone.0153949.g008 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 8 / 14 Ultra-Sensitive Humidity Sensor Fig 9. Contact angle in TiO2 against UV exposure time. doi:10.1371/journal.pone.0153949.g009 Fig 9. Contact angle in TiO2 against UV exposure time. doi:10.1371/journal.pone.0153949.g009 doi:10.1371/journal.pone.0153949.g009 groups are released and the hydrophilic property enhances. Improving the hydrophilic effect in rGO has been studied well in the literature [30] and will not be repeated here again. groups are released and the hydrophilic property enhances. Improving the hydrophilic effect in rGO has been studied well in the literature [30] and will not be repeated here again. In order to measure the performance, the device is examined by disrupted humid air supply with predefined time intervals. The results are shown in Fig 11. In subfigure (a) the output is illustrated for TiO2 and rGO sensors and in subfigure (b), the results of using the differential device are depicted. As can be see, response and recovery times of around 0.75 and 0.93 sec has been observed respectively. The rapid response of the device is attributed to the quick diffusion of the water molecules and the slower recovery time is due to the slow desorption process. To examine the device response time in more detail, specific data are taken and the results are illustrated in Fig 12. Here, breath pulses were applied. As a visional analysis shows, a great reproducibility is resulted. It is worth to mention that the RH is not accurately controlled in this experiment since real breathing is applied however, the range of the humidity is between 50 to 100% RH. Fig 10. Mechanism of enhancing TiO2 hydrophilic properties by exposing UV. doi:10.1371/journal.pone.0153949.g010 Fig 10. Mechanism of enhancing TiO2 hydrophilic properties by exposing UV. Fig 10. Mechanism of enhancing TiO2 hydrophilic properties by exposing UV. d i 10 1371/j l 0153949 010 Fig 10. Mechanism of enhancing TiO2 hydrophilic properties by exposing UV. doi:10.1371/journal.pone.0153949.g010 doi:10.1371/journal.pone.0153949.g010 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 9 / 14 Ultra-Sensitive Humidity Sensor Fig 11. Measured recovery and response and behavior of the proposed humidity sensor rGO and TiO2 devices (a); differential device (b). doi:10.1371/journal.pone.0153949.g011 Fig 11. Measured recovery and response and behavior of the proposed humidity sensor rGO and TiO2 devices (a); differential device (b). doi:10.1371/journal.pone.0153949.g011 doi:10.1371/journal.pone.0153949.g011 From Fig 12, high repeatability can be reported. Nonetheless, to investigate this parameter perfectly, hysteresis of the device is plotted in Fig 13. According to the results, the hysteresis is roughly ~6% for the worst scenario, which is acceptable in most applications. In addition, to show the repeatability in practice, the device was tested for 30 days at two dif- ferent RHs and the results are reported in Fig 14. According to the results the power loss increases by 5% for few days and saturates after 3 days. This phenomenon is attributed to dom- inant water molecules trapped inside the structure of the rGO and TiO2. However, the change in power loss is not significant after 3 days. Furthermore, we investigated the sensor response to several gases such as CO, CO2, butane, nitrogen and oxygen, and no significant power variation was observed. As reported in the liter- ature, a low thickness of coating, in range of a nanometer, is normally used for gas sensing [28, 29], while the thickness we used is in the range of micrometers. Furthermore, the coating here is not functionalized and optimized [23] for gas sensing, and not much interaction can there- fore be seen between TiO2 and gas molecules. Fig 12. Response of the device to the breathing when the sample is located at a distance of around 10 cm to the human mouth. doi:10 1371/journal pone 0153949 g012 Fig 12. Response of the device to the breathing when the sample is located at a distance of around 10 cm to the human mouth. Fig 12. Response of the device to the breathing when the sample is located at a distance of around 10 cm to the human mouth. doi:10 1371/journal pone 0153949 g012 doi:10.1371/journal.pone.0153949.g012 10 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Ultra-Sensitive Humidity Sensor Fig 13. Hysteresis of the proposed sensor for several rising and falling RH cycles in range of 10 to 98% using humidity chamber. doi:10.1371/journal.pone.0153949.g013 Fig 13. Hysteresis of the proposed sensor for several rising and falling RH cycles in range of 10 to 98% using humidity chamber. Fig 13. Hysteresis of the proposed sensor for several rising and falling RH cycles in range of 10 to 98% using humidity chamber. PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Conclusion An optical humidity sensor was presented based on the effect of evanescent field in anastase TiO2 nanoparticle and reduced graphene oxide coatings. The approach to prepare the active material, results in in TiO2 in solution form making the fabrication process fast, easy and cheap. The sensor sensitivity and performance were extensively investigated at different humid conditions. It was observed that the device response is almost linear over a wide dynamic range (35%–98% RH) with high sensitivity of around 0.21 dB/%RH. In addition, the effect of several parameters such as coating thickness and the UV treatment time were studied on repeatability, performance and sensitivity of the device and the attributed mechanisms explained. It was revealed that by increasing the thickness, the sensitivity increases while the performance decreases. With high sensitivity and low response time of ~0.7 s for humidification and 0.95 sec for desiccation, the device is most suitable for real time human breath monitoring applica- tion. It is believed that the approach could be used for other applications such as PH meter, temperature sensor and glucose concentration sensing. Ultra-Sensitive Humidity Sensor Table 3. Comparison of the proposed sensor with other proposed sensors using different coatings and methods including nanoscale materials. Reference Coating material Substrate Range Sensitivity Resp. time Gaston et al. [31] Polyvinyl alcohol ﬁlm SMF 70–90% 0.51 dB∕%RH 40 sec Herrero et al. [32] TiO2 ﬁlm SMF 0–15% 0.49 dB∕%RH - Tan et al. [33] Gelatin LPG 90–99% 1.2 dB∕%RH - Liu et al. [34] Hydrogel LPG 38–100% 0.2 dB∕%RH - Yeo et al. [35] Polyimide FBG 23–97% 5.6 dB∕%RH >5 min Aneesh et al. [36] TiO2 Nano particle PCS ﬁber 24–95% 27 mV∕%RH > 1 sec Aneesh et al. [37] ZnO Nanoparticle PCS 4–96% 0:0012RH−1 > 1 sec Corres et al. [12] SiO2 Nanoparticles SMF 40–98% 0.12 dB%RH 0.15 sec Lim et al. [38] Graphene Oxide SU8 WG 60–100% 0.53 dB%RH ~1 sec Vijayan et al. [6] Co polyaniline Optical ﬁber 20%-95% 6 mV/%RH ~1 min Proposed rGO GO solution SU8 WG 35–95% 0.22 dB/%RH 0.68 sec Proposed TiO2 Nano anatas TiO2 SU8 WG 30–95% 0.25 dB/%RH 0.75 sec Proposed Differential GO + TiO2 Su8 WG 35–95% 0.47 dB/%RH 0.75 sec doi:10.1371/journal.pone.0153949.t003 ensor with other proposed sensors using different coatings and methods including nanoscale materials. omparison of the proposed sensor with other proposed sensors using different coatings and methods inclu Table 3. Comparison of the proposed sensor with other proposed sensors using different co quick response time is superior compared to others. It is worth to mention that there are sen- sors with slightly higher sensitivity that this work but the range or/and their performance are not acceptable. In addition, our work uses coating material in solution from making the fabri- cation process simple and quick while most of the other compared devices use difficult processes. In this design, we used two photo detection devices containing two analog amplifiers and two analog digital converters which would be costly in practice. However, in practical imple- mentation, a CMOS differential amplifier (Diff-Amp) could be designed to amplify the differ- ence between two outputs. In this way, the system can be designed using one amplifier and one analog digital converter to reduce the cost and complexity. doi:10.1371/journal.pone.0153949.g013 doi:10.1371/journal.pone.0153949.g013 In order to give a comprehensive comparison, the proposed method is compared with other state of the art sensors using different techniques and substrates, which include single mode fiber, (SMF), plastic clad silica fiber (PCS), and gratings such as fiber brag grating (FBG) and long period fiber grating (LPG). These methods employ different coating materials which include Polyvinyl alcohol and TiO2 films, Polyimide Gelatin, Hydrogel, TiO2, SiO2 and ZnO Nanoparticles, Graphene Oxide, and CO polyaniline. Comparison results is reported in Table 3. It shows that the proposed device with 0.47 dB/RH sensitivity in wide range of RH and Fig 14. Power loss of the differential sensor for 30 days in 60 and 80%RH. doi:10.1371/journal.pone.0153949.g014 Fig 14. Power loss of the differential sensor for 30 days in 60 and 80%RH. Fig 14. Power loss of the differential sensor for 30 days in 60 and 80%RH. doi:10 1371/journal pone 0153949 g014 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 11 / 14 References 1. Kuang Q., Lao C., Wang Z. L., Xie Z., and Zheng L., "High-Sensitivity Humidity Sensor Based on a Sin- gle SnO2 Nanowire," Journal of the American Chemical Society, vol. 129, pp. 6070–6071, 2007/05/01 2007. 2. Ren Y., Morimile P., Petti L., and Cross G. H., "Optical waveguide humidity sensor with symetric multi- layer configuration " Sensors and actuators B, vol. 75, pp. 76–82, 2001. 3. Lopez-Higuera J. M., Rodriguez Cobo L., Quintela Incera A., and Cobo A., "Fiber Optic Sensors in Structural Health Monitoring," Lightwave Technology, Journal of, vol. 29, pp. 587–608, 2011. 4. J. H. Cocatre-Zilgien, "Aircraft system monitoring air humidity to locate updrafts," ed: Google Patents, 2000. 5. 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Acknowledgments We would like to acknowledge the full support from University of Malaya through their fund- ing LRGS (2015), NGOD/um/KPT, GA010-2014, UM.C/625/1/HIR/MOHE/SCI/29. 12 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 Ultra-Sensitive Humidity Sensor Author Contributions Conceived and designed the experiments: M. Ghadiry. Performed the experiments: CKL. Ana- lyzed the data: M. Gholami CKL HA. Wrote the paper: CWY. Discussions: CWY HA. Device fabrication: CWY. Major revision on the manuscript: HA. Conceived and designed the experiments: M. Ghadiry. Performed the experiments: CKL. Ana- lyzed the data: M. Gholami CKL HA. Wrote the paper: CWY. Discussions: CWY HA. Device fabrication: CWY. Major revision on the manuscript: HA. PLOS ONE \| DOI:10.1371/journal.pone.0153949 April 21, 2016 References Mogera U., Sagade A. A., George S. J., and Kulkarni G. 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https://openalex.org/W2805105777	http://www.scielo.br/pdf/rlae/v26/0104-1169-rlae-26-e2992.pdf	English	null	Spatial pattern and temporal trend of mortality due to tuberculosis	Revista latino-americana de enfermagem	2,018	cc-by	6,436	1 PhD. Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, PAHO/WHO Collaborating Centre for Nursing, Ribeirão Preto, SP, Brazil. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). 2 Master’s student. Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, PAHO/WHO Collaborating Centre for Nursing, Ribeirão Preto, SP, Brazil. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). 3 PhD. Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, PAHO/WHO Collaborating Centre for Nursing, Ribeirão Preto, SP, Brazil. Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). 4 PhD. Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, PAHO/WHO Collaborating Centre for Nursing, Ribeirão Preto, SP, Brazil. Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). 5 Doctoral student. Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, PAHO/WHO Collaborating Centre for Nursing, Ribeirão Preto, SP, Brazil. Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM). 6 Post-doctoral fellow. Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, PAHO/WHO Collaborating Centre for Nursing, Ribeirão Preto, SP, Brazil. Programa Nacional de Pós Doutorado (PNPD) - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). 7 Doctoral student. Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, PAHO/WHO Collaborating Centre for Nursing, Ribeirão Preto, SP, Brazil. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). 8 Doctoral student. Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). 9 PhD. Associate Professor, Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, PAHO/WHO Collaborating Centre for Nursing, Ribeirão Preto, SP, Brazil. 10 Supported by São Paulo Research Foundation 2015/17586-3. patial pattern and temporal trend of mortality due to tuberculosis Ana Angélica Rêgo de Queiroz1 Thaís Zamboni Berra2 Maria Concebida da Cunha Garcia3 Marcela Paschoal Popolin4 Aylana de Souza Belchior5 Mellina Yamamura6 Danielle Talita dos Santos7 Luiz Henrique Arroyo8 Ricardo Alexandre Arcêncio9 Objectives: To describe the epidemiological profile of mortality due to tuberculosis (TB), to analyze the spatial pattern of these deaths and to investigate the temporal trend in mortality due to tuberculosis in Northeast Brazil. Methods: An ecological study based on secondary mortality data. Deaths due to TB were included in the study. Descriptive statistics were calculated and gross mortality rates were estimated and smoothed by the Local Empirical Bayesian Method. Prais- Winsten’s regression was used to analyze the temporal trend in the TB mortality coefficients. The Kernel density technique was used to analyze the spatial distribution of TB mortality. Results: Tuberculosis was implicated in 236 deaths. The burden of tuberculosis deaths was higher amongst males, single people and people of mixed ethnicity, and the mean age at death was 51 years. TB deaths were clustered in the East, West and North health districts, and the tuberculosis mortality coefficient remained stable throughout the study period. Conclusions: Analyses of the spatial pattern and temporal trend in mortality revealed that certain areas have higher TB mortality rates, and should therefore be prioritized in public health interventions targeting the disease. Descriptors: Tuberculosis; Mortality; Spatial Analysis; Time Series Studies; Health Information Systems; Geographic Information Systems. Original Article Rev. Latino-Am. Enfermagem 2018;26:e2992 DOI: 10.1590/1518-8345.2049.2992 www.eerp.usp.br/rlae Spatial pattern and temporal trend of mortality due to tuberculosis10 Original Article Rev. Latino-Am. Enfermagem 2018;26:e2992 DOI: 10.1590/1518-8345.2049.2992 www.eerp.usp.br/rlae Spatial pattern and temporal trend of mortality due to tuberculosis10 Original Article Rev. Latino-Am. Enfermagem 2018;26:e2992 DOI: 10.1590/1518-8345.2049.2992 www.eerp.usp.br/rlae Original Article Original Article Rev. Latino-Am. Enfermagem 2018;26:e2992 DOI: 10.1590/1518-8345.2049.2992 www.eerp.usp.br/rlae Methods This was an ecological study(9). The study was carried out in Natal, the capital of the state of Rio Grande do Norte, in Northeast Brazil. The city is divided into 36 neighborhoods and one area of environmental reserve and healthcare is administered through five health districts: North I, North II, South, East and West(10) (Figure 1). The city has a Human Development Index (HDI) of 0.7 and a Social Exclusion Index (SEI) of about 0.6, a Poverty Index of 40.86% and Gini Index of 0.6(11). The justification for choosing Natal as the study context is that the Ministry of Health has made it a priority city in relation to TB control. In 2015, the incidence of TB in Natal was 37.1 cases per 100,000 inhabitants and the mortality rate was 2.6 deaths per 100,000 inhabitants according to local data from Municipal Health Secretary of the city. This was an ecological study(9). The study was carried out in Natal, the capital of the state of Rio Grande do Norte, in Northeast Brazil. The city is divided into 36 neighborhoods and one area of environmental reserve and healthcare is administered through five health districts: North I, North II, South, East and West(10) (Figure 1). The city has a Human Development Index (HDI) of 0.7 and a Social Exclusion Index (SEI) of about 0.6, a Poverty Index of 40.86% and Gini Index of 0.6(11). There are barriers to achieving these targets; namely the need for new, sustainable diagnostic technologies, new drugs that shorten the treatment time and healthcare systems structured in such a way that it can reach all populations affected by the disease. The justification for choosing Natal as the study context is that the Ministry of Health has made it a priority city in relation to TB control. In 2015, the incidence of TB in Natal was 37.1 cases per 100,000 inhabitants and the mortality rate was 2.6 deaths per 100,000 inhabitants according to local data from Municipal Health Secretary of the city. Several studies have assessed the factors associated with TB mortality, identifying factors related to the patient’s clinical condition such as multiple drug resistance (TB-MDR), infection with the human immunodeficiency virus (HIV), malnutrition, diabetes and its association with hepatitis. Gender is also factor, as mortality is higher in men(3). Introduction important professional in the development of actions to control TB in these places. Tuberculosis (TB) is a global public health problem and the main cause of death due to infectious disease(1). In Brazil, the incidence of TB was around 41.0 cases per 100,000 inhabitants in 2015, and the mortality rate was 2.4 deaths per 100,000 inhabitants. Thus, the study aimed to describe the epidemiological profile of mortality due to tuberculosis (TB), to analyze the spatial pattern of these deaths and to investigate the temporal trend in mortality due to tuberculosis in Northeast Brazil In the last two decades there has been a decline in the incidence of TB and the TB mortality rate, although the disease still influences the economy and the health systems. In 2014, in accordance with the Sustainable Development Goals, the World Health Organization (WHO) established the ‘End TB strategy’, whose target is to reduce TB mortality by 95.0% and TB incidence by 90.0% relative to the 2015 figures by 2035(1-2). How to cite this article Queiroz AAR, Berra TZ, Garcia MCC, Popolin MP, Belchior AS, Yamamura M et al. Spatial pattern and temporal trend of mortality due to tuberculosis. Rev. Latino-Am. Enfermagem. 2018;26:e2992. [Access ___ __ ____]; Available in: ____________________. DOI: http://dx.doi.org/10.1590/1518-8345.2049.2992 day month year Rev. Latino-Am. Enfermagem 2018;26:e2992. 2 www.eerp.usp.br/rlae Methods In addition, cultural factors come into play, for example the social stigma attached to the disease, which means that individuals avoid attending clinics for fear of receiving a TB diagnosis, and then search for alternative therapies(4). The efficiency and quality of healthcare delivery also influence TB mortality(5-6). The study population consisted of all cases of death due to TB for which an underlying and associated cause were registered in the Mortality Information System (MIS) between 2008 and 2014. The study data were collected from death certificates (DC), where the underlying or associated cause of death was any clinical form of TB described according to the International Classification of Diseases version 10 (ICD10) codes A15.0 A19.0, and the death was registered in the MIS of the Natal Municipal Health Department-RN. The Epidemiological Surveillance Division of the Natal Municipal Health Department-RN provided the data. After preliminary contact with the city’s MIS coordinator, a date and time at which the data would be collected was scheduled. The data were collected in March 2015. Due to the available diagnostic and treatment technologies, TB deaths can be considered an unfair event, which highlights the need to reduce its occurrence. Thus, in order to reduce the mortality rate due to TB in Brazil, social as well as political, human and economic changes are needed. Spatial and temporal patterns in TB mortality have been studied(7-8), but have attracted less research attention than TB incidence or prevalence. An important gap in the literature on TB mortality trends is that there is no published evidence on whether Brazil will meet the 2015 goal for the reduction in TB deaths, mainly in Northeast Brazil. The socio-demographic variables studied were: age, gender, ethnic background, marital status, education, and occupation. The operational variables were: place of death and medical care. Descriptive statistics were used to describe the profile of the patients for whom TB was listed as the underlying or associated cause of death. Relative or absolute frequencies were calculated for the categorical variables and position and dispersion measures for the continuous variables. TB mortality is an important issue in nursing, because TB nurses have to select appropriate methods for controlling TB and ensuring that TB patients complete therapy successfully. If vulnerable areas to TB deaths are evidenced in reports, TB nurses can act as an www.eerp.usp.br/rlae 3 Queiroz AAR, Berra TZ, Garcia MCC, Popolin MP, Belchior AS, Yamamura M et al. Methods Figure 1. Geographical location of the municipality under study and its division according to health districts and neighborhoods, Natal, RN, Brazil. Figure 1. Geographical location of the municipality under study and its division according to health districts and neighborhoods, Natal, RN, Brazil. regional rate of the closest neighbors for reference. This rate considered the population density and the local mean rate, starting from a spatial proximity matrix(14). Terraview version 4.2.2 was used to calculate the smoothed rates. Next, ArcGIS version 10.2 was used to produce distribution maps of the local empirical Bayes rates, grouped by quintile. The cases were geocoded using TerraView version 4.2.2 software to enable analysis of the spatial distribution of deaths. Next, the addresses of cases in urban Natal-RN were standardized and matched using a digital address segment map in Latlong/WGS84 projection. The units of analysis were city neighborhood and census sector. In addition, coefficients for mortality due to TB were expressed as logarithms in order to classify the temporal trend in the disease between 2008 and 2014 as downward, stationary or upward. The time series statistics in StataSE 13 were used for calculating this, applying the Prais-Winsten generalized linear regression method. This procedure corrects the first-order temporal autocorrelation in analyses of organized time series. The annual variance in the measure and its 95% confidence intervals (95%CI) were also calculated(15). First we carried out a point density analysis using the Kernel estimation method, an exploratory interpolation method based on defining circular areas of influence around the points where a phenomenon occurs, and then used these to produce a density map that identifies vulnerable areas(12-13). The density map provides an overview of the disease distribution and can be used to guide exploration of the point pattern of the health data. Therefore, a radius of 1,000 meters was considered. The density distribution maps of TB deaths were produced using ArcGIS 10.2 software. Approval for the study was obtained from the Research Ethics Committee at the University of São Paulo at Ribeirão Preto College of Nursing, under CAAE (Certificate of Presentation for Ethical Appreciation) 41398915.6.0000.5393. Next, the mortality rates due to TB were standardized in each neighborhood per gender and age range using the direct method(9) and considering Natal’s population as the standard. The age ranges chosen were based on the disease distribution in the study population: zero to 15 years, 16 to 59 years and 60 years or older. www.eerp.usp.br/rlae Results Annual TB mortality rates were smoothed using a local empirical Bayes model, with a view to minimize the instability caused by oscillations in small population and underreporting of TB deaths. As a result of applying this method, a weighted average was obtained between the gross rate of the neighborhoods and taking the During the study period there were 236 deaths in the study area, and TB was registered as the underlying cause in 154 (65.25%) of them. The minimum age at death was eight years and the maximum was 101 years; age was stratified by group (0 14 years; 15 59 years; ≥ 60 years). 4 Rev. Latino-Am. Enfermagem 2018;26:e2992. Rev. Latino-Am. Enfermagem 2018;26:e2992. Characteristics TB* mortality Underlying cause Associated cause n (%) n (%) Place of death (n=235) Hospital 127 83.00 76 92.70 Other health services 2 1.30 0 0.00 Home 20 13.05 5 6.10 Public road 1 0.65 1 1.20 Others 3 2.00 0 0.00 Medical care (n=163) Yes 90 87.40 56 93.30 No 13 12.60 4 6.70 * TB - Tuberculosis Table 1 - (continuation) The predominant clinical form of TB was pulmonary tuberculosis, without bacteriological or histological confirmation (ICD-10 16.2), specified in 130 cases where TB was the underlying cause of death (84.41%) and 66 cases where it was an associated cause of death (80.49%). In cases where TB was an associated cause of death, the most common underlying cause was infectious and parasitic diseases (n = 51, 62.19%), and most often HIV. Table 1 presents a comparison of the sociodemographic profiles of people for whom TB was listed as the underlying cause of death and those for whom another underlying cause of death was listed. Table 1. Distribution of sociodemographic and clinical characteristics according to the cause of TB mortality. Natal, RN, Brazil, 2015. Taking the sample as a whole, 223 (94.50%) deaths were geocoded, and 215 of which were identified in the cartographic database and processed in TerraView version 4.2.2; the remaining 8 cases were processed using the Batch Geocode tool. Losses in the geocoding process were due to inconsistent addresses in the MIS. Results Characteristics TB* mortality Underlying cause Associated cause n (%) n (%) Age in years (n=236) 014 0 0.00 1 1.20 1559 94 61.03 61 74.40 ≥ 60 60 38.97 20 24.40 Gender (n=236) Male 115 74.70 56 68.30 Female 39 25.30 26 31.70 Ethnicity (n=208) Mixed 78 58.20 47 63.50 White 45 33.60 23 31.10 Black 11 8.20 4 5.40 Marital status (n=221) Single 81 55.90 44 57.90 Married 47 32.40 23 30.30 Widowed 13 9.00 5 6.60 Divorced 3 2.00 3 3.90 Fixed partner 1 0.70 1 1.30 Education (n=159) No education 8 7.60 0 0.00 Unfinished primary education (1 – 3 years) 28 26.70 4 7.40 Finished primary education (4 – 7 years) 32 30.50 17 31.50 Secondary education (8 – 11 years) 22 20.90 17 31.50 Unfinished higher Education (12 – 14 years) 10 9.50 13 24.00 Finished higher education (Over 15 years) 5 4.80 3 5.60 (continue...) Figures 2 and 3 respectively represent density maps for deaths in Natal, RN, for which TB was listed as an underlying or associated cause. Regarding the standardized rates of death per neighborhood, the annual average mortality rate of TB mortality in Natal during the seven-year study period was 2.74 cases per 100,000 inhabitants, with a higher rate in the Areia Preta neighborhood (16.71 cases/100,000 inhabitants). After correction by the local empirical Bayes method, the neighborhood with the highest rate was Praia do Meio (8.53 cases/100,000 inhabitants). Both Areia Preta and Praia do Meio are in the Eastern health district of Natal. Based on the standardized rates for mortality with TB as an associated cause, the neighborhood with the highest coefficient was Areia Preta (7.30 cases/100,000 inhabitants). After correction by the local empirical Bayes method, the highest rates were found in the Mãe Luiza neighborhood (4.19 cases/100,000 inhabitants); once again, both these neighborhoods were located in the Eastern health district. Figure 4 displays the spatial distribution of the annual local empirical Bayesian rates of mortality with TB as an underlying or associated cause. During the study period, the gross TB mortality rate (underlying and associated cause) in the city of Natal was 5.25 per 100,000 inhabitants in 2008, and 4.00 per 100,000 inhabitants in 2014. The coefficient for mortality due to TB (underlying or associated cause) remained stable during the study period, with annual variation of -2.2% (CI95% -4.8%; -0.3%). Discussion level factors associated with increased incidence of TB and with lower treatment compliance, and therefore they may also be related to access to health services and the quality of diagnosis. People with less education and lower incomes are less likely to perceive that they are at risk and to comply with the treatment, because they present individual and unequal access to information, to benefits deriving from knowledge, to consumer goods and mainly to health services(16). The study aimed to describe the epidemiological profile of TB mortality, to analyze the spatial pattern of these deaths and to investigate the temporal trend in TB mortality in Northeast Brazil. The study showed that in most of the cases where TB was listed as the cause of death, the deceased was male, between 15 and 59 years of age, single and of mixed ethnicity. In spatial terms, deaths from TB were concentrated in the West, North and East health districts. The TB mortality rate remained stable throughout the study period, with annual variation of -2.2% (CI95% -4.8%; -0.3%). Moreover, regarding the age of TB mortality as a basic and associated cause, the disease affected patients who were in the economically active age range (15 to 59 years), a finding which is in line with another Brazilian study(17). This is an important issue, since TB mortality affects economic and social development at a regional level, and is both a cause and consequence of poverty(18). Regarding an associate cause of TB mortality, AIDS cases in Brazil are higher in individuals between 25 and 39 years of age for men and women(16). Furthermore, an earlier study showed a larger number of cases of TB/ HIV co-infection among individuals between 30 and 50 years of age. With regard to the epidemiological profile, the results do not differ from those of other studies of TB mortality in Brazil and around the world(6). The gender distribution of mortality (a higher proportion of deaths in males) is similar to the incidence pattern of the disease, with higher morbidity among men(6) and people of mixed ethnicity(3). This may be due to the relationship between biological and social factors, or to gender differences in exposure factors and prevalence of infection with evolution to disease, amongst other issues related to access to health services. Results www.eerp.usp.br/rlae 5 Queiroz AAR, Berra TZ, Garcia MCC, Popolin MP, Belchior AS, Yamamura M et al. Figure 2. Density distribution of deaths for which tuberculosis was listed as the underlying cause, Natal, RN, Brazil, 2015 nsity distribution of deaths for which tuberculosis was listed as the underlying cause, Natal, RN, Brazil, Figure 2. Density distribution of deaths for which tuberculosis was listed as the underlying cause, Natal, RN, Brazil, 2015 Figure 2. Density distribution of deaths for which tuberculosis was listed as the underlying cause, Natal, RN, Brazil, 2015 Figure 3. Density distribution of deaths for which tuberculosis was listed as an associated cause, Natal, RN, Brazil, 2015 Figure 3. Density distribution of deaths for which tuberculosis was listed as an associated cause, Natal, RN, Brazil, Figure 3. Density distribution of deaths for which tuberculosis was listed as an associated cause, Natal, RN, Brazil, 2015 www.eerp.usp.br/rlae 6 Rev. Latino-Am. Enfermagem 2018;26:e2992. 6 Figure 4. Distribution of annual local empirical Bayesian rates of mortality with tuberculosis as the underlying cause (A) or an associated cause (B). Figures are deaths per 100,000 inhabitants per year for neighborhoods of Natal, RN, Brazil, 2015. Figure 4. Distribution of annual local empirical Bayesian rates of mortality with tuberculosis as the underlying cause (A) or an associated cause (B). Figures are deaths per 100,000 inhabitants per year for neighborhoods of Natal, RN, Brazil, 2015. www.eerp.usp.br/rlae Discussion Another aspect verified in the study was the mortality due to the association between TB and HIV. TB is the most frequent opportunistic disease in HIV patients, and several studies have demonstrated that it is also one of the main causes associated with death in that population. A study carried out in Africa(19) reported that 47.8% of all deaths investigated were related to TB/HIV co-infection, which is consistent with global statistics showing that TB is the cause of death in one out of three Acquired Immune Deficiency Syndrome (AIDS) patients. The neighborhoods in the West and North health districts where TB mortality was highest coincided with the regions of the city with the worst social indicators and are areas where incomes are generally low, typically areas on the outskirts of the city. It has been shown that characteristics related to treatment history such as treatment abandonment, multiple drug resistance and TB/HIV co-infection are associated with the death of TB cases( 20). The municipality has organized its health care services by heath district, and for each health district there are enough numbers of services to meet the main needs of the population. The North health district is highly populated, representing 37.77% of the population of municipality and concentrating 40% of the slums and population living on a monthly income below the minimum wage(25); it has coverage by the Family Health Strategy (FHS) of about 77.00% of the population in North I, and North II has 63.00% of the population covered by the FHS(10). In contrast, death from TB may be considered an unfair and avoidable event, as the UHS has all the resources required to diagnose and treat patients, and treatment is freely and universally available(21). An important issue is whether all Brazilian populations affected by TB have access to care; some groups, mostly vulnerable groups (homeless people, prisoners, drugs users and the unemployed, amongst others) still face many barriers to care? (22). The West health district is classified as the poorest based on family income data, and it is also the second most populous and has the highest density of TB cases(25). In addition, it has the highest number of people per household, concentrating the largest number of subnormal clusters, the second highest percentage of slums(25) and 69.00% of the population have FHS coverage(10). Discussion As far as the operational profile is concerned, the largest proportion of deaths happened in hospitals to patients who had been receiving medical care before death. This indicates the weakness of Primary Health Care (PHC) in terms of problem-solving and ability to The study showed that most of the cases had a low level of education. Other authors have reported that low education, unemployment and income are individual- 7 Queiroz AAR, Berra TZ, Garcia MCC, Popolin MP, Belchior AS, Yamamura M et al. Queiroz AAR, Berra TZ, Garcia MCC, Popolin MP, Belchior AS, Yamamura M et al. respond to the needs of TB patients, as this healthcare level should serve as the point of entry to healthcare services, giving patients the opportunity to receive an early diagnosis, and hence a better prognosis. study, it could be proven that the space was relevant in investigating and understanding the occurrence and distribution of mortality in the city, as it is the environment where the infectious agent circulates, and which, under specific conditions, provokes the disease and even death as a result(24). The most frequent clinical form of TB in our sample was pulmonary TB, without bacteriological or histological confirmation; this is in line with WHO estimates reporting that the mean occurrence of pulmonary TB is about 85%(1). The general lack of bacteriological or histological confirmation raises concerns about the reliability of TB diagnoses; bacteriological tests, such as sputum smears and cultures available in the public network of the Brazilian Unified Health System (UHS) possibly in association with bronchoscopy or biopsy, should be used to confirm TB diagnoses(6). The spatial distribution of cases indicated that TB mortality (TB as the underlying or associated cause of death) was most frequent in certain neighborhoods of the East and West health districts. There were fewer TB- related deaths in the South region. Inspection of the density distribution maps of the produced points revealed that TB-related deaths were unevenly distributed across the city, with ‘hot spots’ in the East, West and Northern health districts. The more intense point clusters in the North shown in Figures 2 and 3 can be explained by the fact that the kernel density analysis is based on the counting of points per km2 within influential circulation areas, weighted by the distance of each from the location of interest, without considering the population of the areas(12-13). www.eerp.usp.br/rlae Discussion Considering the Universal health system has been adopted in Brazil under a social right perspective and equality, it would be mandatory to provide actions or TB care according to the needs of the population. Health equality is an important index to verify when each individual has a fair opportunity to achieve his or her full health potential(23). When differential mortality can be linked to differences in social conditions, it is clear that health equality has not been achieved(21). TB mortality in Brazil is more seriously affected by social inequality than by the availability of medical technology for diagnosis and treatment(1). The East health district is part of a region with better social indicators, although some neighborhoods (Rocas, Praia do Meio, Santos Reis and Mãe Luiza) have social indices comparable to those typical of the North and West, and have the highest incidence and TB mortality of anywhere in the city(10). In addition, a feature of the East regions is the social inequality, as areas of low social vulnerability sit very close to areas The spatial distribution of TB is affected by socioeconomic inequalities across the study area. In this Rev. Latino-Am. Enfermagem 2018;26:e2992. Rev. Latino-Am. Enfermagem 2018;26:e2992. 8 8 the data collection period could be a potential limitation of the study (March 2015), since the total number of TB deaths occurred by 2014 may still not have been up-to- date, which could influence the findings, mainly in the analysis of temporal series. of high social vulnerability(25). About 37.00% of the population has FHS coverage in the East health district, which may represent weakness in terms of TB care either for diagnosis or treatment(10). These data show that TB mortality mainly affects the health districts with the worst HDI values, indicating that socioeconomic factors play an important role on the impact of TB. Conclusion This study contributes to knowledge on TB mortality by describing the progress Brazil has made towards achieving the ‘End TB’ targets. Thus, the results of the study through the Geographic Information System technologies evidence relevant aspects to Nursing in terms of care planning and implementation, mainly in the areas with higher death rates. These results may contribute to improve the quality of care by nurses in the Primary Health Care. In addition, the results of the study may not only be relevant to local management, but also to other contexts with a similar epidemiological profile with respect to TB. There is a negative association between HDI and the impact of TB, and the spatial distribution of the disease is affected by multiple factors including the territorial extent, disordered population growth and the concentration of people on the outskirts. In this sense, the geographical space reveals the symbolic dimension of the social relationships, in which the factors associated with the development and dissemination of diseases are expressed, as well as its distribution among the different social groups(20). There is a global downward trend in TB mortality. The number of cases in Brazil has dropped by 2% per year on average over the last ten years(24), which suggests that mortality rates in Brazil follow the WHO proposal concerning the priorities in terms of early case detection, patient treatment and its conclusion with cure as the outcome(2). www.eerp.usp.br/rlae References Available from: http://journals.plos.org/plosone/ article?id=10.1371/journal.pone.0116673 doi http:// dx.doi.org/10.1371/journal.pone.0116673. in a cohort of cases and a research proposal of presumed causes. Cad Saúde Pública. [Internet] 2015;31(4):709-21. [cited Feb 7, 2017]. Available from: http://www.scielo.br/scielo.php?script=sci_ arttext&pid=S0102-311X2015000400709 doi http:// dx.doi.org/10.1590/0102-311X00101214. 14. Magalhães MSFM, Medronho RA. 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The contribution of spatial analysis to understanding HIV/TB mortality in children: a structural equation modelling approach. Glob Health Action. [Internet] 2013 [cited Feb 7, 2017];6. Available from: http://www.globalhealthaction.net/index.php/gha/ article/view/19266 doi http://dx.doi.org/10.3402/gha. v6i0.19266. 8. Musenge E, Vounatsou P, Collinson M, Tollman S, Kahn K. The contribution of spatial analysis to understanding HIV/TB mortality in children: a structural equation modelling approach. Glob Health Action. [Internet] 2013 [cited Feb 7, 2017];6. Available from: http://www.globalhealthaction.net/index.php/gha/ article/view/19266 doi http://dx.doi.org/10.3402/gha. v6i0.19266. 8. Musenge E, Vounatsou P, Collinson M, Tollman S, Kahn K. The contribution of spatial analysis to understanding HIV/TB mortality in children: a structural equation modelling approach. Glob Health Action. [Internet] 2013 [cited Feb 7, 2017];6. Available from: http://www.globalhealthaction.net/index.php/gha/ article/view/19266 doi http://dx.doi.org/10.3402/gha. v6i0.19266. 17. Zenebe Y, Adem Y, Mekonnen D, Derbie A, Bereded F, Bantie M et al. Profile of tuberculosis and its response to anti-TB drugs among tuberculosis patients treated under the TB control programme at Felege-Hiwot Referral Hos pital, Ethiopia. www.eerp.usp.br/rlae References BMC Public Health. [Internet]. 2016 [cited Oct 10, 2017]; 16: 688. Available from: https://bmcpub lichealth.biomedcentral.com/articles/10.1186/s12889- 016-3362-9 doi https://doi.org/10.1186/s12889-016- 3362-9 9. Rothman KJ, Greenland S, Lash T L. Modern Epidemiology. 3nd ed. Philadelphia: Lippincott Williams & Wilkins; 2009. 9. Rothman KJ, Greenland S, Lash T L. Modern Epidemiology. 3nd ed. Philadelphia: Lippincott Williams & Wilkins; 2009. 10. Secretaria Municipal de Saúde da Prefeitura Municipal do Natal. Carteira de serviços da Atenção Básica de Natal. Natal- RN, 2014. 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[Internet]. 2014 Feb [cited May 3, 2017];40(1):61-8. Available from: http://www.scielo.br/scielo.php?pid=S1806- 37132014000100061&script=sci_arttext&tlng=pt doi http://dx.doi.org/10.1590/S1806-37132014000100009 19. Lindoso AABP, Waldman EA, Komatsu NK, Figueiredo SM, Taniguchi M, Rodrigues LC. Profile of tuberculosis patients progressing to death, city of São Paulo, Brazil, 2002. Rev Saúde Pública. [Internet]. 2008;42(5):805-12. [cited Feb 7, 2017]. Available from: http://www.scielo.br/scielo.php?script=sci_artte xt&pid=S0034-89102008000500004 doi http://dx.doi. org/10.1590/S0034-89102008000500004. 20 Pinto ML Silva TC Gomes LCF Bertolozzi MR 11. Programa das Nações Unidas para o Desenvolvimento (PNUD): Atlas do Desenvolvimento Humano no Brasil. PNUD; 2013. [Acesso 7 dez 2016]. Disponível em: http://www.atlasbrasil.org.br/2013/. 12. References 1. World Health Organization. Global Tuberculosis Report 2015: Geneva. [Internet]. World Health Organization; 2015. [cited Feb 7, 2017]. Available from: http://apps.who. int/iris/bitstream/10665/191102/1/9789241565059_ eng.pdf According to the WHO’s Global Tuberculosis Report(1), Brazil reached all the TB-related millennium development goals (stop and reverse the upward trend in TB incidence coefficient by 2015), as well as the ‘Stop TB Partnership’ target of reducing the prevalence of TB and TB mortality by 50% relative to the 1990 figures by 2015. 2. Uplekar M, Weil D, Lonnroth K, Jaramillo E, Lienhardt C, Dias HM et al. WHO’s Global. TB Programme. WHO’s new end TB strategy. Lancet. [Internet] 2015 [cited Feb 7, 2017];385(9979):1799-801. Available from: http://www.sciencedirect.com/science/article/pii/ S0140673615605700 doi http://dx.doi.org/10.1016/ S0140-6736(15)60570-0. However, in order to achieve the WHO goal of reducing TB deaths by 75% by 2025 and 95% by 2035 through the ‘End TB strategy’, the incidence rate would have to drop by 10% per year for the next 20 years(24). 3. Santos-Neto M, Yamamura M, Garcia MCC, Popolin MP, Silveira TRS, Arcêncio RA. Spatial analysis of deaths from pulmonary tuberculosis in the city of São Luís, Brazil. J Bras Pneumol. [Internet]. 2014;40(5):543-51. [cited Feb 7, 2017]. Available from: http://www.scielo.br/scielo. php?pid=S1806-37132014000500543&script=sci_ arttext&tlng=pt doi http://dx.doi.org/10.1590/S1806- 37132014000500011. Various strategies have been proposed to improve Brazilian TB patients’ access to healthcare, and it has been suggested that healthcare decentralization is the best way of achieving health equality and equal access to care for all social strata. Thus, the literature has shown FHS advances in relation to qualification of the system, the regional specificity of FHS policies and programs that represent a way of tackling the fragmented nature of the healthcare system, but as yet it has not had an impact on disease rates(27). 4. Sudhinaraset M, Ingram M, Lofthouse HK, Montagu D. What Is the Role of Informal Healthcare Providers in Developing Countries? A Systematic Review. Plos One. [Internet] 2013 [cited Feb 7, 2017];8(2):e54978. Available from: http://journals.plos.org/plosone/ article?id=10.1371/journal.pone.0054978 doi http:// dx.doi.org/10.1371/journal.pone.0054978. The limitations of this study include the use of secondary data derived from the MIS, as there are weaknesses in these data; for example, underreporting of TB and non-completion of some fields in the DC, which may bias the results of the analyses. Further studies are needed to determine the statistical relationship between TB mortality and social variables. The issue regarding www.eerp.usp.br/rlae 9 9 Queiroz AAR, Berra TZ, Garcia MCC, Popolin MP, Belchior AS, Yamamura M et al. Corresponding Author: Ana Angélica Rêgo de Queiroz University of São Paulo. Escola de Enfermagem de Ribeirão Preto Avenue Bandeirantes, 3900 CEP: 14049-900, Ribeirão Preto, SP, Brazil E-mail: aninha_arego@hotmail.com References Prado JJC, Virgilio TC, Medronho RA. Cure rates for tuberculosis in the municipality of Rio de Ja neiro, Brazil, in 2012 compared with coverage by, and time of establishment of, Family Health units, and socio-economic and demographic factors. Ciênc Saúde Coletiva [Internet]. 2016 [cited Oct 10, 2017]; 21(5):1491-98. Available from: http://www.sci elosp.org/scielo.php?script=sci_arttext&pid=S1413- 81232016000501491&lng=en doi http://dx.doi. org/10.1590/1413-81232015215.03912016. 20. Pinto ML, Silva TC, Gomes LCF, Bertolozzi MR, Villavicencio LMM, Azevedo KMF et al. Occurrence 10 Rev. Latino-Am. 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Available from: http://www. scielo.br/scielo.php?script=sci_arttext&pid=S0104- 12902016000100009&lng=pt&nrm=iso. doi http:// dx.doi.org/10.1590/S0104-12902016146195 24. Maciel ENL. Post-2015 agenda strategies for tu berculosis control in Brazil: challenges and oppor tunities. Copyright © 2018 Revista Latino-Americana de Enfermagem This is an Open Access article distributed under the terms of the Creative Commons (CC BY). This license lets others distribute, remix, tweak, and build upon your work, even commercially, as long as they credit you for the original creation. This is the most accommodating of licenses offered. Recommended for maximum dissemination and use of licensed materials. www.eerp.usp.br/rlae References Epidemiol Serv Saúde. [Internet] 2016 [cited Feb 7, 2017]; 25(2):423-6. Available from: http://www.scielo.br/scielo.php?script=sci_arttext& pid=S2237-96222016000200423 doi http://dx.doi. org/10.5123/s1679-49742016000200021. 25. Medeiros MD, Almeida LQ. 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https://openalex.org/W3094116449	https://scindeks-clanci.ceon.rs/data/pdf/0085-6320/2020/0085-63202003498T.pdf	Serbian	null	The coronavirus crisis as a world-historic event in the digital era	Sociološki pregled/Sociološki pregled	2,020	cc-by-sa	10,379	Charalambos Tsekeris1 616.9 National Centre for Social Research 31 Athens (Greece) Persefoni Zeri2 Panteion University of Social and Political Sciences Athens (Greece) Charalambos Tsekeris1 616.9 National Centre for Social Research 31 Athens (Greece) Persefoni Zeri2 Panteion University of Social and Political Sciences Athens (Greece) 616.98:578.834]:316.774/.776(100)”2020” 316.324.8:[616.98:578.834(100)”2020” Original scientific paper Submitted 27/07/2020 iences Revised 22/08/2020 Accepted 22/08/2020 doi: 10.5937/socpreg54-27687 616.98:578.834]:316.774/.776(100)”2020” 316.324.8:[616.98:578.834(100)”2020” Original scientific paper Submitted 27/07/2020 ences Revised 22/08/2020 Accepted 22/08/2020 doi: 10.5937/socpreg54-27687 THE CORONAVIRUS CRISIS AS A WORLD-HISTORIC EVENT IN THE DIGITAL ERA Abstract: The world-historic event of the COVID-19 pandemic has once more con- firmed that we live in a hyperconnected world society and that, nowadays, epidemics do not count as merely natural phenomena anymore. In such context, the present paper aims to interpret the complex relationship between the society and COVID-19, with emphasis on the role played by different forces in the field of information policy and public percep- tions in general. For this reason, we elaborate on cultural factors, as well as on emotions like responsibility, trust, and fear during the crisis. We also focus on the dynamics of con- temporary media in relation to public images of the pandemic, drawing upon relevant find- ings. Overall, this casts a fresh sociological and interdisciplinary light on the current pan- demic as a relational process and a digital media-driven phenomenon. Keywords: coronavirus crisis, digital media, trust, fear, Greece 1 tsekeris@ekke.gr 2 persazeri@hotmail.com 1 tsekeris@ekke.gr 2 persazeri@hotmail.com Introduction: State, society and the coronavirus crisis A foresight study on the scenario of a great pandemic coming from Asia was given to all members of the German Parliament in 2012 (Kiel, 2020). Interestingly, the study was not taken into account at all. Everybody ignored the weak signals. In the beginning of 2020, the coronavirus (or COVID-19) pandemic came unexpectedly in the West as a superspread- er-driven “black swan”, an extreme fat-tailed process (Taleb, 2020) owing to an increased global connectivity. It seems that all national and supra-national governments were unpre- pared for it, although governing in principle pertains to strategic reflection and anticipa- tion (Tufekci, 2020). Anticipation scenarios assume futures thinking and specific struc- tures for the implementation of emergency measures, based on clear responsibilities, risk assessment, and experts’ participation (Kiel, 2020). What happens now in the West is an open process of defining and containing the coro- navirus pandemic, in which science (epidemiology, infectology, immunology, virology), 498 Sociološki pregled / Sociological Review, vol. LIV (2020), nо. 3, pp. 498–517 under conditions of high uncertainty and unpredictability, plays a leading role, combined with a public debate on the perspectives over the medical treatment of the disease, some- thing that varies over time. In these public deliberations, many diverse actors participate in an active way, such as the printed and digital media, civil society, and other stakehold- ers. A positive role in this information pool about the pandemic is played by the global research network of universities and research institutions, which intensively cooperate on COVID-19 response. This constitutes a general knowledge base that flows in the process of the political decision making (Dianeosis, 2020). The popular belief in the active con- frontation and successful mastery of the crisis helps large-scale behaviour change, or cre- ates the readiness to comply with the required behavioral orientation (Dianeosis, 2020). p y q In such process, people express the readiness to take over the responsibility for them- selves and the others, thus following specific behavioural rules of hygienic conduct, such as handwashing, wearing a mask, and social distancing. In this regard, governments attempt to combine the responsibility shown by the people with the public adherence to the required rules, something that signifies the constitutive role the state plays for the protection of society. Introduction: State, society and the coronavirus crisis The public respect to containment measures (closing schools, banning large pub- lic gatherings and team events, and so on) demonstrates the unconditional acceptance of the political as the supreme responsible of the functioning of a society as a whole. Τhe civil society (or the family) cannot act alone in this situation; it needs the state. This fact becomes evident in the mobilisation of financing measures of immense proportions for the support of economy and society that were deeply affected by the crisis (Strauss-Kahn, 2020, p. 9; Rogoff, 2020). Both the immediate response of the majority of the European states to the crisis and the high approval by the public of the drastic measures has to do with the ultimate “value of the human life that has been considerably reinforced in the collective unconscious of the richest countries” (Strauss-Kahn, 2020, p. 8). Economy and other social activities must therefore sacrifice part of their dynamics in favour of the health system (Bae- cker, 2020a). In this historic occasion, we witnessed the “paradox” for democratic societ- ies that collectivity is inscribed in the service of a simply defensive cause (Gauchet, 2020c). h d l d d d l h f f The coronavirus containment and social distancing measures indicate a radical shift of emphasis from a culture of freedom and self-determination to a culture of the vulnerability of the protection (Bude, 2020) and of collective discipline. This change indicates a weaken- ing of the dominant idea of a strong “I”, of the “identitarian person” (Ladeur, 2007, p. 397), who negates the internalisation (or indoctrination) of social constraints and is focused on itself and its allegedly “authentic” self-presentation, away from the common reality. Instead, we now witness the prevalence of the idea of a weak and vulnerable “I” that needs the state protection. The answer given by the public authorities in Greece, like in the most coun- tries, particularly around the confinement and lockdown measures, has been accepted and respected by the vast majority of the population (Dianeosis, 2020; Papamattheou, 2020). During the confinement period, citizens could reflexively observe the emergence of the perception of a new, effective “We” (Coyle, 2020), bound to an idea of personal pro-so- cial development and broader societal integration and cohesion. This also has to do with the set back of the political polarisation, which is endemic in the Greek political life (Zeri et al., 2019). Introduction: State, society and the coronavirus crisis But is this a dynamical set back? Of course, no one can easily predict whether a revival of the poisonous political climate will take place after the coronavirus crisis, espe- cially in the context of an economic crisis that will probably follow, with unknown extent 499 haralambos Tsekeris, Persefoni Zeri, The coronavirus crisis as a world-historic event in... (European Commission, 2020). In any case, the task of politics, in view of the scientific uncertainties concerning the discovery of a vaccine or effective drugs, is to develop a capac- ity of collective action and to inspire society with a strong belief in the successful collec- tive response to the crisis. The government must also discern and employ communication strategies that strike a sensitive “balance between breaking through optimism bias with- out inducing excessive feelings of anxiety and dread” (Bavel et al., 2020, p. 461). In addi- tion, leadership must coordinate individuals and “help them avoid behaviours that are no longer considered socially responsible” (Bavel et al., 2020, p. 465). The state interference has neither to do with a technocratic, naïve subordination to science, nor with an authoritarian readiness to comply. There was a feeling among citi- zens that we need the democratic state that represents all of us and has sanction powers over the individuals for the common good (Bude, 2020). Public protection is one of the central aims of democratic politics, especially in the strategic effort to express a compel- ling public interest (Gostin & Wiley, 2020) and to make people survive this world-historic phenomenon of the coronavirus with unity and solidarity (Visvizi and Lytras, 2020), as well as with our humanity intact. Interestingly, the Dianeosis (2020) survey results con- firm the image Greeks have about a state that gives priority to public health, as well as to the knowledge and responsibility of the experts. At least temporarily during the confine- ment, we witnessed the spontaneous emergence of a mechanism of solidarity and mutual care (Fanaras, 2020, p. 17) that was not enforced in a top-down way, but based on the feel- ing that, after all, we do need the state. We also witnessed an atmosphere of reserved solidarity (or an idiosyncratic mixture of solidarity and suspicion), whose nature has taken a new dimension; that is, we protect the others not only by helping them, but also through “keeping distance”, so that they will not be infected. Introduction: State, society and the coronavirus crisis The consciousness of a relatively class-neutral vulnerability has stimulated the search for new solidarity forms. Interestingly, 66.8% of Greeks believe that the coronavirus crisis has a positive impact on the solidarity to the Other (Georgakopoulos, 2020, p. 10). However, it is still early to draw final and decisive conclusions about a learning process that refers to the consolidation of new types of solidarity. Recent images of mass demonstra- tions and social gatherings in public spaces, while the rules of social distancing are still in force, may be indicative of a lack of civic mindedness and, perhaps, of a cultural incapac- ity to remember. We now proceed to elaborate on the role played by different forces, such as emotions, culture and the media, in the field of information policy, public perceptions on COVID-19, and the pandemic society in general. Responsibility and trust during the pandemic In the period of implementation of the “stay-at-home” orders and total confinement, the overload of public information (flooded with guidelines managed by the Greek state and broadcasted by television and radio) helped people realize that they are responsible not only for themselves, but also for the others. During the two months of quarantine, social members have learnt to comply to the mandatory rules. The severity of the crisis was made clear after having experienced the media images from Italy and USA showing convoys car- rying coffins of coronavirus victims out of Bergamo or New York and the overwhelmed health-care systems. Given that the pandemic is most dangerous for the elderly and that, 500 Sociološki pregled / Sociological Review, vol. LIV (2020), nо. 3, pp. 498–517 within the Greek family, the grandparents play a vital role (Dianeosis, 2020), there was almost absolute respect to the lockdown measures. The loosening of the confinement measures in May 2020 was connected to strict pre- conditions that obliged people to abide by restrictive measures. But youngsters and the more vulnerable old people have suddenly come to mass gatherings in parks and places by the sea, thus massively breaking the social distancing rules. They acted as if they have erased from their memory the risks of a new COVID-19 outbreak, as well as the media warnings by the highly respected Professor Sotiris Tsiodras, the immunology experts group leader and government’s communications liaison for the health crisis (Kathimerini, 2020). That is, a sudden turn from a collectively disciplined people to a public interest-defying people. It is hereby evident the fragility of the fear-driven unity and cohesion demonstrated in the confinement period, but also the deep desire for normalcy. In the European consciousness, peace and prosperity, combined with high economic performance, have downplayed strategic futures/foresight thinking and diluted the antic- ipation of threat and the fundamental dimension of the political existence. The emphasis on individual rights and material resources has become the only conceivable horizon, thus creating an ill-founded feeling of security (Gauchet, 2020a). Besides, there has been some diversity in people’s attitudes among different European countries regarding the confine- ment measures against the coronavirus. The Catholic countries, such as Italy and France, stand out with a combination of private anarchy and public authoritarianism, while the protestant countries rather rest on a sense of personal responsibility (Gauchet, 2020a). Responsibility and trust during the pandemic In Greece, in particular, we have neither the legacy of the Confucian discipline, nor can we refer back to a heritage of modernity and a deeply enshrined consciousness of per- sonal responsibility. Practicing personal responsibility does not belong to the self-instituting political community “texture” of the Greek society, which is full of deeply rooted pre-mod- ern cultural patterns (Demertzis, 1997, p. 109; Zeri et al., 2019, p. 8). In some way, the Greek individual subject is caged in an unreflected “entrenched self” (Ramfos, 2011) and bears the social and psychological prescriptions as well as the moral assumptions of the family mind- set and strategy embedded in wider networks of interest groups (Pelagidis, 2012). Never- theless, a value change in the post-crisis era is certainly possible and worthy of analysis. Confronting a massive health crisis, such as the COVID-19 pandemic, which pertains to a total social fact (Kasuga, 2020), Greeks fall back on the state for protection. The myriad media images of death have generated a psychological shock that could be soothed only in the pursuit of unity and cohesion. The unity of the people constructs itself around the state and its representatives as the condition of being together, not in the context of Europe or other supranational organisations. However, it is noteworthy that the attitudes of Greeks towards their political institutions have been rather negative (Koniordos, 2018). The suc- cessful management by the Mitsotakis government of the Evros crisis (January 2020) and of the coronavirus crisis (March-April 2020) signified a remarkable change in the image Greeks have about their political institutions, especially about their government. In comparison to the previous Dianeosis surveys, the coronavirus crisis survey of Dia- neosis in April 2020 has showed a conspicuously higher level of people’s trust towards the state institutions, followed by a strengthening of their feelings of optimism, security, and certainty at personal level (Dianeosis, 2020). Also, the state measures taken during the pan- demic for the improvement of the public health care system have ameliorated the trust 501 Charalambos Tsekeris, Persefoni Zeri, The coronavirus crisis as a world-historic event in... towards the state itself (see Kounias, 2020). Notably, trust to (medical) science is at the high- est point of the scale. Fear in the coronavirus era The 9/11/2001 terrorist attacks in New York signified an age of fear. In the course of time, feelings of fear have been variously linked to Islamist terrorism, to the threatening climate change, and now to the massive global health crisis that seriously affects all of us (Bolz, 2020). In the contemporary affluent society, many people perceive the COVID-19 pandemic as an unprecedented fight for survival. Until now, the state was put at the ser- vice of economy, promoting the free circulation of capital, people and goods. After the global spread of the life-threatening coronavirus, the state has slowed down the econ- omy because the human lives were in danger. This is a confirmation of the respect of the humanist dimension claimed by modernity. It is not salvation, in the sense of a religious redemption (Finkielkraut, 2020a), but health that remains from the beginning of the mod- ern times “the first good and the foundation of all the other goods of this life” (Descartes, 1953 [1637], p. 168). In Alain Finkielkraut’s words, we must be “grateful to the politicians for whom human life is more important than money. They have set the right priorities, because they want to save the most weak and vulnerable members of society. […] As long as the life of an old man is only worth as much as the life of a man in full possession of his physical strength, we remain civilized” (Finkielkraut, 2020b). However, within a democratic community, especially during periods of social calm, people give priority to their individual freedoms. Yet, the collective machinery at normal times makes us forget that we are a political community (Gauchet, 2020b). In view of an acute crisis and a collective threat, fear and a sense of general vulnerability energise peo- ple’s capacity to act as citizens who owe their protected existence to their political commu- nity, while the priority of their personal freedoms is relatively reduced. In the same time, unlike the dictatorial or authoritarian regimes, people (internet users) have the freedom to discuss in public and reflect on the motives of the public actions. According to Dianeosis research results (Fanaras, 2020, p. 6), the Greek government’s successful management of the coronavirus crisis has kept the negative feelings of fear and anxiety within limits. The confinement of the whole society for more than six weeks had been surprisingly confronted with patience and creativity. Responsibility and trust during the pandemic According to the corona survey of the Economic University of Athens, 50% of the respondents have trust in the doctors, 88% in the health workers, and 57% in the state, although they have low trust in teachers (45%) and private companies (24%), while 75% believe that the confinement measures were effective (Papamattheou, 2020). Interest- ingly, political trust, clear messages, transparent communication and outreach on all mat- ters at stake is crucial to spur behavioural change (Bavel et al., 2020, p. 466). Moreover, the political management and mastery of the post-COVID-19 economic recession will arguably be a decisive factor for the development of generalised trust in Greek politics. Media and the coronavirus crisis The coronavirus pandemic has made evident the high complexity of the global system and its increased non-linear dynamics (Tufekci, 2020), given that the structural form of the world society (Baecker, 2020b) and the national societies pertains to relational networks which variously differentiate and reproduce themselves (Baecker, 2018, p. 61). In particular, the health crisis we are now experiencing is quite different from what we know from the past, precisely for the reason that every economy nowadays is globally integrated in a wider global context, where China constitutes the “factory of the world” (Strauss-Kahn, 2020, p. 19) and the mem- ory of an infectious pandemic risk is lost. In this analytic setting, it is of crucial importance to elaborate on the fact that, within the network society in which we live, the media, as a form of collective reflection and orientation, have taken the place of politics, economy and religion (Baecker, 2018, p. 192). For this reason, varying degrees of quality information access can greatly influence health perceptions, behaviours, decisions and outcomes in pandemic con- texts, where online disinformation may lead to death and disease in the real world (McGinty & Gyenes, 2020, p. 4). The latter has led the Greek government (in line to other EU govern- ments) to set up a webpage devoted to deconstruct the myths about COVID-19 (covid19.gov. gr/mythoi-covid19/), as part of the official COVID-19 observatory portal (covid19.gov.gr). Moreover, the media are framed in different historical, cultural and institutional con- texts of current societies, which relationally shape their technological systems and patterns of transmission in the era of globally networked communications. This points to the fun- damental role that the printed and digital media play in the information policy of national governments, as well as in the public perception of the pandemic (see e.g. Casero-Ripolles, 2020). According to a recent survey of the University of Chicago (Bertrand et. al, 2020), Americans see the pandemic not through the lens of health and economics, but through the lens of their political beliefs, their opinion on the proper role of government, and their trust in media under the condition of a politically divided society. The trust in media is quite crucial for the assessment of and the response to the pandemic (see Merrefield, 2020). Fear in the coronavirus era Almost similar results were pro- duced by the Coronavirus Disruption Project (Center for the Digital Future, 2020), accord- ing to which Americans, during the pandemic, were enjoying more time with their partners and children, rediscovering hobbies and creative activities. Notably, the corona survey of the Economic University of Athens (Papamattheou, 2020) refers to high levels of fear and anxiety about economy (92%) and less about health (82%). 502 Sociološki pregled / Sociological Review, vol. LIV (2020), nо. 3, pp. 498–517 Media and the coronavirus crisis It is noteworthy that the majority of people do not assess the pandemic on the basis of what they have seen or heard during the public discussions, but according to whom they vote for, what media they follow, or whom television host they prefer (Bursztyn et al., 2020). According to a survey research conducted by the Pew Research Center, the ideolog- ical makeup and the party orientation of the cable network audiences influence the way they assess the coronavirus epidemic (Pew Research Center, 2020a). Although the Ameri- can public says the media have done well covering the crisis, deep partisan divisions about the media’s coronavirus coverage were evident not only between voters of Democrats and Republicans, but also among ideological segments within each party (Pew Research Cen- ter, 2020d). For instance, in a recent US survey (Jamieson & Albarracin, 2020), it was found that mainstream broadcast media use (e.g. NBC News) correlated with accurate informa- tion about the disease’s lethality, and mainstream print media use (e.g. New York Times) correlated with accurate beliefs about protection from infection, while conservative media use (e.g. Fox News) correlated with conspiracy theories. Nowadays, electronic and digital news media are flooded with negative, sensational- ist and misinformation-type news stories about the pandemic. In the mass media, it is well known how journalists turn anything into bad news, because they aim at a mass audience 503 Charalambos Tsekeris, Persefoni Zeri, The coronavirus crisis as a world-historic event in... affected in principle by negative news (Tierney, 2020). Negativity biases and negative infor- mation are one of the most basic psychological principles (Baumeister, 2020). In addition, they constitute a key fact about how the human mind works. People have a propensity to give more weight to things that go wrong than to things that go right, so much that just one negative event can hijack our mind (Baumeister & Tierney, 2019). Negative information puts a kind of stump in the human mind, and it is not easy to remove it (Sunstein, 2020). Evidently, we unconsciously tend to pay attention to bad news (Hancock, 2020). As Gug- lielmo Briscese puts it, the more people are infected, the closer the pandemic gets, and yet people do not change their views (see Merrefield, 2020). Cognitive and negativity biases have great impact upon how we process news (McIn- tyre, 2020). Media and the coronavirus crisis Our brain’s confirmation bias drives us to seek out only information which is aligned with what we already believe and to discredit the rest. Biases like these can prevent us from learning from news about the pandemic and fight it effectively (McIntyre, 2020). But, in the pandemic society, people often live in personal emergency situations, which can overturn in a genuine world of delusion, and reflect the enormous fragility of the psy- che. This helps the flourishing of corona conspiracy theories across all segments of society (Bavel et al., 2020, p. 464ff.). In times of helplessness and lack of stability, because import- ant psychological needs are frustrated, conspiracy theories function as an instrument of personal crisis management (Douglas et al., 2017). Social media architecture somehow facilitates the flourishing and spread of false information and fake news about the corona- virus around the world, despite the coordinated efforts by some digital platform compa- nies (Fraenkel et al., 2020). In addition, Jan Philipp Reemtsma (2020) stresses that politi- cal thinking is overestimated and, actually, affects play a crucial role (see also Demertzis, 2020). People have rarely a political way of thinking (Reemtsma, 2020). The way in which Americans follow news sources for coronavirus news and perceive media’s pandemic coverage differ essentially from the way in which Greeks use information sources for coronavirus news and particularly the news media. Most Americans have paid equal attention to national and local COVID-19 news, whereby 56% national news outlets (including newspapers) are a major coronavirus news source, 51% the public health organ- isations and officials, 46% local news outlets, 31% Donald Trump and the White House coronavirus task force, 25% the international news outlets, 10% online forums and dis- cussion groups, 16% friends, family and neighbors (Pew Research Center, 2020b). Nearly nine-in-ten think that the internet is an essential or important tool during the coronavirus outbreak (Pew Research Center, 2020c). In Greece, the use of coronavirus news sources is accompanied by an increase in trust to the news media (Fanaras, 2020), something that gets in line with the aforementioned increase in the trust to the political institutions and espe- cially to the Greek government. For Greeks, the main coronavirus news source is televi- sion (48%), followed by internet sites (25.4%), health officials (9.1%), social media (7.5%), radio (3.7%), family and friends (2.9%), and newspapers (1.8%). Concluding remarks In some sense, COVID-19 is a digital pandemic in terms of its origin, dynamics and effect: “Governments around the world are resorting to digital instruments to combat the virus. Artificial intelligence and big data analysis play a valuable role herein” (Okano-Hei- jmans, 2020). The pandemic also sheds light on the intricate and intrinsic “connection among the government, citizens and data. This connection is powered by the ever more astounding advances in information and communication technology (ICT) and, even more so, by our ability to make sense of large sets of data” (Visvizi and Lytras, 2020, p. 125). All in all, this massive crisis could be attributed to historic dimensions, as long as his- tory is the product of collective work, or the uncertain outcome of deliberative action of social and political agents (Gauchet, 2016, p. 374). Eventually, it is a multifaceted relational process connected to other crises which emerge in other systems (economy, politics, media, culture, family, etc.), and brings about immense harms on the body, soul, and social life. These crises will deeply imprint themselves in the memory of every society, as well as in the psychic background of the social subjects. The traumatic experience of the pandemic has shown for the first time how dangerous can be a speedy, functionally differentiated and highly networked world society, where the virus spreads far and wide in just a few days or weeks and affects all geographical areas (Esposito, 2020a; see also Esposito 2020b). In this regard, the pandemic can be theorised as a man-made social catastrophe (Leggewie, 2020). g p p ( gg ) Nowadays, the hyperconnected world corresponds to a model of society produced by the new phase of globalisation, which is fundamentally characterised by the constant increase of exchanges and interactions between national digital and virtual systems, as well as by the rapid diffusion of information, knowledge, innovation and services (Bald- win, 2019). This model strives to adapt to turbulences and difficult contexts, such as the COVID-19 context. It is still too early to foresee whether the coronavirus crisis will gener- ate disruptive societal changes in the prospective reality of a serious economic crisis, or the tendency to return to normalcy will prevail. As soon as the virus turns its back, it is highly possible that the reign of the economists will give way to the government of the physicians (Gauchet, 2020b). Media and the coronavirus crisis Finally, a survey of the University of Athens (MediaanalysisGR, 2020) provides us with differentiated results, on the basis of a different sample of respondents, leaning to the left and the centre-left. According to this survey research, 63% follow the news internet sites, 57% the social media, 31% the national television news programs, 25% the news blogs, 13% the radio news, and 4% the newspapers. Although 85% of the participants do not like the news- papers as news source, 26% of them consider the newspaper information as very useful. The 504 Sociološki pregled / Sociological Review, vol. LIV (2020), nо. 3, pp. 498–517 sociohistorical and cultural context of a national society undoubtedly plays the most important role in the way people use the big variety of coronavirus news sources, and also perceive, frame and evaluate information (especially for the case of Italy, see the work of Briscese et al., 2020). (Превод In Extenso) Сажетак: Светски историјски догађај попут пандемије COVID-19 још једном је потврдио да живимо у крајње повезаном светском друштву и да се у данашње време епидемије више не сматрају само природним појавама. У таквом контексту, овај рад има циљ да протумачи сложени однос између друштва и вируса COVID-19, са нагласком на улози коју имају разне силе у области информационе политике и јав- них перцепција уопште. Из тог разлога елаборирамо културолошке факторе, као и емоције попут одговорности, поверења и страха током кризе. Такође смо се усред средили на динамику савремених медија у вези са јавним представама о панде- мији, ослањајући се на релевантне налазе. У целини, тиме се у новом социолошком и интердисциплинарном светлу приказује садашња пандемија као односни процес и феномен покренут дигиталним медијима. Кључне речи: криза изазвана коронавирусом, дигитални медији, поверење, страх, Грчка Concluding remarks In general, we can arguably conclude that the pandemic transforms the government in a dynamical fashion, albeit the scale of that impact is still unknown (Vis- vizi and Lytras, 2020, p. 126; Visvizi and Lytras, 2019). In this analytic setting, it is important to understand that the emergent network soci- ety renders the contemporary media as the main agent of public reflection and orientation, and as the substitute of politics, economy and religion (Baecker, 2018). Given that the cur- rent societies relationally shape their technological systems and patterns of transmission, further empirical and theoretical interdisciplinary research is urgently needed on the role that the digital and printed media (including the diffused cultural formations) play in the information policy of national governments and in the perception of the pandemic by the people, as well as on the media’s impact upon the pandemic society. 505 Хараламбос Цекерис, Персефони Зери, Криза изазвана коронавирусом као светски... Хараламбос Цекерис1 Национални центар за социолошка истраживања Атина (Грчка) Персефони Зери2 Универзитет друштвених и политичких наука „Пантеион” Атина (Грчка) Увод: Држава, друштво и криза изазвана коронавирусом Свим посланицима у немачком парламенту 2012. године дата је прогностичка студија о сценарију велике пандемије потекле из Азије (Kiel, 2020). Занимљиво је то што студија тада уопште није узета у обзир. Сви су игнорисали слабе сигнале. Почетком 2020. године пандемија коронавируса (или COVID-19) неочекивано је стигла до запада као „црни лабуд” који брзо шири заразу, односно као процес који ће највероватније однети многобројне жртве (Taleb, 2020) услед повећане глобалне повезаности. Изгледа да ниједна национална и наднационална влада није била при- премљена иако се управљање у принципу односи на стратешко промишљање и предвиђање (Tufekci, 2020). Прогностички сценарији подразумевају размишљање 506 Социолошки преглед, vol. LIV (2020), nо. 3, стр. 498–517 о будућности и посебне структуре за спровођење ванредних мера на основу јасних обавеза, процене ризика и учешћа стручњака (Kiel, 2020). Оно што се сада дешава на западу јесте отворен процес дефинисања и зау стављања пандемије коронавируса у којем наука (епидемиологија, инфектологија, иму- нологија и вирусологија), у условима велике неизвесности и непредвидивости, има водећу улогу заједно са јавном дебатом о перспективи у погледу медицинског трет- мана болести као нечега што се временом мења. У овим јавним дискусијама активно учествују разни актери, као што су штампани и дигитални медији, цивилно друштво и остале заинтересоване стране. Позитивну улогу у сакупљању информација о пан- демији има глобална истраживачка мрежа универзитета и истраживачких установа који интензивно сарађују како би реаговале на COVID-19. То представља општу базу знања које се размењује у процесу доношења политичких одлука (Dianeosis, 2020). Распрострањено уверење о томе да се треба активно супротставити кризи и успешно је савладати подстиче огромне промене у понашању или ствара спремност на уса глашавање са траженом бихевиоралном оријентацијом (Dianeosis, 2020). У том процесу људи изражавају спремност да преузму одговорност за себе и друге, следећи на тај начин посебна правила понашања када је реч о одржавању хигијене, на пример прање руку, ношење маске и социјална дистанца. У том погледу, владе покушавају да удруже одговорност коју људи показују са јавним поштовањем уведених правила, што наглашава конститутивну улогу државе у заштити друштва. Јавно придржавање мера за спречавање заразе (затварање школа, забрана великих скупова, спортских догађаја итд.) показује безусловно прихватање политике као најодговорнијег елемента у функционисању друштва у целини. Цивилно друштво (или породица) не могу да делују сами у овој ситуацији; то треба да уради држава. Ова чињеница постаје очигледна у предузимању финансијских мера огромних раз- мера како би се подржала привреда и друштво који су снажно погођени кризом (Strauss-Kahn, 2020, str. Хараламбос Цекерис, Персефони Зери, Криза изазвана коронавирусом као светски... Хараламбос Цекерис, Персефони Зери, Криза изазвана коронавирусом као светски... Током периода изолације грађани су могли рефлексивно да посматрају како се јавља перцепција новог, ефикасног „ми” (Coyle, 2020), везано за идеју о личном продруштвеном развоју и широј друштвеној интеграцији и кохезији. Ово је такође повезано са препреком у виду политичке поларизације која је ендемска појава у политичком животу Грчке (Zeri et al., 2019). Али да ли је ово динамичка препрека? Наравно, нико не може лако предвидети да ли ће после кризе изазване коронавиру сом поново доћи до стварања неповољне политичке климе, нарочито у контексту економске кризе непознатих размера која ће највероватније уследити (European Commission, 2020). У сваком случају, задатак политике у вези са научном неизве сношћу када је реч о производњи вакцине или делотворних лекова јесте да развије способност колективне акције и да у друштву учврсти веровање у успешан зајед- нички одговор на кризу. Влада такође мора да смисли и примени стратегије кому- никације које постижу осетљиву „равнотежу између смањивања оптимистичке при страсности не изазивајући претерану анксиозност и страх” (Bavel et al., 2020, str. 461). Поред тога, руководство мора да координише појединце и „да им помогне да избегну понашање које се више не сматра друштвено одговорним” (Bavel et al., 2020, str. 465). Уплитање државе није повезано са технократским, наивним подређивањем науци нити са беспоговорном спремношћу на послушност. Грађани су били миш- љења да нам је потребна демократска држава која заступа све нас и која може да санкционише појединце због општег добра (Bude, 2020). Јавна заштита један је од главних циљева демократске политике, поготово у стратешком напору да се изрази убедљив јавни интерес (Gostin & Wiley, 2020) и да се људима помогне да помоћу јединства и солидарности преживе светско-историјску појаву коронавируса (Visvizi & Lytras, 2020) и да сачувају хуманост људског рода. Занимљиво је то што резултати истраживања института Дианеосис (2020) потврђују представу Грка о држави која даје предност јавном здрављу, као и знању и одговорности стручњака. Током изола- ције смо, мада накратко, били сведоци спонтане појаве механизма солидарности и узајамне бриге (Fanaras, 2020, str. 17) који нам није наметнут из државног врха већ је заснован на осећању да нам је држава ипак потребна. Такође смо доживели атмосферу резервисане солидарности (или идиосинкрат ске мешавине солидарности и сумње), чија је природа добила нову димензију; то јест, штитимо друге не само тако што им помажемо већ и тако што „одржавамо ди станцу” како би се спречила зараза. Увод: Држава, друштво и криза изазвана коронавирусом 9; Rogoff, 2020). Брза реакција на кризу у већини европ ских држава и висок степен одобравања драстичних мера у јавности повезан је са крајњом „вредношћу људског живота која је знатно појачана у колективном несве сном најбогатијих земаља” (Strauss-Kahn, 2020, str. 8). Економија и остале друштвене активности стога морају да жртвују један део своје динамике у корист здравственог система (Baecker, 2020a). У овој историјској ситуацији сведоци смо „парадокса” пред демократским друштвима где се колективитет ставља у службу чисто одбрамбеног циља (Gauchet, 2020c). Мере заустављања коронавируса и одржавања социјалне дистанце показују радикалан заокрет јер се уместо културе слободе и самоодређења наглашава кул- тура осетљивости заштите (Bude, 2020) и колективне дисциплине. Ова промена указује на слабљење доминантне идеје о снажном „ја”, тј. о „идентитарној особи” (Ladeur, 2007, str. 397) која негира интернализацију (или индоктринацију) друштве- них ограничења и усредсређује се на своју наводно „аутентичну” самопрезентацију, далеко од обичне стварности. Уместо тога, сада смо сведоци преовлађујуће идеје о слабом и рањивом „ја” којем је потребна заштита државе. Одговор државних вла сти у Грчкој, као и у већини земаља, нарочито када је реч о мерама затварања и изо- лације, прихватила је с поштовањем огромна већина становника (Dianeosis, 2020; Papamattheou, 2020). 507 Хараламбос Цекерис, Персефони Зери, Криза изазвана коронавирусом као светски... Свест о релативној осетљивости која не познаје класну припадност подстакла је потрагу за новим видовима солидарности. Интере сантно је да 66,8% Грка верује да криза изазвана коронавирусом има позитиван ути- цај на солидарност са другима (Georgakopoulos, 2020, str. 10). Међутим, још увек је рано доносити коначне и чврсте закључке о процесу учења који се односи на изградњу нових типова солидарности. Скорашње слике масовних демонстрација и јавних скупова у периоду када су правила о социјалном дистанцирању и даље била на снази, могу да укажу на недостатак грађанске свести, а могуће и на културолошку неспособност памћења. Сада прелазимо на елаборацију улоге различитих сила као што су емоције, култура и медији у области информационе политике, јавних ставова о вирусу COVID-19 и друштва током пандемије уопште. 508 Социолошки преглед, vol. LIV (2020), nо. 3, стр. 498–517 Одговорност и поверење током пандемије У периоду спровођења наредби о „останку код куће” и тоталне изолације, велики терет јавних информација (преплављених смерницама којима је управљала грчка држава и које су преносили телевизија и радио) допринео је томе да људи схвате да су одговорни не само за себе већ и за друге. Током двомесечног карантина, чланови друштвене заједнице су научили да поштују обавезна правила. Озбиљност кризе пос- тала је сасвим јасна када су се у медијима појавиле слике из Италије и САД-а на којима се виде конвоји како износе сандуке жртава коронавируса из Бергама или Њујорка, као и слике преоптерећених здравствених система. Имајући у виду да је пандемија најопаснија за старије особе и да у грчкој породици деде и баке имају значајну улогу (Dianeosis, 2020), исказано је готово апсолутно поштовање према мерама затварања. Попуштање мера изолације у мају 2020. повезано је са строгим предусловима који су обавезивали људе да поштују рестриктивне мере. Ипак, млади људи и најо сетљивији стари људи одједном су почели да се окупљају у парковима и поред мора, кршећи тако правила социјалног дистанцирања. Понашали су се као да су потпуно избрисали из сећања ризике од новог таласа COVID-19, као и упозорења које је у медијима дао високо цењени професор Сотирис Циодрас, вођа експертског тима имунолога и представник владе за комуникацију са јавношћу током здравствене кризе (Kathimerini, 2020). То је нагли заокрет од колективно дисциплинованог народа у народ који пркоси јавним интересима. Стога је очигледно колико су крхки једин ство и кохезија подстакнути страхом током периода изолације, али и колико је снажна жеља за нормалношћу. У европској свести, мир и просперитет, заједно са високим економским резул- татима, проредили су стратешке прогнозе/размишљање о будућности и потиснули потенцијалну опасност и основну димензију политичког постојања. Истицање права појединаца и материјалних средстава постало је једини видљиви хоризонт, чиме је створено неосновано осећање сигурности (Gauchet, 2020a). Осим тога, постоје разлике у ставовима становника различитих европских земаља када је реч о мерама затва- рања у борби против коронавируса. Католичке земље попут Италије и Француске истичу се својеврсним спојем анархије појединаца и јавног ауторитарности, док се протестантске земље радије ослањају на осећај личне одговорности (Gauchet, 2020a). У Грчкој посебно немамо ни наслеђе конфучијанске дисциплине нити можемо да се позовемо на наслеђе модерности и дубоко усађену свест о личној одговорно сти. Исказивање личне одговорности не припада „текстури” самоинституционалне политичке заједнице у грчком друштву, које обилује чврсто укорењеним предмодер- ним културним обрасцима (Demertzis, 1997, str. 109; Zeri et al., 2019, str. 8). Одговорност и поверење током пандемије На изве стан начин, грчки појединац је заточен у нерефлектованом „укорењеном ја” (Ramfos, 2011) и носи друштвена и психолошка упутства, као и моралне претпоставке поро- дичног начина размишљања и стратегије уграђене у шире мреже интересних група (Pelagidis, 2012). Упркос томе, промена вредности у посткризном периоду свакако је могућа и заслужује анализу. Суочивши се са великом здравственом кризом као што је пандемија COVID-19, која се односи на укупну друштвену чињеницу (Kasuga, 2020), Грци су се ослонили на помоћ државе. Мноштво слика преминулих у медијима изазвало је психолошки шок 509 араламбос Цекерис, Персефони Зери, Криза изазвана коронавирусом као светски... који је могао да се ублажи само тежњом ка јединству и заједништву. Јединство народа гради се око државе и њених представника као стање заједништва, а не у контексту Европе или неке друге наднационалне организације. Међутим, треба напоменути да су ставови Грка према политичким институцијама у њиховој држави прилично нега- тивни (Koniordos, 2018). Успех који је Мицотакисова влада остварила у управљању тзв. Евро кризом (јануар 2020) и кризом изазваном коронавирусом (март–април 2020) означио је упадљиву промену у представи коју Грци имају о својим политич- ким институцијама, нарочито о својој влади. У поређењу са претходним истраживањима института Дианеосис, истраживање кризе изазване коронавирусом које је овај институт спровео у априлу 2020. године показало је упадљиво повећан степен поверења људи у државне институције, после чега следи јачање њиховог осећања оптимизма, сигурности и извесности на личном нивоу (Dianeosis, 2020). Исто тако, мере које је држава предузела током пандемије у циљу унапређивања јавног здравственог система учврстиле су поверење у саму државу (видети Kounias, 2020). Упадљиво је то што је поверење у науку (медицину) на самом врху лествице. Према истраживању коронавируса које је спровео Економ ски факултет у Атини, 50% испитаника имају поверење у лекаре, 88% у здравствене раднике, а 57% у државу, на ниском нивоу је њихово поверење у наставнике (45%) и приватне компаније (24%), док 75% испитаника верује да су мере затварања биле ефикасне (Papamattheou, 2020). Занимљиво је то што су политичко поверење, јасне поруке, транспарентна комуникација и разматрање свих битних питања пресудни за подстицање промене у понашању (Bavel et al., 2020, str. 466). Надаље, политичко руковођење и савладавање економске рецесије после пандемије COVID-19 биће не сумњиво одлучујући фактор у развоју општег поверења у грчку политику. Страх у ери коронавируса Терористички напади на Њујорк 11. септембра 2001. означили су почетак ере страха. С временом су се осећања страха на различите начине повезивала прво са исламистичким тероризмом, затим са угрожавајућим климатским променама, а сада са великом глобалном здравственом кризом која снажно утиче на све нас (Bolz, 2020). У савременом богатом друштву многи људи посматрају пандемију COVID-19 као борбу за опстанак без преседана. До сада се држава налазила у служби економије и подстицала је слободно кретање капитала, људи и добара. Након глобалне појаве коронавируса опасног по живот, држава је успорила привреду због тога што су људ ски животи изложени ризику. Ово је потврда поштовања хуманистичке димензије на коју се позива модерност. Није реч о спасењу у смислу религијског искупљења (Finkielkraut, 2020a), већ о спасењу здравља које је од почетка модерног доба „прво добро и основ свих осталих добара овог живота” (Descartes, 1953 [1637], str. 168). Према речима Алена Финкелкраута, морамо бити „захвални политичарима којима је људски живот важнији од новца. Они су по ставили праве приоритете зато што желе да сачувају најслабије и најрањивије припад- нике друштва. […] Докле год је живот старог човека вредан колико и живот човека на врхунцу физичке снаге, и даље ћемо бити цивилизовани” (Finkielkraut, 2020b). 510 Социолошки преглед, vol. LIV (2020), nо. 3, стр. 498–517 Међутим, у оквиру демократске заједнице, поготово у периодима друштвеног мира, људи дају предност сопственој појединачној слободи. Наиме, колективна машинерија у нормалним временима наводи нас да заборавимо да смо политичка заједница (Gauchet, 2020b). Када је реч о акутној кризи и колективној опасности, страх и осећање опште рањивости подстиче способност људи да поступају као грађани који своју заштићену егзистенцију дугују политичкој заједници, док се релативно потискује приоритет личне слободе. У исто време, за разлику од диктатор ских или ауторитарних режима, људи (корисници интернета) имају слободу да у јавности дискутују и износе мишљења о мотивима јавних мера. На основу резултата истраживања института Дианеосис (Fanaras, 2020, str. 6), успех који је грчка влада постигла у управљању кризом изазваном коронавирусом ограничио је негативна осећања страха и анксиозности. Стрпљење и креативност били су изненађујућа реакција на затварање читавог друштва у периоду дужем од шест седмица. Готово идентичне резултате дао је и Пројекат за спречавање корона- вируса (Center for the Digital Future, 2020), који показују да су Американци током пандемије имали више времена за своје партнере и децу, односно да се поново посвете неком хобију и креативним активностима. Страх у ери коронавируса Истраживање коронавируса које је спровео Економски факултет у Атини (Papamattheou, 2020) истиче висок ниво страха и анксиозности пре свега због економије (92%), а мање због здравља (82%). Медији и криза изазвана коронавирусом Пандемија коронавируса нагласила је високу сложеност глобалног система и његову повећану нелинеарну динамику (Tufekci, 2020), имајући у виду да се структурна форма светског друштва (Baecker, 2020b) и националних друштава односи на релационе мреже које се знатно разликују и репродукују (Baecker, 2018, str. 61). Наиме, здравствена криза коју тренутно доживљавамо потпуно се разли- кује од оних из прошлости, првенствено због тога што је свака економија у данашње време интегрисана у шири глобални контекст, при чему Кина представља „светску фабрику” (Strauss-Kahn, 2020, str. 19), док смо изгубили сећање на ризик од зараза, односно пандемија. У овом аналитичком окружењу, од пресудног је значаја елабори- рати чињеницу да су, у оквиру умреженог друштва у којем живимо, медији као облик колективне рефлексије и оријентације заузели место политике, економије и рели- гије (Baecker, 2018, str. 192). Из тог разлога, различити степени приступа квалитет- ним информацијама могу знатно да утичу на перцепције здравља, одлуке и исходе у контексту пандемије, док дезинформације на интернету могу да доведу до болести и смртног исхода у стварном свету (McGinty & Gyenes, 2020, str. 4). Зато је грчка влада (као и владе осталих земаља чланица ЕУ) направила интернет страницу посвећену деконструисању митова о вирусу COVID-19 (covid19.gov.gr/mythoi-covid19/) у оквиру званичног портала за праћење овог вируса COVID-19 (covid19.gov.gr). Поред тога, медији припадају оквирима различитих историјских, културоло- шких и институционалних контекста садашњих друштава која релационо обликују своје технолошке системе и обрасце преноса у ери глобално умрежених комуника- ција. Ово указује на фундаменталну улогу коју штампани и дигитални медији имају у информационој политици националних влада, као и у јавном поимању пандемије 511 араламбос Цекерис, Персефони Зери, Криза изазвана коронавирусом као светски... (видети нпр. Casero-Ripolles, 2020). Према најновијем истраживању Универзитета у Чикагу (Bertrand et al., 2020), Американци не посматрају пандемију кроз призму здравља и економије већ кроз призму властитих политичких уверења, властитог мишљења о правилној улози владе и поверења у медије у условима политичке поделе друштва. Поверење у медије је изузетно значајно за процену пандемије и одговор на њу (видети Merrefield, 2020). Треба напоменути да већина људи не процењује пан- демију на основу онога што су видели или чули у јавним дискусијама, већ на осно- ву тога за кога гласају, кога медији прате или кога домаћин емисије фаворизује (Bursztyn et al., 2020). Према истраживању које је спровео Истраживачки центар „Пју”, идеолошки састав и партијска оријентација гледалаца кабловске мреже утиче на начин на који они процењују епидемију коронавируса (Pew Research Center, 2020a). Медији и криза изазвана коронавирусом У ери беспомоћности и нестабилности, због тога што су онемо- гућене важне психолошке потребе, теорије завере функционишу као инструмент 512 Социолошки преглед, vol. LIV (2020), nо. 3, стр. 498–517 управљања личном кризом (Douglas et al., 2017). Структура друштвених мрежа у неку руку олакшава процват и ширење лажних информација и вести о коронавирусу широм света, упркос координисаним напорима појединих компанија са дигиталним платформама (Fraenkel et al., 2020). Осим тога, Јан Филип Ремтсма (2020) истиче да је политичко размишљање прецењено и да заправо утицаји имају кључну улогу (видети и Demertzis, 2020). Људи ретко размишљају на политички начин (Reemtsma, 2020). у ју Начин на који Американци прате изворе вести о коронавирусу и сагледавају извештавање медија о пандемији суштински се разликује од начина на који Грци користе изворе информација о коронавирусу, а нарочито вести у медијима. Већина Американаца подједнако поклања пажњу националним и локалним вестима о COVID-19, при чему 56% националних извора вести (укључујући штампу) пред стављају главни извор вести о коронавирусу, 51% чине јавне здравствене организа- ције и представници, 46% локални извори вести, 31% Доналд Трамп и радна група Беле куће задужена за извештавање о коронавирусу, 25% међународни извори вести, 10% форуми и дискусионе групе на интернету, а 16% пријатељи, породица и суседи (Pew Research Center, 2020b). Готово девет од десет испитаника сматра да је интер- нет основно или важно средство информисања током заразе коронавирусом (Pew Research Center, 2020c). У Грчкој је коришћење извора вести о коронавирусу про- праћено повећаним поверењем у вести у медијима (Fanaras, 2020), што је у складу са претходно поменутим повећањем поверења у политичке институције, а нарочито грчку владу. Грцима је главни извор вести о коронавирусу телевизија (48%), затим следе интернет странице (25,4%), здравствени радници (9,1%), друштвене мреже (7,5%), радио (3,7%), породица и пријатељи (2,9%) и новине (1,8%). Напослетку, истраживање Универзитета у Атини (Media analysis GR, 2020) даје нам диференциране резултате на основу различитог узорка испитаника који нагињу левици и левом центру. Према овом истраживању, 63% испитаника прати вести на интернету, 57% на друштвеним мрежама, 31% прати национални дневник, 25% бло- гове са вестима, 13% вести на радију, а 4% чита новине. Иако 85% испитаника не воли новине као извор вести, њих 26% сматра информације из новина веома корисним. Медији и криза изазвана коронавирусом Друштвено-историјски и културолошки контекст националног друштва несумњиво има значајан утицај на начин на који људи користе мноштво разних извора вести о коронавирусу, као и на начин на који сагледавају, формулишу и вреднују информа- ције (поготово у случају Италије, видети рад Briscese et al., 2020). Медији и криза изазвана коронавирусом Иако америчка јавност каже да медији добро обавештавају о кризи, дубоке поделе у погледу начина на који су медији извештавали о коронавирусу биле су очигледне не само између демократа и републиканаца, већ и између идеолошких сегмената унутар ове две странке (Pew Research Center, 2020d). На пример, у истраживању које је недавно спроведено у САД (Jamieson & Albarracin, 2020) утврђено је да је гледање тзв. мејн стрим медија (нпр. NBC News) повезано са тачним информацијама о болести опасној по живот, док је читање мејнстрим штампаних медија (нпр. New York Times) пове- зано са тачним мишљењем о заштити од заразе, док су конзервативни медији (нпр. Fox News) у корелацији са теоријама завере. Данас су електронски и дигитални медији преплављени негативним, сензацио налистичким вестима и дезинформацијама о пандемији. У масовним медијима добро је познато како новинари све могу да претворе у лоше вести зато што су усмерени на масовну публику која у начелу реагује на лоше вести (Tierney, 2020). Негативне предрасуде и негативне информације су један од основних психолошких принципа (Baumeister, 2020). Осим тога, оне представљају кључну чињеницу о начину на који функционише човеков ум. Људи су склони да више значаја придају стварима које су пошле по злу него онима које су добре, до те мере да је довољан само један догађај да потпуно заокупи наше мисли (Baumeister & Tierney, 2019). Негативне информације у човековом уму остављају својеврсан печат који није лако избрисати (Sunstein, 2020). Очигледно у својој подсвести тежимо да пажњу обраћамо на лоше вести (Hancock, 2020). Наиме, Гуљелмо Бришезе (Guglielmo Briscese) тврди да је много људи зара- жено и да нам се пандемија све више приближава, али људи и даље не мењају своје погледе (видети Merrefield, 2020). Когнитивне и негативне предрасуде имају велики утицај на наш начин обраде вести (McIntyre, 2020). Пристрасност коју у свом мозгу потврђујемо подстиче нас да тражимо само оне информације које су у складу са оним у шта већ верујемо, а да одбацимо остале информације. Оваква пристрасност може нас спречити да сазнамо новости о пандемији и да се делотворно боримо против ње (McIntyre, 2020). Ипак, у друштву погођеном пандемијом, људи често живе у личном ванредном стању које може да прерасте у прави свет заблуде и да покаже изузетну крхкост психе. То доприноси ширењу теорија завере о корони у свим сегментима друштва (Bavel et al., 2020, str. 464ff.). Хараламбос Цекерис, Персефони Зери, Криза изазвана коронавирусом као светски... араламбос Цекерис, Персефони Зери, Криза изазвана коронавирусом као светски... У сваком случају, овој великој кризи могле би се приписати историјске димен- зије ако се сматра да је историја производ колективног рада или неизвесни исход делиберативног деловања друштвених и политичких агената (Gauchet, 2016, str. 374). На крају, ово је вишезначни релациони процес повезан са кризама које се јављају у другим системима (економија, политика, медији, култура, породица итд.) нано сећи штету телу, души и друштвеном животу. Те кризе оставиће дубок траг у сећању сваког друштва, као и у психичкој позадини друштвених субјеката. Трауматично искуство изазвано пандемијом показало је први пут колико потенцијалне опасно сти носи брзо, функционално диференцирано и високо умрежено светско друштво, где се вирус назаустављиво шири за свега неколико дана или недеља и захвата сва географска подручја (Esposito, 2020a; видети и Esposito 2020b). У том погледу, пан- демија може да буде теоретизована као вештачки створена друштвена катастрофа (Leggewie, 2020). У данашње време хиперповезани свет одговара моделу друштва који је створен у новој фази глобализације, а чија је основна одлика стални пораст размене и инте- ракција између националних дигиталних и виртуелних система, као и брзо ширење информација, знања, иновација и услуга (Baldwin, 2019). Овај модел има за циљ прилагођавање турбуленцијама и тешким контекстима као што је контекст вируса COVID-19. Још је рано за прогнозе да ли ће криза изазвана коронавирусом довести до драстичних друштвених промена у перспективи озбиљне економске кризе или ће преовладати тежња ка повратку у нормалност. Када вирус буде нестао, сасвим је могуће да ће владавина економиста уступити место влади састављеној од лекара (Gauchet, 2020b). Уопштено узев, можемо без сумње закључити да пандемија транс- формише владу на динамичан начин, иако је обим тог утицаја и даље непознат (Visvizi and Lytras, 2020, str. 126; Visvizi and Lytras, 2019). У оваквом аналитичком окружењу важно је схватити да новонастало умре- жено друштво савремене медије чини главним агентом јавног промишљања и усмерености, те да представља замену за политику, економију и религију (Baecker, 2018). 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https://openalex.org/W4288844461	https://espace.curtin.edu.au/bitstream/20.500.11937/89024/3/88848.pdf	English	null	Balancing food waste and sustainability goals in online food delivery: Towards a comprehensive conceptual framework	Technovation	2,022	cc-by	14,482	A B S T R A C T Keywords: Digitalization Eco-friendly packaging Food delivery apps (FDAs) Hospitality Leftover reuse Sustainable development goals Increasing food waste is a major threat to sustainability and food security. Recognizing the issue, the United Nations Sustainable Development Goal (SDG) 12 mandates reducing global food waste by 50 percent by 2030. This situation has also given impetus to academic research on consumer food waste in both household and out-of- home settings. However, food delivery apps (FDAs) remain under-researched from this perspective. This is a serious gap because operating under a business model that inherently facilitates food over-ordering, FDAs can be a major source of food waste. Understanding the demand-side factors that drive consumers to order more food than required to satiate their hunger can be useful in mitigating such wasteful indulgences. Noting this, we extend the seminal theory of planned behavior (TPB) to propose direct and intervening mechanisms that can better explicate why consumers indulge in a ‘shopping routine’ of ordering more food than required. We analyzed data from 487 FDA users to test the proposed hypotheses. Our results reveal the positive associations of (a) attitude and subjective norms with usage intentions and (b) trust, intentions, and leftover reuse routine with shopping routine. In addition, both proposed moderators—i.e., willingness to pay for eco-friendly packaging and number of years of FDA usage—do, in fact, moderate the associations of leftover reuse routine with both usage intentions and shopping routine. These findings can help marketers and policymakers devise appropriate stra tegies to promote pro-environmental green behaviors among FDA users without harming the commercial in terests of the sector. mit Shankar a, Amandeep Dhir b,c,d,, Shalini Talwar e, Nazrul Islam f, Piyush Sharma g a Indian Institute of Management Visakhapatnam, Andhra Pradesh, 530003, India b Department of Management, School of Business & Law, University of Agder, Kristiansand, Norway c Norwegian School of Hotel Management, Faculty of Social Sciences, Stavanger, Norway d Optentia Research Focus Area, North-West University, Vanderbijlpark, South Africa e S.P. Jain Institute of Management and Research (SPJIMR), Mumbai, India f Department of Science, Innovation, Technology and Entrepreneurship, University of Exeter Business School, England, UK g School of Management and Marketing, Faculty of Business and Law, Curtin University, Australia Technovation journal homepage: www.elsevier.com/locate/technovation Corresponding author. Department of Management, School of Business & Law, University of Agder, Kristiansand, Norway. E-mail addresses: ashankar@iimv.ac.in (A. Shankar), amandeep.dhir@uia.no (A. Dhir), shalini.talwar@spjimr.org (S. Talwar), n.islam@exeter.ac.uk (N. Islam), Piyush.Sharma@curtin.edu.au (P. Sharma). 0166-4972/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.technovation.2022.102606 Received 3 March 2022; Received in revised form 9 July 2022; Accepted 15 July 2022 0166-4972/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. Department of Management, School of Business & Law, University of Agder, Kristiansand, Norway. E-mail addresses: ashankar@iimv.ac.in (A. Shankar), amandeep.dhir@uia.no (A. Dhir), shalini.talwar@spjimr.org (S. Talwar), n.islam@exeter.ac.uk (N. Islam), Piyush.Sharma@curtin.edu.au (P. Sharma). https://doi.org/10.1016/j.technovation.2022.102606 Received 3 March 2022; Received in revised form 9 July 2022; Accepted 15 July 2022 Technovation xxx (xxxx) xxx Contents lists available at ScienceDirect Technovation journal homepage: www.elsevier.com/locate/technovation Technovation xxx (xxxx) xxx Contents lists available at ScienceDirect Technovation journal homepage: www.elsevier.com/locate/technovation Balancing food waste and sustainability goals in online food delivery: Towards a comprehensive conceptual framework Shankar a, Amandeep Dhir b,c,d,, Shalini Talwar e, Nazrul Islam f, Piyush Sharma g 2.1. Food delivery apps (FDAs): the digital face of hospitality As information technology has advanced, the combination of the Internet and mobile phones has produced a new avenue for the sharing economy—shared platforms, usually called food delivery aggregators, which provide a marketplace for online food ordering and delivery (e.g., Zomato, Uber Eats, foodpanda, and Foodora; e.g., Xu and Huang, 2019). A review of the literature indicates that these aggregators, available via FDAs, have emerged as innovative and popular platforms for delivering foods for three key reasons. First, the number of working parents and single-person households has increased, and this shift toward a working parent lifestyle has reduced the home cooking of daily meals (Roh and Park, 2019). By ordering food on FDAs, working parents can save the time and effort that goes into meal preparation (Cho et al., 2019). Sec ond, the increase in internet access and smartphone use has motivated restaurants, third-party delivery platforms, and other intermediaries (e. g., Uber Eats drivers) to provide delivery services, thereby supporting the FDA ecosystem. Third, the interactivity and efficiency of such apps have improved significantly in the recent past (Correa et al., 2019), making FDAs an integral part of the restaurant industry (Cho et al., 2019). In addition to consumer behavior, many recent studies have explored technological advancements related to FDAs (e.g., AI) and their positive impact on consumer behavior (e.g., He et al., 2018; Correa et al., 2019; Xu and Huang, 2019; Dhir et al., 2020; Hwang et al., 2020; Lee et al., 2020; Kaur et al., 2021b). Our study addresses these gaps using the well-established theory of planned behavior (TPB), which has been widely deployed to predict consumer intentions across various contexts (e.g., Wu et al., 2015; Alzubaidi et al., 2021). Scholars have also considered TPB a suitable theoretical lens to study the complexity of factors influencing con sumers’ behavioral decision-making (Arora and Sahney, 2018). The classic TPB postulation links attitude (ATT), subjective norms (SN), and perceived behavioral control (PBC) with intentions to use, which, in turn, drive behavior (Ajzen, 1991). However, scholars are increasingly acknowledging the need to extend and expand the classic TPB concep tualization to capture contemporary nuances and variances in the associated outcome variables (Yadav et al., 2019; Dhir et al., 2020). 1. Introduction We answered these questions by analyzing cross-sectional, single- wave data from 478 FDA users. i Our findings make three noteworthy contributions. First, our study is among the pioneering research attempts to examine demand-side be haviors towards FDAs with food waste as the fulcra. The uniqueness of this contribution is further enhanced by the entire study’s focus on and alignment with SDG 12. Specifically, our study extends the food waste literature by focusing on the impact of the increasing usage of FDAs on food waste generation and thereby initiating debate regarding the ways in which multiple stakeholders in the sector can balance commercial interests and sustainability concerns. Second, the study extends the seminal principles of TPB by including variables of contemporary in terest and pertinence, such as trust, leftover reuse routine, and shopping routine, to better capture the FDA usage behaviors that promote the possibility of food waste generation. Finally, our study makes a novel contribution by examining the moderating effects of two relevant yet under-explored variables—willingness to pay for eco-friendly packaging and number of years of FDA usage. In doing so, it presents more nuanced insights into consumer behavior toward FDAs and FDAs’ impact on food waste generation. g The scholarly literature on FDAs has also grown substantially, with past studies exploring consumers’ usage intentions (Cai and Leung, 2020; Lee et al., 2020; Raza et al., 2020; Song et al., 2021), FDAs’ service quality dimensions (Chen et al., 2020), consumers’ perceived risks (Hwang and Choe, 2019; Choe et al., 2021), consumers’ perceived values (Kaur et al., 2021b; Tandon et al., 2021), barriers to usage (Talwar et al., 2021), and service failures (Furunes and Mkono, 2019; Kaur et al., 2022). The existing scholarship has, moreover, made some attempts, albeit limited, to examine the sustainability-oriented aspects of FDA usage, such as food waste reduction intentions (Olavarria-Key et al., 2021), strategies to reduce food waste (Vizzoto et al., 2021), the effect of message framing on food waste reduction (Zhang et al., 2020), and food ordering behavior that can lead to food waste (Sharma et al., 2021). 1. Introduction Offering testimony to the rapid growth of FDA usage globally, recent estimates suggest that FDAs’ market volume is likely to reach USD 217.80 billion by the end of 2022 (Statista, 2022). Although the anticipated growth figures are commercially exciting, this digital success story also has a dark side in the form of the sustainability con cerns that arise from the potential for such high FDA usage to increase food waste generation. has—despite FDAs’ growing popularity—ignored the possibility of food waste being generated when consumers order prepared food through such apps (Lee et al., 2020). FDAs offer many advantages, such as con venience, localization, promotional offers, last-minute discounts, authentic reviews, 24/7 access, an exhaustive list of cuisines, menus, and restaurants, and rapid delivery (Kapoor and Vij, 2018; Cho et al., 2019; Xu and Huang, 2019; Shankar et al., 2022; Talwar et al., 2022a). These advantages have led to an exponential increase in the number of active FDA users around the world. This growth is expected to continue, with the number of FDA users anticipated to surpass 1.2 billion by 2023 (Statista, 2018). Offering testimony to the rapid growth of FDA usage globally, recent estimates suggest that FDAs’ market volume is likely to reach USD 217.80 billion by the end of 2022 (Statista, 2022). Although the anticipated growth figures are commercially exciting, this digital success story also has a dark side in the form of the sustainability con cerns that arise from the potential for such high FDA usage to increase food waste generation. consumers’ behavioral intentions and usage decisions (M. K. Kim et al., 2016; Sharma et al., 2021). Similarly, the sustainability-focused litera ture has discussed the willingness to pay for more environmentally friendly options—representing consumers’ readiness to sacrifice for the greater good—as a key construct (e.g., Talwar et al., 2022c). Summarizing the preceding discussion, we propose to address the following research questions (RQs): RQ1. How are attitude, subjective norms, and perceived behavioral control associated with consumers’ intentions to use FDAs? RQ2. How are trust and leftover reuse routines associated with FDA users’ intentions to use and shopping routine? RQ3. How are intentions to use FDAs associated with users’ shopping routine? and RQ4. Do willingness to pay for eco-friendly packaging and number of years of FDA usage moderate the associations of trust and leftover reuse routine with intentions to use and shopping routine, and if so, how? 1. Introduction However, a comprehensive review of the literature regarding FDAs and food waste reveals three persistent gaps: (a) Shopping routine—a significant contemporary measure due to its association with food waste—remains less examined in the FDA context despite being well investigated in household settings (e.g., Grandhi and Appaiah Singh, 2016; Nabi et al., 2021); (b) Scholars have yet to investigate the role of trust—an important variable in the online context, including mobile app usage (Shankar et al., 2020; Chakraborty et al., 2022) and buying and shopping behavior (Patil et al., 2020; Dhir, Sadiq et at., 2021; Dhir, Sadiq et at., 2021)—in driving FDA usage that can lead to food waste generation; and (c) Despite being acknowledged as an important variable in this context, leftover reuse routine—an important food waste reduction strategy examined in the household context (Hamerman et al., 2018; Talwar et al., 2022b)—remains under-explored in terms of FDA usage and its role in driving over-ordering behavior (Talwar et al., 2022a). 1. Introduction contributors to food waste globally (Dhir et al., 2020; Petit et al., 2020; Sharma et al., 2021). The scholarly literature has termed this tendency to purchase food in excess (i.e., over and above the quantity required to satiate hunger) a ’shopping routine’ (Stefan et al., 2013; Samsioe and Fuentes, 2022). i Food waste poses serious social, economic, and environmental sus tainability challenges globally (Stancu et al., 2016; De Visser-Amundson, 2020; Dhir et al., 2020; Carolan, 2021). According to a recent report, 1.3 billion tons of food worth USD 2.6 trillion is wasted around the globe each year; this amount of wasted food would be suf ficient to feed up to 815 million people (FAO, 2019). Approximately 61 percent of global food waste comes from households while 26 percent comes from food services and 13 percent comes from retail shops (UNEP, 2021). Because they tend to purchase excessive food, which often re mains unconsumed and, ultimately, wasted, consumers are the main Past research on shopping routines identifies cash rewards, pro motions, and free delivery (e.g., Talwar et al., 2021a) as well as the desire to save time, effort, and costs of food shopping (Wilson et al., 2017; Petit et al., 2020; Schmitt et al., 2021) as key drivers of this wasteful behavior. These findings, however, derive from prior research, which has focused primarily on shopping behavior in the context of household food items. With this narrow focus, the extant research Corresponding author. Department of Management, School of Business & Law, University of Agder, Kristiansand, Norway. E-mail addresses: ashankar@iimv.ac.in (A. Shankar), amandeep.dhir@uia.no (A. Dhir), shalini.talwar@spjimr.org (S. Talwar), n.islam@exeter.ac.uk (N. Islam), Piyush.Sharma@curtin.edu.au (P. Sharma). Please cite this article as: Amit Shankar, Technovation, https://doi.org/10.1016/j.technovation.2022.102606 Technovation xxx (xxxx) xxx A. Shankar et al. has—despite FDAs’ growing popularity—ignored the possibility of food waste being generated when consumers order prepared food through such apps (Lee et al., 2020). FDAs offer many advantages, such as con venience, localization, promotional offers, last-minute discounts, authentic reviews, 24/7 access, an exhaustive list of cuisines, menus, and restaurants, and rapid delivery (Kapoor and Vij, 2018; Cho et al., 2019; Xu and Huang, 2019; Shankar et al., 2022; Talwar et al., 2022a). These advantages have led to an exponential increase in the number of active FDA users around the world. This growth is expected to continue, with the number of FDA users anticipated to surpass 1.2 billion by 2023 (Statista, 2018). A. Shankar et al. Technovation xxx (xxxx) xxx examined this aspect (e.g., Sharma et al., 2021; Talwar et al., 2021b), variables of interest in the food waste literature, such as leftover reuse routine and shopping routine, require deeper examination in the context of FDAs. Because these factors are related to food over-ordering through FDAs and the possibility of subsequent waste recovery, a lack of insights may hinder the ability of concerned stakeholders to act in alignment with the United Nations Sustainable Development Goal 12.3, which calls for the reduction of retail and consumer food waste by 50 percent by 2030 (UNSDG, 2018). thought process, which might allow them to justify ordering more food than required to satiate their hunger through their intention to reuse any unconsumed food. Finally, our study models over-ordering behavior itself as a shopping routine. Overall, as presented in Fig. 1, our conceptual model proposes atti tude, subjective norms, and perceived behavioral control as antecedents of intentions to use FDAs and trust and leftover reuse routine as ante cedents of both intentions to use FDAs and shopping routine. To better illustrate the complex mechanism through which the identified ante cedents affect wasteful behavior on FDAs, we further propose the moderating effect of willingness to pay for eco-friendly packaging and number of years of FDA usage on the hypothesized associations. Table 1 presents the operational descriptions of all variables under study. 2.2. Theory of planned behavior (TPB) According to the TPB (Ajzen, 1991), attitude, subjective norms, and perceived behavior control are the basic predictors of consumers’ adoption intentions. The TPB (Ajzen, 1991) is considered the most influential theory for explaining consumer adoption behavior (Ajzen, 2011). Scholars identify TPB as an ideal theory to study consumer behavior because it provides sensible explanations for consumers’ deliberate choices (Chen et al., 2020). Recognizing TPB’s popularity and efficacy, past studies have used it to examine food choices and con sumption behavior in various contexts, including over-ordering behavior at restaurants (Yu et al., 2021), green food consumption (Qi and Ploeger, 2019), fruit consumption (Canova et al., 2020; Carfora et al., 2016), organic food consumption (Yadav and Pathak, 2016), healthy eating behavior (Lim et al., 2020), sugar-sweetened beverage consumption intentions (Gregorio-Pascual and Mahler, 2020), adher ence to gluten-free diets (Xhakollari et al., 2021), sugar-free consump tion (Phipps et al., 2020), consumption of non-perishable food (Lehberger et al., 2021), and fast food consumption (Sharifirad et al., 2013). Recent studies have also utilized TPB in the specific context of FDAs to confirm attitude (Tandon et al., 2021), subjective norms (Troise et al., 2020), and perceived behavioral control (J. J. Kim et al., 2021) as key variables associated with behavioral intentions. Going beyond the classic TPB constructs, scholars have noted that the extended TPB, which accommodates context-specific variables, has greater predict ability, comprehensiveness, and parsimony in explaining consumer behavior (Kumar, 2017). For these reasons, this study extends the TPB theory to explore consumer behavior in the context of FDAs. i 2.1. Food delivery apps (FDAs): the digital face of hospitality Guided by this view, we not only model the three original TPB variables but also include two contemporary variables—trust (TR) and leftover reuse routine (LR), which are relevant to the FDA and food waste context. We also extend the classic TPB framework by including two moderating variables—willingness to pay for eco-friendly packaging and number of years of FDA usage. These choices are consistent with the recent literature on FDAs and sustainability. Past studies on FDAs have, in particular, emphasized the role of the number of years of FDA use on Despite the growing research on sustainability, SDGs, and food waste in out-of-home and household settings (e.g., Kaur et al., 2021a), how ever, the scholarly literature has left the linkage between FDA usage and food waste almost entirely unexplored. While some recent studies have 2 3.3. Leftover reuse routine, intentions to use FDAs, and shopping routine Wasting food refers to the disposal of food that is fit for human consumption (Dhir et al., 2020). Food waste is high across the world, making it a major environmental, social, and economic concern. Increasing concerns regarding food waste have stimulated debate about ways to reduce it. One such solution, which many households already likely practice, is the reuse of unconsumed food for future meals. Aca demic researchers have formalized this approach with the term “leftover reuse routine,” and they identify this routine as one of the most effective strategies to reduce food waste (Stancu et al., 2016; Talwar et al., 2022a). Scholars have observed that the increase in awareness about food waste and its negative impact on the environment and society has made consumers more willing to reuse leftovers for future consumption (e.g., Romani et al., 2018). i In the context of the present study, i.e., food-related behavior, the literature has found subjective norms to influence consumers’ food choices and food consumption behavior (e.g., Mullan et al., 2013; Stefan et al., 2013; Stancu et al., 2016; Raab et al., 2018). Given the strong theoretical support for the impact of subjective norms on consumers’ choices, we suggest that subjective norms are also likely to impact their intentions to use FDAs. More specifically, if individuals believe that their social circle and family approve of their decision to use FDAs to order food, their intentions to use FDAs for that purpose will increase. Hence, we propose the following hypothesis: H2. Subjective norms are positively associated with intentions to use FDAs. PBC represents an individual’s perception that he or she possesses the necessary knowledge and resources to perform a specific task (Ajzen, 1991). Conceptualized as an individual’s ability to control a situation and his or her self-confidence in undertaking a behavior (Shankar and Datta, 2018), PBC enhances the perceived safety and reduces the perceived risks associated with the behavior (Ajzen, 2011). Several studies have identified PBC as an important variable in the classic TPB model, which impacts adoption choice in various contexts of consumer behavior, such as luxury consumption (Shankar and Jain, 2021). Noting the same impact of PBC in the case of technology adoption, scholars have reasoned that consumers tend to adopt new technology when they believe they possess the required resources and capabilities to use such services (Arora and Sahney, 2018). H1. Attitude is positively associated with intentions to use FDAs. H1. Attitude is positively associated with intentions to use FDAs. Subjective norms refer to the impact of family members, friends, peer groups, and society’s opinions regarding an individual’s choice to exhibit a certain behavior (Ajzen, 1991). Subjective norms have a crucial impact on consumers’ adoption behavior in various contexts, including technology adoption (Shankar and Datta, 2018), social media use (Raza et al., 2020), and the choice of green hotels (Yadav et al., 2019). Explaining this impact, scholars note that consumers value the suggestions of the people they follow (Arora and Sahney, 2018) and their behavior is influenced by the people around them (Shankar and Datta, 2018). Explaining further, past studies have reported that con sumers feel more confident in making purchase decisions when the people around them support their choice (e.g., Shankar and Jain, 2021). Subjective norms refer to the impact of family members, friends, peer groups, and society’s opinions regarding an individual’s choice to exhibit a certain behavior (Ajzen, 1991). Subjective norms have a crucial impact on consumers’ adoption behavior in various contexts, including technology adoption (Shankar and Datta, 2018), social media use (Raza et al., 2020), and the choice of green hotels (Yadav et al., 2019). Explaining this impact, scholars note that consumers value the suggestions of the people they follow (Arora and Sahney, 2018) and their behavior is influenced by the people around them (Shankar and Datta, 2018). Explaining further, past studies have reported that con sumers feel more confident in making purchase decisions when the people around them support their choice (e.g., Shankar and Jain, 2021). In the context of the present study, i.e., food-related behavior, the literature has found subjective norms to influence consumers’ food choices and food consumption behavior (e.g., Mullan et al., 2013; Stefan et al., 2013; Stancu et al., 2016; Raab et al., 2018). Given the strong theoretical support for the impact of subjective norms on consumers’ choices, we suggest that subjective norms are also likely to impact their intentions to use FDAs. More specifically, if individuals believe that their social circle and family approve of their decision to use FDAs to order food, their intentions to use FDAs for that purpose will increase. Hence, we propose the following hypothesis: H4. Trust in FDAs is positively associated with a) intentions to use FDAs and b) shopping routine. Table 1 i Table 1 Description of study constructs. Table 1 Description of study constructs. Study constructs Brief description Reference Attitude An individual’s firm belief regarding the value of performing or not performing a particular behavior Fishbein and Ajzen (1975) Subjective norms The social pressure a person faces while performing a particular behavior Ajzen and Madden (1986) Perceived behavioral control A consumer’s perception that he or she has the necessary knowledge and resources to perform a specific task Ajzen (1991) Trust A consumer’s positive belief about the reliability and quality of the services offered Shankar et al. (2020) Leftover reuse routine An individual’s method of reusing and storing food leftovers Stancu et al. (2016) Intentions to use A consumer’s positive intent toward FDAs usage Ajzen (1991) Shopping routine The consumer practice of purchasing more food than required Evans (2012) Willingness to pay extra for eco-friendly packaging A consumer’s willingness to pay extra for socially responsible products Yadav et al. (2019) Number of years of usage A consumer’s affiliation tenure or duration of usage with FDAs service providers M. K. Kim et al. (2016) The TPB’s proven efficacy in explaining food consumption behavior in general as well as consumer behavior in the specific context of FDAs makes it a suitable theoretical framework for our study. In the interest of exploring the typicality of food waste behavior in the FDA context effectively, we go a step further and extend the TPB with relevant con structs. In particular, we extend the TPB framework by including con sumers’ trust in FDAs to capture their motivation to use FDAs and leftover reuse routine to capture their internal offsetting mechanism/ Fig. 1. Conceptual model. Fig. 1. Conceptual model. Fig. 1. Conceptual model. 3 A. Shankar et al. Technovation xxx (xxxx) xxx consumption (Mullan et al., 2013). Extending this evidence to the FDA context, we argue that consumers are more likely to use FDAs when they believe in their own ability to do so. Hence, we hypothesize as follows: H3. Perceived behavioral control is positively associated with in tentions to use FDAs. 3.1. Attitude, subjective norms, perceived behavioral control, and intentions to use FDAs 3.1. Attitude, subjective norms, perceived behavioral control, and intentions to use FDAs H3. Perceived behavioral control is positively associated with in tentions to use FDAs. Attitude refers to an individual’s positive or negative assessment of performing a behavior (Ajzen, 1991). Previous studies have confirmed that consumers who exhibit a positive attitude will engage in a behavior toward a subject (Ajzen, 2011), such as technology or brand use (Tan et al., 2018). Scholars have also found attitude to be significantly associated with consumers’ adoption behavior in several contexts, including green buying behavior (Yadav and Pathak, 2017), web-rooming behavior (Arora and Sahney, 2018), and food waste behavior (Stefan et al., 2013) and food over-ordering behavior (Talwar et al., 2022a). Past findings further suggest that personal characteristics, situational factors, experience, and product performance are crucial elements contributing to consumers’ attitudes toward an online service and subsequently to their responses (Shankar and Datta, 2018). 3.2. Trust, intentions to use FDAs, and shopping routine 3. Hypotheses development consumption (Mullan et al., 2013). Extending this evidence to the FDA context, we argue that consumers are more likely to use FDAs when they believe in their own ability to do so. Hence, we hypothesize as follows: 3.2. Trust, intentions to use FDAs, and shopping routine Trust refers to an individual’s positive belief about the quality and reliability of a product or service based on past experiences (Shankar et al., 2020). Recent scholarly literature has identified trust as a crucial determinant of consumers’ adoption of online services and of their perception of online service providers (Shankar and Jebarajakirthy, 2019; Patil et al., 2020; Talwar et al., 2021a). Past studies suggest that due to the absence of face-to-face interactions between online service providers and consumers, trust plays an important role in reducing consumers’ perceived risks and forming a positive response toward service providers in mobile commerce (Matute et al., 2016; Shaw, 2014). It is, therefore, hardly surprising that trust is not only positively asso ciated with consumers’ intentions to use online services (Li´ebana-Cabanillas et al., 2014) but also has a significant positive as sociation with positive word-of-mouth in online settings (Talwar et al., 2021a). The above-presented evidence provides sufficient basis for us to contend that in the specific case of FDAs, too, trust is likely to play a positive role in enhancing consumers’ intentions to use. In addition, we extrapolate the existing findings regarding the positive role of trust in the online environment to suggest that consumers’ trust in FDAs may lead them to order more food than they require. In other words, trust in FDAs, which we expect to increase consumers’ intentions to use, is also likely to encourage consumers to indulge in shopping routines that enable them to enjoy the benefits offered of FDAs, such as free delivery loyalty programs, quantity discounts, and so on (Yeo et al., 2017; Xu and Huang, 2019). Hence, we propose the following hypothesis: In the context of our study, we propose the effect of attitude on FDA usage. Given past findings regarding the effect of attitude on outcomes, we expect attitude to influence consumers’ decisions about using FDAs for food delivery. More specifically, we expect a positive attitude to increase consumers’ intentions to use FDAs to order food. In contrast, consumers who exhibit a negative attitude toward FDAs will be less likely to use FDAs to order food. Based on the preceding discussion, we propose the following association: 3.3. Leftover reuse routine, intentions to use FDAs, and shopping routine PBC has also been found to have a significant impact on consumers’ behavior in the context of food We interpret past findings to suggest that leftover reuse routine is not only a viable food waste mitigation strategy but also an approach that many consumers have begun adopting to reduce food waste. Extrapo lating this thought to the present context, we posit that consumers may—encouraged by their well-established leftover reuse routi nes—find it even more convenient and cost-effective to have food delivered to their location than to visit a restaurant. Because many consumers do have food delivered to their homes, which increases the probability and ease of reusing unconsumed food, the presence of a well- established leftover reuse routine is also likely to encourage them to order more food than required—whether out of temptation or the desire to take advantage of deals and offers. In addition, past studies have observed that most consumers fail to predict their required food intake accurately when ordering food online to consume at home (Yeo et al., 4 A. Shankar et al. Technovation xxx (xxxx) xxx 2017; Kapoor and Vij, 2018). This could also result in over-ordering. Thus, although a priori evidence is lack, the preceding discussion pro vides us sufficient rationale to assume a positive correlation between leftover reuse routine and shopping routine. Thereby, we propose the following association: instance, consumers who have been ordering food from FDAs for some time may develop a sense of trust toward them, which may also increase their usage intentions (Sharma et al., 2021). Furthermore, due to their long-standing positive experience using FDAs, such consumers might not only have stronger use intentions but also have an established and effective leftover reuse routine if the need arises. At the same time, consumers with a long history of ordering food using FDAs may have developed an ability to better plan their order size, which would help them avoid ordering an excessive amount of food. In other words, experienced FDA users are less likely to impulsively order too much, creating the possibility of food waste. Consumers with a long history of using FDAs are also likely to have more knowledge about the quantity likely to be delivered per food item, which can also help them better manage their order size/quantity. 3.5. Control variables We include four control variables to capture the socio-demographic profile of the consumer—age, gender, educational background, and economic background. Our choice of these variables as controls is rooted in prior findings that suggest a significant impact of various socio- demographic variables on consumers’ behavioral intentions toward food consumption (Stancu et al., 2016; Ray et al., 2019). H6. Intentions to use FDAs are positively associated with shopping routine. 4.1. Sampling and data collection We collected the data from consumers of leading FDAs providers in India, including Zomato, Swiggy, and Uber Eats. We employed a struc tured questionnaire comprised of items adapted from the prior literature on FDAs in the context of food waste. To ensure that we collected the data from active FDA consumers, we asked two screening questions: “Do you order food through FDAs?” and “During the last three months, how many times have you ordered food using FDAs?” The study utilized a shopping mall intervention study to collect the data from individuals in two major cities in India. Our research assistants approached 1000 re spondents and collected a total of 525 completed surveys (for a response rate of 52.5 percent). During the data screening process, we identified 38 responses as incomplete; hence, we advanced the remaining 487 re sponses (n = 329, 67.6% males) for further analysis. The collected data were normally distributed and free from outlier issues. The age of the respondents ranged from 24 to 32 years (mean age = 28.65 years, SD = 3.13 years), and 46% of the respondents (n = 227) had been using FDAs for more than one year. Prior studies have noted that an increasing awareness of environ mental issues (Prakash et al., 2019) and concern for environmental protection has made consumers more willing to pay extra for eco-friendly packaging (e.g., Yadav et al., 2019). This willingness to pay an additional cost for eco-friendly packaging is a crucial indicator of consumers’ behavioral intentions toward environment-friendly prod ucts (Yadav et al., 2019). We extrapolate from this finding to assume that the same environmental awareness that causes consumers to pay extra for eco-friendly packaging also inclines them to order only as much food as they require. In other words, consumers’ willingness to pay extra for eco-friendly packaging symbolizes their environmental concern, which is also likely to make them sensitive to avoiding food waste. We also expect consumers who are willing to pay extra for eco-friendly packaging to have a well-planned routine to reuse any food that re mains unconsumed after a meal. In addition, we suggest that environ mentally conscious consumers may have stronger intentions to use FDAs because they perceive such apps to be a more environmentally friendly way of procuring food. In sum, we suggest that willingness to pay for eco-friendly packaging may moderate the proposed associations. Hence, we hypothesize as follows: 3.3. Leftover reuse routine, intentions to use FDAs, and shopping routine Based on the logic of these arguments and even in the absence of any prior examination of these effects, we propose the following hypothesis: 2017; Kapoor and Vij, 2018). This could also result in over-ordering. Thus, although a priori evidence is lack, the preceding discussion pro vides us sufficient rationale to assume a positive correlation between leftover reuse routine and shopping routine. Thereby, we propose the following association: H5. Leftover reuse routine is positively associated with a) intentions to use FDAs and b) shopping routine. Consumers’ routines play an important role in their food consump tion decisions. For instance, planning routines and shopping routines are relevant factors in explaining consumers’ food waste behavior. Planning routine refers to checking one’s inventory, making a list of required items, and planning a meal in advance (Stefan et al., 2013), whereas shopping routine refers to purchasing more food than one requires (Stefan et al., 2013). Shopping routine is the variable of interest in the current study because it represents a behavior that entails the possibility of food waste generation. Past studies have noted that consumers, including those ordering via FDAs (Sharma et al., 2021), tend to pur chase excessive amounts of food so that they can take advantage of bulk order discounts and reduce/save on delivery charges (Stancu et al., 2016). In fact, FDAs use discounts as incentives to attract consumers and encourage them to order large quantities (Sharma et al., 2021). In sum, we speculate that FDAs’ pricing schemes and offers are structured so that consumers with positive intentions to use FDAs are likely to order more food than they require to satiate their hunger. Accordingly, we propose the following hypothesis: H8. Number of years of FDA usage positively moderates the relation ships between a) trust in FDAs and intentions to use FDAs, b) trust in FDAs and shopping routine, c) leftover reuse routine and intentions to use FDAs, and d) leftover reuse routine and shopping routine. 4. Methodology Taking both the sustainability dimension and the consumer perspective into consideration, we include two moderating varia bles—willingness to pay for eco-friendly packaging and number of years of FDA usage—to capture the effect of individual differences in the hypothesized associations. Our choice of these variables is grounded in the extended literature on consumer behavior. Table 2 Table 2 Study measures, items, and factor loadings. Scale items λ M SD Attitude (ATT; Stancu et al., 2016) 3.55 0.93 ATT1: Ordering food via FDAs is gratifying. .80 ATT2: Ordering food via FDAs is pleasant. .87 ATT3: Ordering food via FDAs is satisfying. .89 ATT4: Ordering food via FDAs is good. .82 Subjective norm (SN; Ajzen, 1991) 3.43 1.07 SN1: Most people who are important to me would approve of my ordering food via FDAs instead of conventional restaurants. .87 SN2: Most people who are important to me want me to order food via FDAs instead of conventional restaurants. .90 SN3: Most people who are important to me think that I should order food via FDAs instead of conventional markets. .85 Perceived behavioral control (PBC; Stefan et al., 2013) 4.00 0.89 PBC1: Ordering food from FDAs is easy for me. .90 PBC2: If I wanted to, I could easily order food from FDAs. .89 PBC3: Ordering food from FDAs depends entirely on me. .58 Shopping routine (SR; Stefan et al., 2013) 3.04 1.21 SR1: I often buy unintended food items when ordering via FDAs. .76 SR2: I often buy too much food when ordering via FDAs. .94 SR3: I usually buy more food than required if FDAs offer good value for the money. .80 Trust (TR; Yadav et al., 2019) 3.47 1.21 TR1: I perceive ordering food via FDAs to be reliable. .88 TR2: Ordering food via FDAs appears trustable to me. .90 TR3: No risk is involved in ordering food via FDAs. .84 Leftovers reuse routine (LR; Stancu et al., 2016) 3.53 1.02 LR1: Leftovers are usually eaten as such or just reheated when used again. .79 LR2: Leftovers are usually transformed into a different dish by adding some ingredients before eating them. .77 LR3: Leftovers are stored in appropriate conditions so they will last. .74 Intentions to use (IU; Ajzen, 1991) 3.49 1.05 IU1: I intend to order food via FDAs in the coming week. .84 IU2: I plan to order food via FDAs in the coming week. .88 IU3: I am willing to order food via FDAs in the coming week. .79 Note. λ = Standardized factor loadings; M = Mean; SD = Standard deviation. 5.4. Structural model 5.4. Structural model all scale items and sources. The SEM we used to validate the hypotheses also indicated a good fit (χ2/df = 2.42, CFI = 0.96, TLI = 0.94, RMSEA = 0.05; Hair et al., 2010). The model explained 71.6% of the variance in consumers’ intentions to use FDAs and 34.4% of the variance in their shopping routine. Fig. 2 presents the results of the statistical analysis. i 5.3. Control variables Previous studies have demonstrated that including control variables may improve the robustness of a study’s findings (Talwar et al., 2020). Hence, we controlled for the confounding effects of the respondents’ age, gender, educational background, and economic background on our structural model. None of the control variables exerted any significant influence on consumers’ usage intentions. Similarly, gender and educational background had no significant influence on shopping routine. However, age and economic background did significantly in fluence shopping routine. 4.3. Data analysis method We employed a two-step approach to analyze the data (Anderson and Gerbing, 1988). We performed a confirmatory factor analysis (CFA) to examine the reliability and validity of the measurement scales. Recog nizing that structural equation modeling (SEM) is the most suitable tool to access complex multiple latent constructs and relationships (Hair et al., 2010), we utilized SEM in AMOS 26 to examine the proposed hypotheses. We also considered SEM the most appropriate tool for data analysis because our study proposes intention to use FDAs as both an antecedent and outcome variable. Finally, we utilized PROCESS macro for SPSS to validate the hypotheses related to moderation. The results for the hypothesis testing indicated the significant posi tive associations of attitude (H1: β = 0.68, p < 0.001) and subjective norms (H2: β = 0.16, p < 0.05) with intentions to use FDAs. Next, shopping routine exhibited a positive association with trust in FDAs (H4b: β = 0.22, p < 0.01), leftover reuse routine (H5b: β = 0.30, p < 0.001), and intentions to use FDAs (H6: β = 0.18, p < 0.05). Thus, H1, H2, H4b, H5b, and H6 received support. Surprisingly, we observed no significant associations of intentions to use with PBC (H3: β = −0.08, p > 0.05), trust in FDAs (H4a: β = 0.10, p > 0.05), and leftover reuse routine (H5a: β = −0.02, p > 0.05). Hence, we rejected H3, H4a, and H5a. 5. Results 5. Results 5.2. Measurement model We examined the validity, reliability, and dimensionality of the constructs via a CFA. The CFA indicated a good model fit (χ2/df = 2.64, CFI = 0.96, TLI = 0.95, RMSEA = 0.06). The average variance extracted (AVE) values for all study constructs exceeded the threshold of 0.5, while their composite reliability (CR) values exceeded the threshold of 0.7, suggesting convergent validity (Hair et al., 2010). As Table 2 shows, the factor loadings for all constructs were significant (p < 0.01) and above 0.7, which is an additional indicator of convergent validity (Hair et al., 2010). Moreover, the square root of the AVE value of each construct exceeded the correlation coefficient of the corresponding construct, indicating the constructs’ discriminant validity (Fornell and Larcker, 1981). Furthermore, the correlation among study constructs was significant and below 0.90 (Tabachnick and Fidell, 2012). Finally, the CR values for the study constructs, which exceeded 0.70, established the study’s reliability. Table 3 presents these values. 4.2. Measures and questionnaire development The items measuring the study constructs were taken from previ ously validated scales. However, we modified the wording where appropriate to make the items suitable for the study context. A group of experts, which included three professors and three researchers special izing in the field of marketing, consumer behavior, and hospitality, content-tested the survey instrument. In addition, we conducted a pilot study with 10 participants representing the target population of FDA consumers. We then made the above-mentioned minor modifications of the survey items based on the feedback from the expert group and the pilot study. We used a five-point Likert scale (1 = “Strongly disagree” to 5 = “Strongly agree”) to operationalize all constructs. Table 2 presents H7. Willingness to pay for eco-friendly packaging positively moder ates the relationships between a) trust in FDAs and intentions to use FDAs, b) trust in FDAs and shopping routine, c) food leftover reuse routine and intentions to use FDAs, and d) food leftover reuse routine and shopping routine. i Because consumers’ past experiences and affiliation tenure are likely to affect their usage decisions, FDA usage experience has a crucial impact on consumers’ behavioral intentions (M. K. Kim et al., 2016). For 5 A. Shankar et al. Technovation xxx (xxxx) xxx respondents, common method bias (CMB) also posed a threat. There fore, consistent with Podsakoff et al.’s (2003) recommendation, we performed Harman’s single-factor test to examine the data for CMB. The analysis revealed that a single factor accounted for only 46.07% of the total variance, which is below the recommended threshold value of 50 percent and thus indicates that the data were free from CMB. Table 3 TR→ ITU .05 .69 .49 −.0957 .1998 No TR → SR .16 1.50 .13 −.0489 .3646 No LR → ITU .24 2.44 .02 .0461 .4260 Yes LR → SR .27 2.46 .01 .0531 .4773 Yes Moderator 2: Number of years of FDA usage В t p LLCI ULCI Moderation? TR→ ITU .02 1.04 .30 −.0201 .0650 No TR → SR .05 1.55 .12 −.0125 .1059 No LR → ITU .12 4.25 .00 .0640 .1740 Yes LR → SR .08 2.64 .01 .0217 .1469 Yes Moderator 1: Willingness to pay for eco-friendly packaging β t p LLCI ULCI Moderation? TR→ ITU .05 .69 .49 −.0957 .1998 No TR → SR .16 1.50 .13 −.0489 .3646 No LR → ITU .24 2.44 .02 .0461 .4260 Yes LR → SR .27 2.46 .01 .0531 .4773 Yes Moderator 2: Number of years of FDA usage В t p LLCI ULCI Moderation? TR→ ITU .02 1.04 .30 −.0201 .0650 No TR → SR .05 1.55 .12 −.0125 .1059 No LR → ITU .12 4.25 .00 .0640 .1740 Yes LR → SR .08 2.64 .01 .0217 .1469 Yes Moderator 1: Willingness to pay for eco-friendly packaging We offer two explanations for the lack of statistical support for H3, which had proposed a positive association between PBC and intentions to use. First, because people now use technology, including mobile apps (such as FDAs), extensively and instinctively (i.e., they need not engage in any conscious thought regarding their capability or control), PBC may no longer be an essential element for their intentions to use (Zhao et al., 2018). Another possible reason for the lack of support for the association of PBC with intentions to use could be the interactive features of FDAs, which make ordering food easy and ensure that they need not possess any specific technical knowledge or expertise. FDAs usage positively moderated the relationship of leftover reuse routine with intentions to use (β = 0.12, p < 0.01) and shopping routine (β = 0.08, p < 0.05). Thus, H8c and H8d received support, while H8a and H8b did not. i Unexpectedly, our results also fail to support H4a and H5a, sug gesting that trust in FDAs and leftover reuse routine exert no significant impact on consumers’ intentions to use FDAs. Table 3 Based on the extended literature, one would expect that consumers’ perceptions that FDAs are reliable, trustworthy, and relatively safe to use would increase their intentions to use FDAs. Our results, however, contradict this expecta tion. Because past studies have demonstrated a crucial role for trust in consumers’ adoption behavior (e.g., Shankar et al., 2020), we find it difficult to explain the apparent lack of association between trust and intentions. We thus call for further exploration with data collected from Fig. 3a–d also illustrate the results. Fig. 3a–d also illustrate the results. 5.5. Moderation analysis After ascertaining that the data were normally distributed, we computed the variance inflation factors (VIF) for the independent vari ables to determine whether the issue of multicollinearity affected the collected data. The test produced VIF values below the recommended threshold of 5.0 (Hair et al., 2010), thus indicating that all five inde pendent variables were free from any issues of multicollinearity. Spe cifically, the VIF values for the study variables ranged from 1.13 to 2.88. The moderation analysis conducted using Model 1 in PROCESS macro involved bootstrapping the effects 5000 times, resulting in the production of interaction terms and their 95% confidence intervals. As the results in Table 4 reveal, willingness to pay for eco-friendly pack aging positively and significantly moderated the relationship between leftover reuse routine with intentions to use FDAs (β = 0.24, p < 0.05) and shopping routine (β = 0.27, p < 0.05). Thus, H7c and H7d received support, while H7a and H7b were rejected. Next, number of years of i Because we measured the dependent and independent variables through responses to a single instrument from the same set of 6 Technovation xxx (xxxx) xxx A. Shankar et al. Table 3 Table 3 Validity and reliability analysis. CR AVE MSV ASV LR IU ATT SN PBC SR TR LR .81 .59 .20 .13 .77 IU .88 .70 .70 .38 .32 .84 ATT .91 .72 .70 .45 .36 .83 .85 SN .91 .76 .60 .39 .36 .72 .78 .87 PBC .84 .65 .39 .23 .24 .50 .62 .57 .80 SR .87 .70 .23 .16 .44 .43 .42 .39 .15 .84 TR .91 .76 .69 .43 .44 .74 .83 .78 .57 .48 .87 Note. Composite reliability = CR, Average variance extracted = AVE, Maximum shared variance = MSV, Average shared variance = ASV, LR = Leftovers reuse routine, IU = Intentions to use, ATT = Attitude, SN = Subjective norm, PBC= Perceived behavioral control, SR = Shopping routine, TR = Trust; Square root of AVE in bold. Note. Composite reliability = CR, Average variance extracted = AVE, Maximum shared variance = MSV, Average shared variance = ASV, LR = Leftovers reuse routin IU = Intentions to use, ATT = Attitude, SN = Subjective norm, PBC= Perceived behavioral control, SR = Shopping routine, TR = Trust; Square root of AVE in bol Fig. 2. Results of hypothesis testing. Fig. 2. Results of hypothesis testing. previous studies (Yadav et al., 2019). To elaborate, support for H1 confirms our expectation that consumers who perceive ordering food via FDAs to be gratifying, pleasant, satisfying, and good will exhibit strong usage intentions in the near future. The results also confirm the crucial influence of subjective norms on consumers’ usage intentions toward FDAs. This finding likewise aligns with the results of past studies, albeit in different contexts (Yadav et al., 2019; Shankar and Jain, 2021). This finding implies that if most people who are important to consumers approve of and encourage them to order food via FDAs, those consumers will be more likely to use FDAs for food delivery in the near future. Table 4 Results of moderation analysis. FDAs usage positively moderated the relationship of leftover reuse routine with intentions to use (β = 0.12, p < 0.01) and shopping routine (β = 0.08, p < 0.05). Thus, H8c and H8d received support, while H8a and H8b did not. Fig. 3a–d also illustrate the results. y Moderator 1: Willingness to pay for eco-friendly packaging β t p LLCI ULCI Moderation? 6. Discussion The results for the main effects indicate that attitude and subjective norms significantly impact intentions to use FDAs. In contrast, PBC, the third classic TPB construct, did not significantly influence intentions to use. Hence, H1 and H2 stand supported, but H3 does not. This outcome implies that consumers who have a positive attitude toward FDAs tend to adopt such apps for online food ordering, a finding that aligns with 7 A. Shankar et al. Technovation xxx (xxxx) xxx Fig. 3a. Moderating effect of willingness to pay for eco-friendly packaging. Fig. 3a. Moderating effect of willingness to pay for eco-friendly packaging. Fig. 3b. Moderating effect of willingness to pay for eco-friendly packaging. Fig. 3c. Moderating effect of number of years of FDA usage. Fig. 3a. Moderating effect of willingness to pay for eco-friendly packaging. Fig. 3a. Moderating effect of willingness to pay for eco-friendly packaging. Fig. 3b. Moderating effect of willingness to pay for eco-friendly packaging. Fig. 3b. Moderating effect of willingness to pay for eco-friendly packaging. Fig. 3b. Moderating effect of willingness to pay for eco-friendly packaging. Fig. 3c. Moderating effect of number of years of FDA usage. Fig. 3c. Moderating effect of number of years of FDA usage. A. Shankar et al. Technovation xxx (xxxx) xxx Fig. 3d. Moderating effect of number of years of FDA usage. Fig. 3d. Moderating effect of number of years of FDA usage. a larger, more varied sample to confirm that trust plays no role in driving FDA usage intentions. indicates that at low intensity of leftover reuse routine, consumers who are willing to pay extra for eco-friendly packaging exhibit a lower in tensity of shopping routine than do those who are unwilling to pay extra for eco-friendly packaging. In contrast, at high intensity of leftover reuse routine, consumers who are willing to pay more for eco-friendly pack aging exhibit higher intensity of shopping routine than do those who are unwilling to pay these extra costs. The results also do not support the anticipated positive association between leftover reuse routine and intentions to use. 6. Discussion Although past studies have acknowledged leftover reuse routine as a significant and valuable consumer food waste reduction strategy (Stefan et al., 2013; Stancu et al., 2016; Talwar et al., 2022a), we offer the fact that con sumers do not believe their FDA usage will result in food waste as a possible explanation for the apparent lack of association between left over reuse routine and intentions to use FDA. In other words, in con sumers’ minds, FDA usage and food waste are not related; therefore, any thoughts of leftover reuse fail to impact their usage intentions. ii Next, while H8c and H8d stand supported, H8a and H8b do not. Support for H8c demonstrates that number of years of FDA usage exerts the same effect on the usage intentions of consumers with a high in tensity of leftover reuse routine. However, it has a varied effect on the usage intentions of consumers with a low intensity of leftover reuse routine. To elaborate, the results suggest that consumers who have been using FDAs for more than one year exhibit stronger intentions to use FDAs than do those who have been using FDAs for less than one year. At the same time, support for H8d implies that the number of years of FDA usage has varied effects on the shopping routines of consumers who exhibit different intensities of leftover reuse routine. Explaining further, the results can be interpreted to mean that at a low intensity of leftover reuse routine, consumers who have been using FDAs for more than one year exhibit a higher intensity of shopping routine than do those who have been using FDAs for less than one year. In comparison, at high intensity of leftover reuse routine, consumers who have been using FDAs for more than one year exhibit lower intensity of shopping routine than do those who have been using FDAs for less than one year. In contrast, H4b and H5b stand supported, confirming the significant positive associations of trust in FDAs and leftover reuse routine with shopping routine. These findings are consistent with previous studies (Patil et al., 2020; Sharma et al., 2021). 6. Discussion The support we find for the positive association between trust and shopping routine suggests that FDA users who perceive such apps to be a reliable and safe avenue for ordering food will tend to order food items they did not originally intend to order, especially if those items offer good value for the money. Similarly, support for the positive association between leftover reuse routine and shopping routine indicates that consumers’ well-established routine of storing and subsequently using leftovers—either by simply reheating them or transforming them into a different dish—increases their tendency to over-order food from FDAs, whether instinctively or in response to the deals these apps offer. i 7.2. Practical implications Our study offers four academic implications. First, the study con tributes to a keener understanding of consumer adoption and usage in tentions toward FDAs, a rapidly emerging digital form of hospitality (Rivera, 2019). Thus far, the related literature has remained skewed toward the technology acceptance perspective, i.e., the opportunities and threats associated with FDAs as a technology tool (e.g., Correa et al., 2019). Meanwhile, it offers fewer insights on consumer choice behavior and preferences (e.g., Kaur et al., 2021). The findings of this study illuminate the antecedents of consumers’ usage intentions toward FDAs, including socio-psychological factors, such as attitude, subjective norms, and perceived behavioral control. A deeper knowledge of the mechanism through which these drivers impact consumers’ positive intentions toward FDAs enriches the existing literature and provides a platform for future research in the area. Our study also offers four implications for the tourism and hospitality sector. Because FDAs are an integral part of the restaurant industry (Cho et al., 2019), our findings are particularly crucial to managers therein. The study likewise offers insights for online food delivery service pro viders as they craft strategies to improve the consumer experience. The findings of our study can also help restaurants and online food delivery service providers to better understand the key factors affecting con sumer responses to FDAs. First, with regard to the implications for restaurant managers, the findings of our study uncover the drivers of consumers’ intentions to use FDAs to order prepared food for delivery to their preferred location. Past scholars have identified app-related factors, such as information quality, design, convenience, and others (Cho et al., 2019; Ray et al., 2019; Sharma et al., 2021), with the potential to increase FDA usage—for example, by improving the ordering interface. Our study builds upon these efforts to reveal the ways in which consumer-related variables, such as attitude and trust, impact usage intentions and over-ordering behavior. Our findings demonstrate that restaurants, which operate in a hyper-competitive segment, can enhance their profit margins by engaging consumers, building their trust, and encouraging them to develop positive attitudes, through collaborations with FDAs. Further more, restaurants can—by emphasizing leftover reuse—overcome the ethical dilemma involved in the association of over-ordering and food waste. 7.2. Practical implications Two possible strategies for this could be (a) to coordinate with FDAs to create a platform on which FDA users can share leftover reuse recipes and experiences (similar to the currently available option of online reviews) and (b) to place a sticker on food packages to indicate the date through which food is safe to eat/use. i Our second contribution involves shifting the focus of all stake holders to the possibility that FDA usage has a dark side in the form of food waste generation. Given their advantages and inherent features, FDAs may cause consumers to order more food than they require, which has the potential to generate food waste. This is a quite serious aspect because the United Nations has identified food waste as a key threat to sustainable development (UNSDG, 2018). Our study utilizes variables such as trust, leftover reuse routine, and shopping routine to illuminate the complex relationships among FDA usage, the possibility of food waste generation and mitigation, and other factors, including the packaging of food for delivery. Previous research largely focuses on the positive behavioral outcomes of consumers’ trust (Patil et al., 2020; Shankar et al., 2020). Scholars have also acknowledged leftover reuse routine as a crucial way of reducing food waste (Talwar et al., 2022a). Presenting a finer-grained and deeper understanding of the associated complexities, our findings challenge conventional thinking by high lighting the hidden negative effects of consumers’ trust in FDAs and their leftover reuse routine, which cause them to order more food than they require to satiate their hunger (i.e., to indulge in a shopping routine). Here, we find that trust and leftover reuse routines can increase consumers’ tendency to indulge in shopping routines, thereby increasing the possibility of food waste. To elaborate, we reveal that consumers’ trust in FDAs, which engenders positive feelings and rationalizes their indulgence in over-ordering through their leftover reuse routine, drives them to exhibit a higher intensity of shopping routine. In sum, our study advances the debate on food waste in the digital context, which has thus far remained confined to food-sharing platforms (e.g., Mazzucchelli et al., 2021; Nica-Avram et al., 2020). Second, our findings that subjective norms and attitudes are posi tively associated with usage intentions provide valuable insights for FDA service providers and marketers seeking to promote the use of their apps by improving customer engagement. 7. Conclusion Regarding the last direct effect, our findings support H6, indicating that intentions to use have a significant impact on shopping routine. This result aligns with the findings of previous studies regarding food over- ordering via FDAs (e.g., Sharma et al., 2021). Support for this associa tion confirms that consumers who intend to use FDAs to order food in the near future also tend to purchase more food than they can imme diately consume. This study aimed to investigate the ways in which FDA usage con tributes to food waste. The study employed an extended TPB model to understand various factors that drive consumers’ intentions to use FDAs. These intentions drive food over-ordering behavior, which, in turn, creates the possibility that such food will remain unconsumed and, ul timately, be wasted. In addition to the direct associations, which we grounded in the TPB framework, we also contemplated and investigated the effects of two relevant moderators—willingness to pay for eco- friendly packaging and number of years of FDA usage. Testing these moderators enabled us to develop a fuller understanding of the dy namics of the proposed associations. Specifically, we sought answers to four research questions by analyzing data from 478 FDA users. In response to RQ1, which examined the association of the classic TPB constructs (attitude, subjective norms, and PBC) with FDA usage in tentions, we found the positive associations of attitude and subjective norms with FDA usage intentions but no statistically significant In terms of the moderation effects, H7c and H7d are supported, but H7a and H7b are not. Support for H7c indicates that willingness to pay for eco-friendly packaging positively moderates the associations of leftover reuse routine with both intentions to use and shopping routine. To elaborate, consumers who are willing to pay for eco-friendly pack aging exhibit stronger intentions to use FDAs than do those who are unwilling to pay extra for eco-friendly packaging at both low and high leftover reuse routine intensities. However, the difference between the intentions of consumers who are willing and unwilling to pay extra for eco-friendly packaging is greater for consumers with a higher rather than lower level of leftover reuse routine. Similarly, support for H7d 9 A. Shankar et al. 7. Conclusion Technovation xxx (xxxx) xxx constructs to investigate variables that could promote behavior that generates food waste but also extended the theory by identifying con textually relevant constructs, such as trust in FDAs, leftover reuse routine, and shopping routine. association between PBC and FDA usage intentions. In the case of RQ2, which examined the associations of trust and leftover reuse routine with intentions to use and shopping routine, we observed the positive asso ciations of the former two variables only with shopping routine. Regarding RQ3, our results confirmed the proposed positive association between intentions and shopping routine. Finally, in response to RQ4, which examined the moderation effects of willingness to pay for eco- friendly packaging and number of years of FDA usage on the associa tions of trust and leftover reuse routine with intentions to use and shopping routine, our results confirmed all moderation effects. Our findings offer noteworthy theoretical and practical implications, which we discuss below. Finally, our study examines the moderating effects of relatively less explored yet contextually relevant variables—willingness to pay for eco- friendly packaging and number of years of FDA usage—on the associa tions of trust and leftover reuse routine with usage intentions and shopping routine. Although the existing literature has found these var iables to be crucial (M. K. Kim et al., 2016; Stefan et al., 2013; Yadav et al., 2019), scholars have made only limited efforts to examine their moderating effects. Thus, our study contributes to the existing literature by offering newer and fuller perspectives. References Mindset matters in purchasing online food deliveries during the pandemic: the application of construal level and regulatory focus theories. Int. J. Hospit. Manag. 91, 102677. Arora, S., Sahney, S., 2018. 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A multi-stakeholder partnership to fight food waste in the hospitality industry: a contribution to the United Nations Sustainable Development Goals 12 and 17. J. Sustain. Tourism 1–28. Finally, our study’s conceptualization and findings highlight the need for policymakers to contemplate the formulation of some regula tions or at least guidelines to sensitize FDAs to the ways in which their promotional approaches contribute to food waste and hinder the achievement of sustainability targets, especially SDG 12. Dhir, A., Sadiq, M., Talwar, S., Sakashita, M., Kaur, P., 2021. Why do retail consumers buy green apparel? A knowledge-attitude-behaviour-context perspective. J. Retailing Consum. Serv. 59, 102398. Dhir, A., Talwar, S., Kaur, P., Malibari, A., 2020. Food waste in hospitality and food services: a systematic literature review and framework development approach. J. Clean. Prod. 270, 122861. Evans, D., 2012. Beyond the throwaway society: Ordinary domestic practice and a sociological approach to household food waste. Sociology 46 (1), 41–56. 7.2. Practical implications Because intentions to use increase under the influence of peers and friends’ opinions, FDA providers can enlist existing users to influence their social circle. To this end, they can request that users post testimonials for their apps. Ideally, FDA service providers should keep these testimonials distinct from more traditional online reviews and ratings. FDAs can, moreover, encourage users to tag their friends in these testimonials. Overall, our findings regarding the positive drivers of FDA usage can guide service providers to develop effective marketing strategies to increase their market share and profits. At the same time, by highlighting the importance of leftover reuse routine and the ways it can be leveraged not only to persuade users to order more but also to educate them to avoid food waste, we help service providers to advance their commercial interests in ways that align with the UN’s Sustainable Development Goals. i Third, our study deepens the scholarly literature in the area by extending the TPB to examine and explicate consumer behavior and decision-making in hospitality settings while also accounting for the sustainability perspective. Scholars have made scant efforts thus far to examine the consumer response to FDAs by invoking the TPB or extending it to make it more hospitality-specific (J. J. Kim et al., 2021). By using the TPB as our theoretical framework, we thus contribute to the advancement of the TPB in contemporary and diverse consumer behavior domains. Our study not only modeled the classic TPB Third, our findings can aid the marketers on whom FDAs rely to promote their apps’ usage. For instance, our findings regarding the positive moderating effects of willingness to pay for eco-friendly pack aging and number of years of FDA usage can help marketers to develop effective strategies to positively impact existing consumers’ continued 10 A. Shankar et al. Technovation xxx (xxxx) xxx Choe, J.Y., Kim, J.J., Hwang, J., 2021. Innovative marketing strategies for the successful construction of drone food delivery services: merging TAM with TPB. J. Trav. Tourism Market. 38 (1), 16–30. usage intentions. One means to this end could be to introduce a loyalty program to enhance the user experience, provide promotional offers to increase order frequency, and provide high-quality and hygienic pack aging to enhance consumers’ willingness to pay for eco-friendly pack aging, which will, in turn, increase food ordering through FDAs. 7.3. Limitations and future research directions S i Si l S 2020 A li i f i l i l Hwang, J., Lee, J.S., Kim, J.J., Sial, M.S., 2020. Application of internal environmental locus of control to the context of eco-friendly drone food delivery services. J. Sustain. Tourism 1–19. Kapoor, A.P., Vij, M., 2018. Technology at the dinner table: ordering food online through mobile apps. J. Retailing Consum. Serv. 43, 342–351. Kaur, P., Dhir, A., Talwar, S., Alrasheedy, M., 2021a. Systematic literature review of food waste in educational institutions: setting the research agenda. Int. J. Contemp. Hospit. Manag. 33 (4), 1160–1193. Kaur, P., Dhir, A., Talwar, S., Ghuman, K., 2021b. The value proposition of food delivery apps from the perspective of theory of consumption value. Int. J. Contemp. Hospit. Manag. 33 (4), 1129–1159. Kaur, P., Talwar, S., Islam, N., Salo, J., Dhir, A., 2022. The effect of the valence of forgiveness to service recovery strategies and service outcomes in food delivery apps. J. 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The present study makes important empirical contributions to the literature on hospitality, online food delivery services, and food waste. However, its findings must be interpreted in light of certain limitations. First, this study’s sample included only FDAs users in India. Hence, the findings are not generalizable to other countries and cultures. However, the robustness of our findings supports our conceptualization, suggest ing that future researchers can replicate our model in different contexts. Second, our study used cross-sectional data and thus can only detect associations among variables. Future researchers should conduct lon gitudinal survey-based studies or experimental studies to overcome this limitation and explore causality among the variables. This recommen dation aligns with those of recent scholars who have underscored the need to inject methodological variety into investigations of such be haviors (De Visser-Amundson, 2020). 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https://openalex.org/W4220672028	https://appliednetsci.springeropen.com/track/pdf/10.1007/s41109-022-00456-0	English	null	Distribution of labor, productivity and innovation in collaborative science	Applied network science	2,022	cc-by	8,167	© 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 mate- rial. 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/. Distribution of labor, productivity and innovation in collaborative science Floriana Gargiulo1* , Maria Castaldo2, Tommaso Venturini3 and Paolo Frasca2 Correspondence: floriana.gargiulo@cnrs.fr 1 CNRS, Université Paris- Sorbonne - Paris IV, GEMASS, Paris, France Full list of author information is available at the end of the article Abstract In this paper, we investigate the process of scientific discovery using an under- exploited source of information: the Polymath projects. Polymath projects are an origi- nal attempt to solve a series mathematical problems collectively and in a collaborative online environment. To investigate the Polymath experiment, we analyze all the posts related to the projects that have resulted in a peer-reviewed publication. We focus in particular on the organization of the scientific labor and on the innovations that result from the contributions of the different authors. We find that a high presence of occa- sional contributors increases the productivity of the most active users and the overall productivity of the forums (i.e., the number of posts grows super-linearly with the number of contributors). We argue that, in large-scale collaborations, the serendipitous interaction between occasional contributors can be crucial to the scientific process, and individual contributions from occasional participants can open new directions of research. Keywords: Polymath, Collaborative science, Innovation Gargiulo et al. Applied Network Science (2022) 7:19 https://doi.org/10.1007/s41109-022-00456-0 Gargiulo et al. Applied Network Science (2022) 7:19 https://doi.org/10.1007/s41109-022-00456-0 Applied Network Science Open Access Introduction With the advent of Internet technologies, we have witnessed the emergence of large- scale collaborative platforms that enable the creation of open-source content through the voluntary participation of large numbers of users. These platforms have fundamen- tally changed the practices of knowledge production and consumption, turning into pub- lic goods a number of cultural and scientific resources. The best-known example of these collective cultural platforms is the online encyclopedia Wikipedia, which has become one of the most important and trusted sources of information worldwide (Anthony et al. 2009; Fallis 2008). Other examples of online collaboration can be found in software pro- duction, from the Linux movement, to GitHub, to Q&A systems, like Stack Overflow, and in scientific task-sharing projects, like Galaxy Zoo. i Platforms such as the ones mentioned above foster interactions and allow a multiplic- ity of participants with different expertise to come together without a rigid and hier- archical role structure and collectively performed together simple and complex tasks. These collaborative systems have proved that spontaneous interactions between users can produce a shared knowledge that goes beyond the mere aggregation of individual Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science Page 2 of 15 contributions, a process referred to as “collective intelligence” (Straub et al. 2021; Grasso and Convertino 2012). Despite the success of these platforms and even though academic institutions have long insisted on the idea of open and participatory science, there are few actual exam- ples of large-scale collective production in science. The conceptual framework of online collaborative structures raises several important questions when applied to scientific production: Is science the craft of many or of few? Can research be conducted in a large-scale open collaborative environment? Can sci- ence be based on collaboration rather than competition? These questions are discussed in the book “Reinventing discovery” by Nielsen (2011), which presents several examples of collective problem solving. Among the cases described by Nielsen, one—the Polymath project—has attracted our attention, because it can be studied not only ethnographically but also computationally, since all its contributions are available as digital records. The first Polymath project was proposed in 2009 by mathematician Tim Gowers, who, with a post on his blog, invited mathematicians to find a combinatorial proof of the den- sity version of the Hales-Jewett Theorem, using a dedicated thread of discussion. Introduction Since then, fifteen other Polymath projects have been launched, six of which have resulted in one or more peer-reviewed publications signed with the collective name “Polymath Col- laboration”. The Polymath blogs not only enable the study of an important project in col- laborative science, but also provide an unprecedented playground for the in-depth study of discovery processes. In our work, we present a comprehensive statistical analysis of the Polymath eco- system, looking specifically at the activities of participants and the content that they produce. Our findings are based on all projects that have resulted in a peer-reviewed publication (projects 1,4,5,8,15). We did not examine the other projects because they were abandoned by their contributors at an early stage and their data are insufficient to support a robust analysis. After discussing some related work in Sects. , 1 we present the data and methods used in our analysis. In Sect. 1 we present our results. In Sects. 3.1 and 3.2 we analyze the internal structure of collaboration and its role in productivity patterns. We identify a clear hierarchy in participation patterns with a hyperactive elite responsible for 80% of the work. At the same time, we show that collaborative architecture plays an important role in promoting individual production: Indeed, we observe a dynamic of super-pro- duction in which the presence of occasional participants helps to increase the productiv- ity of the elite. After analyzing the organization of work in open science, we focus on the mechanisms of scientific discovery. A mathematical discovery is the rigorous verification of a formal statement, realized by bringing together a set of pre-existing theorems, conjectures, axi- oms, and so on. It is therefore part of a larger category of innovation processes in which the introduction of new ideas and concepts is crucial to intellectual progress. Innovation processes can be described by the notion of “adjacent possible expansion” introduced by Kauffman (2000). This term refers to the expansion or restructuring of the possible knowledge space, triggered by the introduction of novel concepts. This type of process has been shown to leave distinctive traces in the statistical properties of the knowledge produced, expressed by two key laws first observed in linguistics: Zipf’s law and Heaps’ Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science Page 3 of 15 law (Tria et al. 2018). In Sect. Introduction 3.3 we show that Polymath’s discovery dynamics exhibit the markers of adjacent possible expansion processes, similar to literary production and musical innovation. Finally, in Sect. 3.4 we examine the triggering factors for innovation and show that no rule determines a priori who the key innovators will be, as peripheral users can sometimes steer collective work in new directions. Related workh There is an extensive and multidisciplinary literature on online collaborative systems, and on interaction patterns in large-scale collaborations: the case of Linux is, for exam- ple, analyzed in Maillart et al. (2008), Wikipedia in Voß (2005), Yasseri and Kertész (2013), Ciampaglia and Vancheri (2010), Yasseri et al. (2012), Kittur and Kraut (2008) and GitHub in Thung et al. (2013), Guzman et al. (2014), Sornette et al. (2014). h However, few works focus on large-scale scientific collaboration and even less on Pol- ymath projects. In addition to the reflections by Gowers himself (Gowers and Nielsen 2009), a descriptive analysis of Polymath 1 project can be found in Barany (2010), where the authors provide a qualitative discussion of the rules that Polymath contrib- utors developed to organize their work. For a more quantitative analysis of the initia- tive, but limited to the first project, see Cranshaw and Kittur (2011). Kloumann et al. (2016) are—to our knowledge—the only authors that have presented a statistical analysis of multiple Polymath projects. Their analysis compares full Polymath projects with the side initiatives of “Mini-Polymath projects”, which are smaller collaborations concern- ing Math Olympics questions that, while quite difcult, have known solutions. A detailed description of the collective problem-solving approach in the third Mini-Polymath has also been provided by Pease and Martin (2012). Drawing on this literature, our paper develops in three directions. First, we confirm and extend the results on the distribution of labor obtained by Cranshaw and Kittur (2011) on Polymath 1, for the five projects that achieved a final peer-review publication. We also extend this research by considering interaction patterns among contributors. Second, we investigate the productivity of collective intelligence in collaborative sys- tems. Following Sornette’s work on GitHub (Sornette et al. 2014), we study the super- linearity of production as a function of the number of users. Finally, based on innovation studies in online systems, such as the one presented in Tria et al. (2018), we introduce an innovation measure for the mathematical production process and identify the actors responsible for introducing innovations in the Polymath ecosystem. Demography of the projects The five projects we analyzed contain between 545 and 3363 posts and the number of contributors varies from 57 to 199. Detailed information about each project can be found in Fig. 1. The network in Fig. 1 represents the bipartite network of contributors and projects: In the graph, every edge represents an author’s participation in a pro- ject. The size of the contributors’ nodes represents the number of their contributions. The graph shows that there is a small core of very active authors who have partici- pated in almost all projects, and a periphery of occasional contributors working on a single project. Data and methods We collected all the posts from Polymath projects 1, 4, 5, 8, and 15, starting from the links listed on the Polymath project wiki page (The Polymath Wiki 2021). The corpus for each project consists of a collection of posts identified by publication date, author, text, and parent post (for posts written in response to another contribution). The posts were published primarily on three blogs: Timothy Gowers’s blog (2021), Ter- ence Tao’s blog (2021) and The Polymath blog (2021). Each of these blogs entails dif- ferent technical restrictions on author interaction. On Gowers’s blog, comments can only be posted in the main threads, limiting the depth of discussion and preventing authors from responding to comments in sub-threads. In contrast, Tao’s blog allows Gargiulo et al. Applied Network Science (2022) 7:19 Page 4 of 15 Gargiulo et al. Applied Network Science Fig. 1 The table on the left shows the number of posts, the number of contributors and the periods of development for each project. The network on the right represents the participation of each author to the projects Fig. 1 The table on the left shows the number of posts, the number of contributors and the periods of development for each project. The network on the right represents the participation of each author to the projects comments up to a depth of 4. The Polymath blog does not appear to limit nested com- ments at any level and shows comments up to a depth of 10. comments up to a depth of 4. The Polymath blog does not appear to limit nested com- ments at any level and shows comments up to a depth of 10. 1 A Planar Maximally Filtered Graph is a filtered graph obtained by subsequently adding edges to a graph that was originally deprived of all its edges. Edges are added in a descending order according to their weights if and only if the resulting graph can still be embedded in a planar surface. A Planar Maximally Filtered Graph preserves the hierarchical organization of the Minimum Spanning Tree but contains a larger amount of information in its structure and proves to be efficient in filtering relevant information about the clustering of the system. Topic extraction We then aggregate different mathematical concepts into topics. This aggregation allows us to study the collaboration between authors and the structure of the collective labor. To define topics, we create a co-occurrence network for each Polymath project, where the nodes represent different mathematical concepts. In this network, two concepts are connected if there is at least one post in which they were discussed together. The net- work is weighted according to the number of co-occurrences in different posts. Since this network is highly connected and extremely complex, we filter the edges to highlight relevant structures. In order to do so, we compute the Planar Maximally Filtered Graph1 (PMFG) proposed in Tumminello et al. (2005). We then define our topics as the clus- ters of mathematical concepts identified by the Louvain community detection algorithm (Blondel et al. 2008) over the PMFG graph. Table 2 shows the number of topics extracted for each project and the modularity of the partition of keywords in the filtered co-occur- rences network. Using this definition of topics, we label each post with the topic whose keywords appear most frequently in the text. In case of a tie, no label is assigned to the post. Therefore, in addition to its publication time (t), author ( α ), and content (K), each post is also characterized by a topic label (T): i = (ti, αi, Ki, Ti). Contents’ identification Since we are interested in reconstructing the collaborative processes that led to the discovery of a mathematical a solution, we need to identify the mathematical objects used in the posts. Natural language processing techniques performed poorly on this task and tended to identify non-mathematical terms, such as features of each par- ticipant’s personal language patterns. Therefore, we built a mathematical vocabulary by means of a two-steps protocol. First, we collected the titles of all Wikipedia pages labeled as “mathematics” (Lists of mathematics topics 2021). Second, we added to the list all the expressions “Theorem of ”, “’s conjecture”, etc. extracted from the cor- pora. The dictionary we obtained contains 25.035 mathematical concepts. Table 1 shows the number of independent mathematical concepts retrieved in the projects: in expressions like “theorem of ”, the sub-string “theorem” is not considered as an inde- pendent concept. Through this mathematical dictionary, the content of each post can be qualified by the set of mathematical concepts that it contains: Ki = {kw1, . . . , kwm} . A post will thus be generally characterized by its time (t), its author ( α ) and its con- tent (K): i = (ti, αi, Ki). Page 5 of 15 Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science Table 1 Number of independent mathematical concepts in the corpora Project Concepts 1 1040 4 768 5 1089 8 1116 15 661 Table 1 Number of independent mathematical concepts in the corpora Project Concepts 1 1040 4 768 5 1089 8 1116 15 661 Table 2 Number of topics per project Project Topics Modularity 1 9 0.70 4 9 0.75 5 10 0.72 8 12 0.72 15 9 0.69 Similarity and innovation We first define the semantic similarity between two posts using the Jaccard measure between their contents: Jij = (Ki ∩Kj)/(Ki ∪Kj) . We tweak this similarity by consid- ering the temporal distance among the posts, thus introducing the semantic-temporal similarity: Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science Page 6 of 15 (1) i,j = Ji,je−\|ti−tj\|/τ0, i,j = Ji,je−\|ti−tj\|/τ0, (1) where τ0 is the average time distance among all the pairs of posts (within each project). According to this measure, two posts that are similar in content but distant in time will be less similar than according to the standard Jaccard measure. where τ0 is the average time distance among all the pairs of posts (within each project). According to this measure, two posts that are similar in content but distant in time will be less similar than according to the standard Jaccard measure. We use the semantic-temporal similarity measure to define an innovation index for each post. First we define two separate indicators for each post: • The in-debate index measures the similarity between a post and the contents pub- lished before it. It is calculated as the average of the semantic-temporal similarity from the previous posts: (2) νi = j\|tj<ti ij j\|tj<ti 1 . νi = j\|tj<ti ij j\|tj<ti 1 . (2) • The impact index measures how much a post content is reproduced in the posts fol- lowing it. It is calculated as the average Jaccard similarity with the following posts: • The impact index measures how much a post content is reproduced in the posts fol- lowing it. It is calculated as the average Jaccard similarity with the following posts: ξi = j\|tj>ti Jij j\|tj>ti 1 . (3) ξi = j\|tj>ti Jij j\|tj>ti 1 . (3) An innovative post is characterized by a low in-debate index (i.e., it is different from the earlier content) and a high impact (i.e., it influences the following contents that are therefore similar to it). For this reason we define the innovation index for each post as: (4) Ii = −ξilog(νi). Ii = −ξilog(νi). (4) Organization of labor As usual in collaborative systems, only a few contributors do most of the work (Bara- basi 2003). When we analyze the number of contributions made by each author, we find a power-law distribution (Fig. 2B) and high Gini indices (Fig. 2C). In Fig. 2A we rep- resented this distribution in the form of the Lorenz curve: authors are ordered by the number of contributions and curves represent the cumulative fraction of posts produced by the corresponding fraction of ranked authors. From the figure, we can see that the most active 10% produce the 80% of the posts (with the exception of project 4, which is characterized by a lower Gini index, where the 20% of contributors produce the 80% of the posts). Following the procedure described in Bassolas et al. (2019), we use the Lorenz curve to categorize authors hierarchically: We take the derivative of the Lorenz curve at the point (1,1) and set an initial threshold at the point where the derivative crosses the horizontal axis (as you can see in Fig. 2A). The authors after this threshold represent the most productive elite of the project. We remove these elite contributors and we repeat the procedure recur- sively, identifying a group we define as the first shell (highly active authors but outside the hyperactive elite) at the first iteration, and the peripheral shells (namely shells E3, E4, E5, E6 in Fig. 2D) at subsequent iterations. In Fig. 2C, we display the number of contributors Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science (2022) 7:19 Page 7 of 15 Fig. 2 A Lorenz curve for authors’ contributions to the projects. B Empirical probability distribution of the number of contributions by author. C Statistics about contributors activity. D Elite–peripheries structure of the projects Fig. 2 A Lorenz curve for authors’ contributions to the projects. B Empirical probability distribution of the number of contributions by author. C Statistics about contributors activity. D Elite–peripheries structure of the projects in the elite group and in the first shell, while Fig. 2D shows the percentage of authors in each hierarchical category. We can see that, according to this classification, the elite group contains less than 10% of the authors while the peripheral shells are consistently the most represented. In the following we will refer to authors belonging to the elite and the first shell as the active core. Interactions between the authors 3 Interactions through comments: A Boxplots represent the distribution of the fraction of in-core links after 100 simulations. ⋄ represents the fraction of in-core links in the Polymath dataset. B Boxplots represent the distribution of the fraction of in-periphery links after 100 simulations. ⋄ represents the fraction of in-periphery links in the Polymath dataset Fig. 3 Interactions through comments: A Boxplots represent the distribution of the fraction of in-core links after 100 simulations. ⋄ represents the fraction of in-core links in the Polymath dataset. B Boxplots represent the distribution of the fraction of in-periphery links after 100 simulations. ⋄ represents the fraction of in-periphery links in the Polymath dataset in-core links in our dataset. Figure 3B shows the same comparison for the in-periphery links, i.e., the fraction of messages written by peripheral contributors directed to other peripheral contributors. Both plots show a peculiar division of labor in the Polymath project: both core-to-core and periphery-to-periphery links are more represented than in random simulations, underlining that authors are more likely to reply to contributors who participate in the discovery process to a similar extent. As mentioned in the Data and Methods section, some of the blogs we studied limit the depth of response structures. We qualitatively observed a shift from a non-hierarchical structure in the very first project (i.e., only the presence of second-level comments and no deeper structures) to a more structured organization of posts in later projects. To evaluate the robustness of the results presented in Fig. 3, we compared them with the results obtained with a different definition of network interactions. We define a topic interaction network TIN(T) = (V, E(T), W(T)) with the following properties: the node set V still represents the set of authors, an edge (i, j) ∈E represents the fact that authors i and j published a post on the same topic at a distance no bigger than T posts (when posts are ordered chronologically). The weight Wij(T) , associated to the edge (i, j), rep- resents the number of times author i and j published a post on the same topic in the time window defined by parameter T. Notice that, by definition, such a network is undirected. Interactions between the authors To better understand the division of labor in Polymath, we investigated the distribu- tion of interactions between authors. In particular, we focused on how the active core authors, as defined in Sect. 3.1, interact with the peripheral shells. i In order to do so, based on the dependencies between posts, we defined a comments interaction network CIN = (V, E, W) with the following properties: each node i ∈V rep- resents an author, an edge (i, j) ∈E represents the existence of at least one comment by author i to a post of author j, and the weight Wij associated to the edge (i, j) represents the number of times author i replied to a post of author j. To understand whether such interactions are highly concentrated in the active core of elite authors or more spread towards peripheral contributors, we compared the obtained graphs with a stochas- tic network model preserving, on average, the activity level of each node. Similarly to Roth et al. (2013), we hence simulate K networks {Ŵk = (V, Ek, Qk)}k∈{1,...,K} with an expected degree for each node equal to the one of the authors of our dataset, keeping the same number of nodes n = \|V\| and edges m = \|E\| = \|Ek\| for all k ∈{1, . . . , K} . To do so we draw the weights Qk ij from a multinomial distribution with parameters m and p = {pij}i,j∈V such that pij = dout i · din j m2 where dout i is the out-degree of node i and din j is the in-degree of node j in the com- ments interaction network. Figure 3A shows the distribution of the fraction of in-core links (i.e., the fraction of messages from elite contributors to other elite contributors) in our K = 100 simulations and compares these distributions with the actual fraction of where dout i is the out-degree of node i and din j is the in-degree of node j in the com- ments interaction network. Figure 3A shows the distribution of the fraction of in-core links (i.e., the fraction of messages from elite contributors to other elite contributors) in our K = 100 simulations and compares these distributions with the actual fraction of Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science (2022) 7:19 Page 8 of 15 Fig. Interactions between the authors A Percentage of links stemming from a core node and ending in a core node over the total number of edges stemming from core nodes. B Percentage of links stemming from peripheral nodes and ending in peripheral nodes over the total number of edges stemming from peripheral nodes. The increasing transparency corresponds to the increase of the time window regardless the definition of the window, in-core and in-periphery interactions are higher in the actual Polymath projects than in simulated networks. Moreover, results obtained on the topic interaction network confirm the ones obtained on the comment interaction network. We can thus conclude that in the Polymath collaborations, elite actors inter- act more with other elite actors, while peripheral actors preferentially respond to other peripheral actors. This result is consistent with the forms of status-based homophily observed by McPherson in social networks (McPherson and Smith-Lovin 1987). This is however surprising in a scientific context where interactions are generally assumed to be based on cumulative advantage processes (Merton 1968). Interactions between the authors Once again, in order to study authors interactions, we need to compare them with a set of simulated networks {Ŵk = (V, Ek, Qk)}k∈{1,...,K} where m = \|E\| = \|Ek\|, k ∈{1, . . . , K} . To do so, it is now sufficient to draw the solely values {Qij}i,j∈V,j≥i from a multinomial random distribution, as we want the network to be undirected and Qk ij = Qk ji for all the K simulations. Therefore, we draw the values {Qij}i,j∈V,j≥i from a multinomial distribution of parameters m and p = {pij}i,j∈V,j≥i such that pij = 2di · dj m2 if i = j pii = di · di m2 otherwise, pij = 2di · dj m2 if i = j pii = di · di m2 otherwise, where di is the degree of node i in the topic interaction network. The resulting distri- bution of in-core and in-periphery interactions is shown in Fig. 4. We notice that, where di is the degree of node i in the topic interaction network. The resulting distri- bution of in-core and in-periphery interactions is shown in Fig. 4. We notice that, Page 9 of 15 Gargiulo et al. Applied Network Science (2022) 7:19 (2022) 7:19 Gargiulo et al. Applied Network Science Fig. 4 Interactions on the same topic: boxplots represent the outcome of simulations while ⋄ markers represents real values coming from data. A Percentage of links stemming from a core node and ending in a core node over the total number of edges stemming from core nodes. B Percentage of links stemming from peripheral nodes and ending in peripheral nodes over the total number of edges stemming from peripheral nodes. The increasing transparency corresponds to the increase of the time window Fig. 4 Interactions on the same topic: boxplots represent the outcome of simulations while ⋄ markers represents real values coming from data. A Percentage of links stemming from a core node and ending in a core node over the total number of edges stemming from core nodes. B Percentage of links stemming from peripheral nodes and ending in peripheral nodes over the total number of edges stemming from peripheral nodes. The increasing transparency corresponds to the increase of the time window Fig. 4 Interactions on the same topic: boxplots represent the outcome of simulations while ⋄ markers represents real values coming from data. Collective intelligence at work Several studies on collaborative systems have shown a super-linear effect of collabora- tion: The very expression “collective intelligence” suggests that the collective productivity (in our case the number of posts) is higher than the sum of the individual productions. Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science Page 10 of 15 Fig. 5 A Superlinear growth of the number of posts with the number of users. B Average daily individual productivity as a function of the number of users. C Average daily individual productivity of core authors as a function of the number of peripheral contributors Fig. 5 A Superlinear growth of the number of posts with the number of users. B Average daily individual productivity as a function of the number of users. C Average daily individual productivity of core authors as a function of the number of peripheral contributors Fig. 5 A Superlinear growth of the number of posts with the number of users. B Average daily individual productivity as a function of the number of users. C Average daily individual productivity of core authors as a function of the number of peripheral contributors To test this feature dynamically, we count the daily number of posts and the daily num- ber of participants for all projects: npost(t) = [npost(t0), npost(t1), . . .] nuser(t) = [nuser(t0), nuser(t1), . . .], npost(t) = [npost(t0), npost(t1), . . .] nuser(t) = [nuser(t0), nuser(t1), . . .], nuser(t) = [nuser(t0), nuser(t1), . . .], nuser(t) = [nuser(t0), nuser(t1), . . .], where t0, t1, . . . represent different days. To reduce noise, we smooth these time series with a 7-days rolling window. By plotting the pairs (nuser(t), npost(t)) , we obtain the curves representing the relationship between the number of users and the number of posts. Figure 5A shows a pronounced superlinear growth of the number of posts with the number of users, aggregated for all projects: npost = nγ user (with exponent γ = 1, 46 ). Our results are similar to those of Sornette et al. (2014) for GitHub. Figure 5B, C suggest that contributions have positive super-linear effects, even when they are relatively marginal. In Fig. 5B, we show that the average individual daily pro- duction (for all contributors with more than 10 posts in all the projects) grows with the number of users active on that day. Collective intelligence at work Figure 5C displays the average daily productivity of the active core as a function of the number of users in the peripheral shells. We observe that an important presence of peripheral users boosts the productivity of the most active users. In Fig. 5, we show the results obtained by aggregating all the projects. The individ- ual analysis of each blog shows similar trends with very small variations in the growth exponents (blog1: γ = 1.30 , blog4: γ = 1.22 , blog5: γ = 1.46 , blog8: γ = 1.65 , blog15: γ = 1.50 ). Since the blog platforms are diverse, the robustness of these results suggests super-productivity to be an intrinsic characteristic of collaborative science, regardless the communication medium. Statistical properties of scientific discoveries While in the previous sections we analyzed collaborative patterns in open science, we now focus on the analysis of the scientific discovery process itself. Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science (2022) 7:19 Page 11 of 15 Fig. 6 Left plot: the rank-frequency relationship respects the Zipf’s law with the exponents indicated in the plot. Right plot: the relationship between the text length and the vocabulary size respect the Heaps’ law with the exponents indicated in the plot Fig. 6 Left plot: the rank-frequency relationship respects the Zipf’s law with the exponents indicated in the plot. Right plot: the relationship between the text length and the vocabulary size respect the Heaps’ law with the exponents indicated in the plot First, we analyze the statistical properties of the mathematical concepts used in the projects. As described in the Methods Section, we have assigned a set of mathematical concepts to each post. We first test whether our corpus follows the basic laws of linguis- tic patterns: Zipf’s Law and Heaps’ Law. Zipf’s law expresses the relationship between the frequency and the ranking of words. It states that the frequency of a word is inversely correlated with its rank, f ∼r−α . For example, looking at the Gutenberg Project corpus (a large sample of English literature), one can observe a value α ∼−1 for low values of r and α ∼−2 for high values of r. Heaps’ law concerns the entry of innovative concepts into a text and expresses the relationship between the number of different words (i.e., the vocabulary size) and the total number of words used (i.e., the length of the text). It describes an initial linear growth followed by an asymptotic behavior according to the power law l = vα : in the Gutenberg corpus α ∼1 for low values of l and α ∼0.44 for high values of l have been observed. In Fig. 6, we see that not only are these laws respected in our corpus, but also all projects have the same behavior and exponents, Zipf’s exponents being α = −0.36 and α = −2 and Heaps’ exponents being α = 0.9 and α = 0.4 . These values are consistent with those from Gutenberg corpus (Tria et al. Statistical properties of scientific discoveries 2018), although the first exponent of Zipf’s law in our corpora is lower, due to the fact that we removed the non-mathematical expressions and stop words. This consistency means that, statistically, the creative process of scientific discovery follows the same basic rules that characterize literary production. Second, we focused on the typical timing of the discovery process, based on the the hypothesis that posts that are close in time would also tend to be similar in terms of content. In Fig. 7 we show the average Jaccard similarity between all pairs of posts pub- lished within a given time delay. We observe a power law decay of similarity with time, J ∼t−γ (with γ = 0.2 ), once again similar for all projects. Thus, for all projects, there exists a typical time window in which the debate remains focused on the same topic before switching to new one. Conclusion Over the past few decades, we have witnessed the rise of large online collaborations such as Linux, GitHub, and Wikipedia. In 2009, the first Polymath project was launched with the goal of exploiting online collaborative environments to solve mathematical problems. In this work, we have investigated how the path to scientific discovery develops in this collaborative environment, how the labor is organized between authors, and which actors are the main innovators. Our results, which are consistent with previous works, show that productivity is highly skewed between contributors and that there is a small hyper-productive elite that publishes the bulk of the contributions. Nonetheless, periph- eral contributors also play a significant role, as content production grows super-linearly with the number of discussants. Our analysis shows that, in Polymath projects, peripheral contributions boost the activity of other authors in a rather indirect way. Although interactions between the elite and the rest of the participants are relatively limited (as both peripheral and hyper-pro- ductive authors tend to interact mainly with authors with similar levels of activity), we have demonstrated that peripheral authors often play a crucial role in bringing new and innovative ideas to the debate. Our analysis has also shown that innovators cannot be defined only by their productivity level. Sometimes, occasional contributors can play a key role in innovation and be responsible for steering the research in new directions. In this exploration of the Polymath ecosystem, we focused on four main directions: classifying contributors by their involvement in the projects, analyzing interactions among contributors, examining the impact of large-scale collaboration on productivity, and finally identifying the actors responsible for innovation. We conducted only a lim- ited analysis of the content of the posts and of the semantic relationships between them. In a follow-up study, it would be interesting to examine the relationships between con- tributions based on the similarity of the content they produced rather than considering only their direct interactions in the response network. This structure would allow us to analyze the thematic cooperation patterns between contributors. This similarity network would also allow us to characterize the internal composition of users’ “opinions” on solu- tion techniques and their complex dynamics. Funding Funding This research has been supported by CNRS through the 80 PRIME MITI project “Disorders of Online Media” (DOOM). This work has been partially supported by MIAI@Grenoble Alpes (ANR-19-P3IA-0003) and by ANR grant HANDY (ANR-18-CE40-0010). Innovation patterns Finally, we analyze how innovations affects the discovery mechanism, by using the innovation measure we defined in Sect. 2.4. As observed in Fig. 8A, the innovation Page 12 of 15 Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science Fig. 7 Average distance between posts at a certain temporal distance Fig. 8 Plot A Distributions of innovation for each project. Plot B Innovation z-score for each activity shell Fig. 7 Average distance between posts at a certain temporal distance Fig. 7 Average distance between posts at a certain temporal distance Fig. 8 Plot A Distributions of innovation for each project. Plot B Innovation z-score for each activity shell Fig. 8 Plot A Distributions of innovation for each project. Plot B Innovation z-score for each activity shell values’ distributions are long-tailed, meaning that few posts have a much larger innovative content compared to the others: high innovation is rare, but statistically significant. i We define posts in the top quartile of the innovation distribution as innovative. Then, referring to the definition of activity shells introduced in Sect. 3.1, we examine which actors lead innovation. Since the groups vary in size, we compare the number of innova- tions observed in each class with their multinomial expectation, namely the probability that a post is innovative (25%) multiplied by the number of posts produced by the group. We calculate the z-score between the observed and expected values. While the previ- ous results showed a fairly homogeneous behavior between the different projects, here we observe significant differences. In projects 1,4,8, the elite produces more innovation than expected. In project 15, the first shell is the main driver of innovation. Finally, in project 5, the peripheral shells are the largest producers of innovation. This result high- lights that in large-scale collaborations no rule determines a priori who will be the main innovators. An innovator can be a member of the hyper-active elite, but sometimes ser- endipitous interactions of peripheral participants can also have a large impact on the discovery process: an isolated contribution of an occasional participant can be responsi- ble for opening a large adjacent possible and giving a new direction to the work. Gargiulo et al. Applied Network Science (2022) 7:19 Page 13 of 15 Gargiulo et al. Applied Network Science (2022) 7:19 Gargiulo et al. Applied Network Science Abbreviations l Abbreviations PMFG: Planar maximally filtered graph; CIN: Comments interaction network; TIN: Topic interaction network. Authors’ contributions G d h h F.G. conceived the research, collected and analyzed the data, discussed the results, wrote the manuscript. M.C. collected and analyzed the data, discussed the results, wrote the manuscript. T.V. discussed the results, wrote the manuscript. P.F. discussed the results, wrote the manuscript. All authors have read and approved the manuscript. Conclusion Finally, it would be interesting in future works to compare the results obtained from the Polymath dataset with other online col- laborative environments, in particular, to analyze the relationship between the level of participation and innovation, which remains largely unexplored in the literature. Received: 20 September 2021 Accepted: 6 March 2022 Received: 20 September 2021 Accepted: 6 March 2022 Author details Author details 1 CNRS, Université Paris-Sorbonne - Paris IV, GEMASS, Paris, France. 2 CNRS, Inria, Grenoble INP, GIPSA‑Lab, Univ. Grenoble Alpes, Grenoble, France. 3 CNRS, CIS-lab, Paris, France. References Anthony D, Smith SW, Williamson T (2009) Reputation and reliability in collective goods: the case of the online encyclopedia Wikipedia. Ration Soc 21(3):283–306 Anthony D, Smith SW, Williamson T (2009) Reputation and reliability in collective goods: the case of the online encyclopedia Wikipedia. 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